@article{nguyen_jameson_baldwin_nardini_smith_haugh_flores_2024, title={Quantifying collective motion patterns in mesenchymal cell populations using topological data analysis and agent-based modeling}, volume={370}, ISSN={["1879-3134"]}, DOI={10.1016/j.mbs.2024.109158}, abstractNote={Fibroblasts in a confluent monolayer are known to adopt elongated morphologies in which cells are oriented parallel to their neighbors. We collected and analyzed new microscopy movies to show that confluent fibroblasts are motile and that neighboring cells often move in anti-parallel directions in a collective motion phenomenon we refer to as "fluidization" of the cell population. We used machine learning to perform cell tracking for each movie and then leveraged topological data analysis (TDA) to show that time-varying point-clouds generated by the tracks contain significant topological information content that is driven by fluidization, i.e., the anti-parallel movement of individual neighboring cells and neighboring groups of cells over long distances. We then utilized the TDA summaries extracted from each movie to perform Bayesian parameter estimation for the D'Orsgona model, an agent-based model (ABM) known to produce a wide array of different patterns, including patterns that are qualitatively similar to fluidization. Although the D'Orsgona ABM is a phenomenological model that only describes inter-cellular attraction and repulsion, the estimated region of D'Orsogna model parameter space was consistent across all movies, suggesting that a specific level of inter-cellular repulsion force at close range may be a mechanism that helps drive fluidization patterns in confluent mesenchymal cell populations.}, journal={MATHEMATICAL BIOSCIENCES}, author={Nguyen, Kyle C. and Jameson, Carter D. and Baldwin, Scott A. and Nardini, John T. and Smith, Ralph C. and Haugh, Jason M. and Flores, Kevin B.}, year={2024}, month={Apr} }
@article{rahman_zhou_deiters_haugh_2023, title={Dissection of MKK6 and p38 Signaling Using Light-Activated Protein Kinases}, volume={11}, ISSN={["1439-7633"]}, DOI={10.1002/cbic.202300551}, abstractNote={Abstract}, journal={CHEMBIOCHEM}, author={Rahman, Shah Md. Toufiqur and Zhou, Wenyuan and Deiters, Alexander and Haugh, Jason M.}, year={2023}, month={Nov} }
@article{appalabhotla_butler_bear_haugh_2023, title={G-actin diffusion is insufficient to achieve F-actin assembly in fast-treadmilling protrusions}, volume={122}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2023.08.022}, abstractNote={Abstract
To generate forces that drive migration of a eukaryotic cell, arrays of actin filaments (F-actin) are assembled at the cell's leading membrane edge. To maintain cell propulsion and respond to dynamic external cues, actin filaments must be disassembled to regenerate the actin monomers (G-actin), and transport of G-actin from sites of disassembly back to the leading edge completes the treadmilling cycle and limits the flux of F-actin assembly. Whether or not molecular diffusion is sufficient for G-actin transport has been a long-standing topic of debate, in part because the dynamic nature of cell motility and migration hinders the estimation of transport parameters. In this work, we applied an experimental system in which cells adopt an approximately constant and symmetrical shape; they cannot migrate but exhibit fast, steady treadmilling in the thin region protruding from the cell. Using fluorescence recovery after photobleaching, we quantified the relative concentrations and corresponding fluxes of F- and G-actin in this system. In conjunction with mathematical modeling, constrained by measured features of each region of interest, this approach revealed that diffusion alone cannot account for the transport of G-actin to the leading edge. Although G-actin diffusion and vectorial transport might vary with position in the protruding region, good agreement with the fluorescence recovery after photobleaching measurements was achieved by a model with constant G-actin diffusivity ∼2 μm2/s and anterograde G-actin velocity less than 1 μm/s.}, number={18}, journal={BIOPHYSICAL JOURNAL}, author={Appalabhotla, Ravikanth and Butler, Mitchell T. and Bear, James E. and Haugh, Jason M.}, year={2023}, month={Sep}, pages={3816–3829} }
@article{zhou_ryan_janosko_shoger_haugh_gottschalk_deiters_2023, title={Isoform-specific optical activation of kinase function reveals p38-ERK signaling crosstalk}, volume={8}, ISSN={["2633-0679"]}, DOI={10.1039/d2cb00157}, journal={RSC CHEMICAL BIOLOGY}, author={Zhou, Wenyuan and Ryan, Amy and Janosko, Chasity P. and Shoger, Karsen E. and Haugh, Jason M. and Gottschalk, Rachel A. and Deiters, Alexander}, year={2023}, month={Aug} }
@article{zhou_ryan_janosko_shoger_haugh_gottschalk_deiters_2023, title={Isoform-specific optical activation of kinase function reveals p38-ERK signaling crosstalk}, volume={4}, ISSN={["2633-0679"]}, DOI={10.1039/d2cb00157h}, abstractNote={Protein isoforms are difficult to differentiate in a cellular context. Here, we describe the acute light control of individual isoforms of the mitogen-activated protein kinase (MAPK) p38, revealing a novel point of crosstalk between two MAPK pathways.}, number={10}, journal={RSC CHEMICAL BIOLOGY}, author={Zhou, Wenyuan and Ryan, Amy and Janosko, Chasity P. and Shoger, Karsen E. and Haugh, Jason M. and Gottschalk, Rachel A. and Deiters, Alexander}, year={2023}, month={Oct}, pages={765–773} }
@article{rahman_haugh_2023, title={On the inference of ERK signaling dynamics from protein biosensor measurements}, volume={34}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.E22-10-0476}, abstractNote={ Live-cell microscopy is used to infer dynamics in single cells. Here, ERK signaling was monitored using four different biosensors in a common cell context. Each responds with unique kinetics, reflecting differences in ERK localization and activity. Mathematical modeling offers an interpretation of the data and may be used to guide biosensor design. }, number={6}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Rahman, Shah Md. Toufiqur and Haugh, Jason M.}, year={2023}, month={May} }
@article{baldwin_haugh_2023, title={Semi-autonomous wound invasion via matrix-deposited, haptotactic cues}, volume={568}, ISSN={["1095-8541"]}, DOI={10.1016/j.jtbi.2023.111506}, abstractNote={Proper wound healing relies on invasion of fibroblasts via directed migration. While the related experimental and mathematical modeling literature has mainly focused on cell migration directed by soluble cues (chemotaxis), there is ample evidence that fibroblast migration is also directed by insoluble, matrix-bound cues (haptotaxis). Furthermore, numerous studies indicate that fibronectin (FN), a haptotactic ligand for fibroblasts, is present and dynamic in the provisional matrix throughout the proliferative phase of wound healing. In the present work, we show the plausibility of a hypothesis that fibroblasts themselves form and maintain haptotactic gradients in a semi-autonomous fashion. As a precursor to this, we examine the positive control scenario where FN is pre-deposited in the wound matrix, and fibroblasts maintain haptotaxis by removing FN at an appropriate rate. After developing conceptual and quantitative understanding of this scenario, we consider two cases in which fibroblasts activate the latent form of a matrix-loaded cytokine, TGFβ, which upregulates the fibroblasts' own secretion of FN. In the first of these, the latent cytokine is pre-patterned and released by the fibroblasts. In the second, fibroblasts in the wound produce the latent TGFβ, with the presence of the wound providing the only instruction. In all cases, wound invasion is more effective than a negative control model with haptotaxis disabled; however, there is a trade-off between the degree of fibroblast autonomy and the rate of invasion.}, journal={JOURNAL OF THEORETICAL BIOLOGY}, author={Baldwin, Scott A. and Haugh, Jason M.}, year={2023}, month={Jul} }
@article{nosbisch_bear_haugh_2022, title={A kinetic model of phospholipase C-?1 linking structure-based insights to dynamics of enzyme autoinhibition and activation}, volume={298}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2022.101886}, abstractNote={Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain-pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.}, number={5}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Nosbisch, Jamie L. and Bear, James E. and Haugh, Jason M.}, year={2022}, month={May} }
@article{chandra_butler_bear_haugh_2022, title={Modeling cell protrusion predicts how myosin II and actin turnover affect adhesion-based signaling}, volume={121}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2021.11.2889}, abstractNote={Orchestration of cell migration is essential for development, tissue regeneration, and the immune response. This dynamic process integrates adhesion, signaling, and cytoskeletal subprocesses across spatial and temporal scales. In mesenchymal cells, adhesion complexes bound to extracellular matrix mediate both biochemical signal transduction and physical interaction with the F-actin cytoskeleton. Here, we present a mathematical model that offers insight into both aspects, considering spatiotemporal dynamics of nascent adhesions, active signaling molecules, mechanical clutching, actin treadmilling, and nonmuscle myosin II contractility. At the core of the model is a positive feedback loop, whereby adhesion-based signaling promotes generation of barbed ends at, and protrusion of, the cell's leading edge, which in turn promotes formation and stabilization of nascent adhesions. The model predicts a switch-like transition and optimality of membrane protrusion, determined by the balance of actin polymerization and retrograde flow, with respect to extracellular matrix density. The model, together with new experimental measurements, explains how protrusion can be modulated by mechanical effects (nonmuscle myosin II contractility and adhesive bond stiffness) and F-actin turnover.}, number={1}, journal={BIOPHYSICAL JOURNAL}, author={Chandra, Ankit and Butler, Mitchell T. and Bear, James E. and Haugh, Jason M.}, year={2022}, month={Jan}, pages={102–118} }
@article{baldwin_van bruggen_koelbl_appalabhotla_bear_haugh_2021, title={Microfluidic devices fitted with "flowver" paper pumps generate steady, tunable gradients for extended observation of chemotactic cell migration}, volume={15}, ISSN={["1932-1058"]}, DOI={10.1063/5.0054764}, abstractNote={Microfluidics approaches have gained popularity in the field of directed cell migration, enabling control of the extracellular environment and integration with live-cell microscopy; however, technical hurdles remain. Among the challenges are the stability and predictability of the environment, which are especially critical for the observation of fibroblasts and other slow-moving cells. Such experiments require several hours and are typically plagued by the introduction of bubbles and other disturbances that naturally arise in standard microfluidics protocols. Here, we report on the development of a passive pumping strategy, driven by the high capillary pressure and evaporative capacity of paper, and its application to study fibroblast chemotaxis. The paper pumps—flowvers (flow + clover)—are inexpensive, compact, and scalable, and they allow nearly bubble-free operation, with a predictable volumetric flow rate on the order of μl/min, for several hours. To demonstrate the utility of this approach, we combined the flowver pumping strategy with a Y-junction microfluidic device to generate a chemoattractant gradient landscape that is both stable (6+ h) and predictable (by finite-element modeling calculations). Integrated with fluorescence microscopy, we were able to recapitulate previous, live-cell imaging studies of fibroblast chemotaxis to platelet derived growth factor (PDGF), with an order-of-magnitude gain in throughput. The increased throughput of single-cell analysis allowed us to more precisely define PDGF gradient conditions conducive for chemotaxis; we were also able to interpret how the orientation of signaling through the phosphoinositide 3-kinase pathway affects the cells’ sensing of and response to conducive gradients.}, number={4}, journal={BIOMICROFLUIDICS}, author={Baldwin, Scott A. and Van Bruggen, Shawn M. and Koelbl, Joseph M. and Appalabhotla, Ravikanth and Bear, James E. and Haugh, Jason M.}, year={2021}, month={Jul} }
@article{nosbisch_rahman_mohan_elston_bear_haugh_2020, title={Mechanistic models of PLC/PKC signaling implicate phosphatidic acid as a key amplifier of chemotactic gradient sensing}, volume={16}, ISSN={["1553-7358"]}, DOI={10.1371/journal.pcbi.1007708}, abstractNote={Chemotaxis of fibroblasts and other mesenchymal cells is critical for embryonic development and wound healing. Fibroblast chemotaxis directed by a gradient of platelet-derived growth factor (PDGF) requires signaling through the phospholipase C (PLC)/protein kinase C (PKC) pathway. Diacylglycerol (DAG), the lipid product of PLC that activates conventional PKCs, is focally enriched at the up-gradient leading edge of fibroblasts responding to a shallow gradient of PDGF, signifying polarization. To explain the underlying mechanisms, we formulated reaction-diffusion models including as many as three putative feedback loops based on known biochemistry. These include the previously analyzed mechanism of substrate-buffering by myristoylated alanine-rich C kinase substrate (MARCKS) and two newly considered feedback loops involving the lipid, phosphatidic acid (PA). DAG kinases and phospholipase D, the enzymes that produce PA, are identified as key regulators in the models. Paradoxically, increasing DAG kinase activity can enhance the robustness of DAG/active PKC polarization with respect to chemoattractant concentration while decreasing their whole-cell levels. Finally, in simulations of wound invasion, efficient collective migration is achieved with thresholds for chemotaxis matching those of polarization in the reaction-diffusion models. This multi-scale modeling framework offers testable predictions to guide further study of signal transduction and cell behavior that affect mesenchymal chemotaxis.}, number={4}, journal={PLOS COMPUTATIONAL BIOLOGY}, author={Nosbisch, Jamie L. and Rahman, Anisur and Mohan, Krithika and Elston, Timothy C. and Bear, James E. and Haugh, Jason M.}, year={2020}, month={Apr} }
@article{rahman_zhou_deiters_haugh_2020, title={Optical control of MAP kinase kinase 6 (MKK6) reveals that it has divergent roles in pro-apoptotic and anti-proliferative signaling}, volume={295}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.RA119.012079}, abstractNote={The selective pressure imposed by extrinsic death signals and stressors adds to the challenge of isolating and interpreting the roles of proteins in stress-activated signaling networks. By expressing a kinase with activating mutations and a caged lysine blocking the active site, we can rapidly switch on catalytic activity with light and monitor the ensuing dynamics. Applying this approach to MAP kinase 6 (MKK6), which activates the p38 subfamily of MAPKs, we found that decaging active MKK6 in fibroblasts is sufficient to trigger apoptosis in a p38-dependent manner. Both in fibroblasts and in a murine melanoma cell line expressing mutant B-Raf, MKK6 activation rapidly and potently inhibited the pro-proliferative extracellular signal–regulated kinase (ERK) pathway; to our surprise, this negative cross-regulation was equally robust when all p38 isoforms were inhibited. These results position MKK6 as a new pleiotropic signal transducer that promotes both pro-apoptotic and anti-proliferative signaling, and they highlight the utility of caged, light-activated kinases for dissecting stress-activated signaling networks.}, number={25}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Rahman, Shah Md. Toufiqur and Zhou, Wenyuan and Deiters, Alexander and Haugh, Jason M.}, year={2020}, month={Jun}, pages={8494–8504} }
@article{tiruthani_mischler_ahmed_mahinthakumar_haugh_rao_2019, title={Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation}, volume={12}, ISSN={["1937-9145"]}, DOI={10.1126/scisignal.aap7584}, abstractNote={Engineered biosensors accurately report the kinetics of EGFR activation.}, number={584}, journal={SCIENCE SIGNALING}, author={Tiruthani, Karthik and Mischler, Adam and Ahmed, Shoeb and Mahinthakumar, Jessica and Haugh, Jason M. and Rao, Balaji M.}, year={2019}, month={Jun} }
@article{miller_lafosse_asokan_haugh_bear_elston_2019, title={Emergent spatiotemporal dynamics of the actomyosin network in the presence of chemical gradients}, volume={11}, ISSN={["1757-9708"]}, DOI={10.1093/intbio/zyz023}, abstractNote={Abstract}, number={6}, journal={INTEGRATIVE BIOLOGY}, author={Miller, Callie J. and LaFosse, Paul K. and Asokan, Sreeja B. and Haugh, Jason M. and Bear, James E. and Elston, Timothy C.}, year={2019}, month={Jun}, pages={280–292} }
@article{miller_lafosse_asokan_haugh_bear_elston_2019, title={Simulating Emergent Spatiotemporal Actomyosin Dynamics to Understand Spatial Regulation of Non-Muscle Myosin II}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.1372}, DOI={10.1016/J.BPJ.2018.11.1372}, abstractNote={The punctuated dynamics of cortical actomyosin are critical for cell migration in applications like cancer, wound healing, or morphogenesis, however the mechanical role and organization of cortical actomyosin is not well understood. We developed a Monte Carlo, particle-based computer simulation that resulted in emergent actomyosin asters. In particular, we were interested to understand the role of non-muscle myosin II's (motor) activation in emergent f-actin (filament) aster formation. Our model incorporates activation of individual motors through the transition from an inactive folded state to an active unfolded state, and the bundling of two active motors to form a processive motor capable of binding to filaments. In addition to considering how changing a single parameter affected the emergent filament asters, we introduced a spatial gradient of model parameters to mimic spatially controlled activation of motors, or filament polymerization. Recent studies have shown that regulation of motor activity is critical for directed fibroblast migration in response to a gradient of platelet derived growth factor, and we found that spatially inhibiting motor-filament binding resulted in spatial variations in filament aster formation. Additionally, motivated from studies with the small molecule ROCK inhibitor, Y-27632, and Calyculin A, which either disrupt or enhance the ability of non-muscle myosin II to exert force, and work from the Sellers lab on the biochemical properties of different types of co-assembled myosin isoforms, we simulated spatially controlled motor stiffness which directly affects the ability of motors to exert force to reorganize filaments. We found that not only was there a change in where asters emerged, but that we were able to generate a dynamic pulsatile aster structure where filament asters would dissipate and new asters would emerge. Results from our simulation will guide future experimentation for cortical actomyosin.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Miller, Callie J. and LaFosse, Paul and Asokan, Sreeja and Haugh, Jason and Bear, James E. and Elston, Timothy C.}, year={2019}, month={Feb}, pages={251a} }
@article{miller_asokan_haugh_bear_elston_2018, title={A Computational Investigation of Asymmetric Emergent Structures in Actomyosin Dynamics During Chemotaxis}, volume={114}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2017.11.2111}, DOI={10.1016/J.BPJ.2017.11.2111}, abstractNote={We have developed a particle-based computer simulation to study emergent properties of the actomyosin cytoskeleton. In particular, our model accounts for biophysical interactions between filamentous actin (f-actin) and non-muscle myosin II (NM II). Our investigations were motivated by recent studies that demonstrate regulation of myosin activity is critical for directed migration of fibroblasts responding to gradients of platelet derived growth factor so we have incorporated the dynamics for NM II formation. Individual NM II transition from a folded inactive state to an active unfolded state. Once active, two NM II bundle together to create a processive NM II mini-filament capable of binding to f-actin. We performed a parametric analysis that led to the identification of biophysical parameters that control the formation of f-actin asters. We identified that aster formation was sensitive to filament length and the ability of motors to exert a spring-like force via changes in the spring constant for motors, or the maximum stretch allowed. When we considered the steps for NM II assembly, we found that inhibiting motor-filament binding and not motor activation or motor bundling was responsible for disrupting the actin morphology. Extending the bulk parameter analysis, we simulated chemotaxis by introducing the parameters to the computational simulation in a spatial gradient. We found that spatially regulating the ability of NM II to bind to f-actin resulted in a significant variation in actomyosin morphology in space. Additionally, we were able to generate a dynamic pulsatile aster structure through spatially regulating motor stiffness. Our identification of spatial regulators with the computational simulation will help guide future experimentation.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Miller, Callie J. and Asokan, Sreeja and Haugh, Jason and Bear, James E. and Elston, Timothy C.}, year={2018}, month={Feb}, pages={381a} }
@article{mohan_nosbisch_elston_bear_haugh_2017, title={A Reaction-Diffusion Model Explains Amplification of the PLC/PKC Pathway in Fibroblast Chemotaxis}, volume={113}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2017.05.035}, abstractNote={Abstract
During the proliferative phase of cutaneous wound healing, dermal fibroblasts are recruited into the clotted wound by a concentration gradient of platelet-derived growth factor (PDGF), together with other spatial cues. Despite the importance of this chemotactic process, the mechanisms controlling the directed migration of slow-moving mesenchymal cells such as fibroblasts are not well understood. Here, we develop and analyze a reaction-diffusion model of phospholipase C/protein kinase C (PKC) signaling, which was recently identified as a requisite PDGF-gradient-sensing pathway, with the goal of identifying mechanisms that can amplify its sensitivity in the shallow external gradients typical of chemotaxis experiments. We show that phosphorylation of myristoylated alanine-rich C kinase substrate by membrane-localized PKC constitutes a positive feedback that is sufficient for local pathway amplification. The release of phosphorylated myristoylated alanine-rich C kinase substrate and its subsequent diffusion and dephosphorylation in the cytosol also serves to suppress the pathway in down-gradient regions of the cell. By itself, this mechanism only weakly amplifies signaling in a shallow PDGF gradient, but it synergizes with other feedback mechanisms to enhance amplification. This model offers a framework for a mechanistic understanding of phospholipase C/PKC signaling in chemotactic gradient sensing and can guide the design of experiments to assess the roles of putative feedback loops.}, number={1}, journal={BIOPHYSICAL JOURNAL}, author={Mohan, Krithika and Nosbisch, Jamie L. and Elston, Timothy C. and Bear, James E. and Haugh, Jason M.}, year={2017}, month={Jul}, pages={185–194} }
@article{rahman_haugh_2017, title={Kinetic Modeling and Analysis of the Akt/Mechanistic Target of Rapamycin Complex 1 (mTORC1) Signaling Axis Reveals Cooperative, Feedforward Regulation}, volume={292}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m116.761205}, abstractNote={Mechanistic target of rapamycin complex 1 (mTORC1) controls biosynthesis and has been implicated in uncontrolled cell growth in cancer. Although many details of mTORC1 regulation are well understood, a systems-level, predictive framework synthesizing those details is currently lacking. We constructed various mathematical models of mTORC1 activation mediated by Akt and aligned the model outputs to kinetic data acquired for growth factor-stimulated cells. A model based on a putative feedforward loop orchestrated by Akt consistently predicted how the pathway was altered by depletion of key regulatory proteins. Analysis of the successful model also elucidates two dynamical motifs: neutralization of a negative regulator, which characterizes how Akt indirectly activates mTORC1, and seesaw enzyme regulation, which describes how activated and inhibited states of mTORC1 are controlled in concert to produce a nonlinear, ultrasensitive response. Such insights lend quantitative understanding of signaling networks and their precise manipulation in various contexts.}, number={7}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Rahman, Anisur and Haugh, Jason M.}, year={2017}, month={Feb}, pages={2866–2872} }
@article{johnson_haugh_2016, title={Are Filopodia Privileged Signaling Structures in Migrating Cells?}, volume={111}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2016.09.022}, abstractNote={Filopodia are thin, fingerlike structures that contain bundled actin filaments and project from the cell periphery. These structures are dogmatically endowed with the ability to sense cues in the microenvironment, implying that filopodia foster local signal transduction, yet their small diameter hampers the imaging of dynamic processes therein. To overcome this challenge, we analyzed total internal reflection fluorescence images of migrating fibroblasts coexpressing either a plasma membrane marker or tagged AktPH domain, a translocation biosensor for signaling through the phosphoinositide 3-kinase pathway, along with a cytosolic volume marker. We devised a scheme to estimate the radii of filopodia using either the membrane marker or volume marker data, and we used that information to account for geometry effects in the biosensor data. With conservative estimates of relative target molecule abundance, it is revealed that filopodia typically harbor higher densities of 3' phosphoinositides than adjacent regions at the cell periphery. In this context at least, the analysis supports the filopodial signaling hypothesis.}, number={9}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Johnson, Heath E. and Haugh, Jason M.}, year={2016}, month={Nov}, pages={1827–1830} }
@article{king_asokan_haynes_zimmerman_rotty_alb_tagliatela_blake_lebedeva_marston_et al._2016, title={Lamellipodia are crucial for haptotactic sensing and response}, volume={129}, ISSN={["1477-9137"]}, DOI={10.1242/jcs.184507}, abstractNote={Haptotaxis is the process by which cells respond to gradients of substrate-bound cues such as extracellular matrix proteins (ECM), however the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECM of different concentrations. To study haptotaxis on gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin (FN), and imaged cell migration response. Lamellipodia are fan-shaped protrusions common in migrating cells. Here we define a novel function for lamellipodia and the cellular mechanism required for haptotaxis; differential actin and lamellipodial protrusion dynamics leading to biased cell migration. Modest differences in lamellipodial dynamics occurring over seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of FN. We identify a specific subset of lamellipodia regulators as being critical for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis, and consistent with this we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.}, number={12}, journal={JOURNAL OF CELL SCIENCE}, publisher={The Company of Biologists}, author={King, Samantha J. and Asokan, Sreeja B. and Haynes, Elizabeth M. and Zimmerman, Seth P. and Rotty, Jeremy D. and Alb, James G., Jr. and Tagliatela, Alicia and Blake, Devon R. and Lebedeva, Irina P. and Marston, Daniel and et al.}, year={2016}, month={Jun}, pages={2329–2342} }
@article{liu_asokan_bear_haugh_2016, title={Quantitative analysis of B-lymphocyte migration directed by CXCL13}, volume={8}, ISSN={["1757-9708"]}, DOI={10.1039/c6ib00128a}, abstractNote={B-lymphocyte migration, directed by chemokine gradients, is essential for homing to sites of antigen presentation. B cells move rapidly, exhibiting amoeboid morphology like other leukocytes, yet quantitative studies addressing B-cell migration are currently lacking relative to neutrophils, macrophages, and T cells. Here, we used total internal reflection fluorescence (TIRF) microscopy to characterize the changes in shape (morphodynamics) of primary, murine B cells as they migrated on surfaces with adsorbed chemokine, CXCL13, and the adhesive ligand, ICAM-1. B cells exhibited frequent, spontaneous dilation and shrinking events at the sides of the leading membrane edge, a phenomenon that was predictive of turning versus directional persistence. To characterize directed B-cell migration, a microfluidic device was implemented to generate gradients of adsorbed CXCL13 gradients. Haptotaxis assays revealed a modest yet consistently positive bias of the cell's persistent random walk behavior towards CXCL13 gradients. Quantification of tactic fidelity showed that bias is optimized by steeper gradients without excessive midpoint density of adsorbed chemokine. Under these conditions, B-cell migration is more persistent when the direction of migration is better aligned with the gradient.}, number={8}, journal={INTEGRATIVE BIOLOGY}, publisher={Royal Society of Chemistry (RSC)}, author={Liu, Xiaji and Asokan, Sreeja B. and Bear, James E. and Haugh, Jason M.}, year={2016}, pages={894–903} }
@article{lauffenburger_haugh_byrne_linding_2016, title={Systems Cell Biology: An Overview}, DOI={10.1016/b978-0-12-394447-4.40044-1}, journal={Encyclopedia of Cell Biology}, publisher={Elsevier BV}, author={Lauffenburger, D.A. and Haugh, J.M. and Byrne, H.M. and Linding, R.}, year={2016}, pages={1–3} }
@article{herring_grant_blackburn_haugh_goshe_2015, title={Development of a tandem affinity phosphoproteomic method with motif selectivity and its application in analysis of signal transduction networks}, volume={988}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2015.02.017}, abstractNote={Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, publisher={Elsevier BV}, author={Herring, Laura E. and Grant, Kyle G. and Blackburn, Kevin and Haugh, Jason M. and Goshe, Michael B.}, year={2015}, month={Apr}, pages={166–174} }
@article{johnson_king_asokan_rotty_bear_haugh_2015, title={F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling}, volume={208}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.201406102}, abstractNote={Mesenchymal cells such as fibroblasts are weakly polarized and reorient directionality by a lamellipodial branching mechanism that is stabilized by phosphoinositide 3-kinase (PI3K) signaling. However, the mechanisms by which new lamellipodia are initiated and directed are unknown. Using total internal reflection fluorescence microscopy to monitor cytoskeletal and signaling dynamics in migrating cells, we show that peripheral F-actin bundles/filopodia containing fascin-1 serve as templates for formation and orientation of lamellipodia. Accordingly, modulation of fascin-1 expression tunes cell shape, quantified as the number of morphological extensions. Ratiometric imaging reveals that F-actin bundles/filopodia play both structural and signaling roles, as they prime the activation of PI3K signaling mediated by integrins and focal adhesion kinase. Depletion of fascin-1 ablated fibroblast haptotaxis on fibronectin but not platelet-derived growth factor chemotaxis. Based on these findings, we conceptualize haptotactic sensing as an exploration, with F-actin bundles directing and lamellipodia propagating the process and with signaling mediated by adhesions playing the role of integrator.}, number={4}, journal={JOURNAL OF CELL BIOLOGY}, publisher={Rockefeller University Press}, author={Johnson, Heath E. and King, Samantha J. and Asokan, Sreeja B. and Rotty, Jeremy D. and Bear, James E. and Haugh, Jason M.}, year={2015}, month={Feb}, pages={443–455} }
@article{haynes_asokan_king_johnson_haugh_bear_2015, title={GMF beta controls branched actin content and lamellipodial retraction in fibroblasts}, volume={209}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.201501094}, abstractNote={The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMFβ, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMFβ decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMFβ in vivo. Furthermore, depletion or overexpression of GMFβ disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFβ plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration.}, number={6}, journal={JOURNAL OF CELL BIOLOGY}, publisher={Rockefeller University Press}, author={Haynes, Elizabeth M. and Asokan, Sreeja B. and King, Samantha J. and Johnson, Heath E. and Haugh, Jason M. and Bear, James E.}, year={2015}, month={Jun}, pages={803–812} }
@article{liu_welf_haugh_2015, title={Linking morphodynamics and directional persistence of T lymphocyte migration}, volume={12}, ISSN={["1742-5662"]}, DOI={10.1098/rsif.2014.1412}, abstractNote={T cells play a central role in the adaptive immune response, and their directed migration is essential for homing to sites of antigen presentation. Like neutrophils, T lymphocytes are rapidly moving cells that exhibit amoeboid movement, characterized by a definitive polarity with F-actin concentrated at the front and myosin II elsewhere. In this study, we used total internal reflection fluorescence (TIRF) microscopy to monitor the cells' areas of contact with a surface presenting adhesive ICAM-1 and the chemokine, CXCL12/SDF-1. Our analysis reveals that T-cell migration and reorientation are achieved by bifurcation and lateral separation of protrusions along the leading membrane edge, followed by cessation of one of the protrusions, which acts as a pivot for cell turning. We show that the distribution of bifurcation frequencies exhibits characteristics of a random, spontaneous process; yet, the waiting time between bifurcation events depends on whether or not the pivot point remains on the same side of the migration axis. Our analysis further suggests that switching of the dominant protrusion between the two sides of the migration axis is associated with persistent migration, whereas the opposite is true of cell turning. To help explain the bifurcation phenomenon and how distinct migration behaviours might arise, a spatio-temporal, stochastic model describing F-actin dynamics is offered.}, number={106}, journal={JOURNAL OF THE ROYAL SOCIETY INTERFACE}, author={Liu, Xiaji and Welf, Erik S. and Haugh, Jason M.}, year={2015}, month={May} }
@article{rotty_wu_haynes_suarez_winkelman_johnson_haugh_kovar_bear_2015, title={Profilin-1 Serves as a Gatekeeper for Actin Assembly by Arp2/3-Dependent and -Independent Pathways}, volume={32}, ISSN={["1878-1551"]}, DOI={10.1016/j.devcel.2014.10.026}, abstractNote={Cells contain multiple F-actin assembly pathways, including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay and/or competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2(-/-) cells lack lamellipodia and migrate more slowly than WT cells but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2(-/-) cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. Antagonism of the Arp2/3 complex by profilin-1 in cells appears to maintain actin homeostasis by balancing Arp2/3 complex-dependent and -independent actin assembly pathways.}, number={1}, journal={DEVELOPMENTAL CELL}, publisher={Elsevier BV}, author={Rotty, Jeremy D. and Wu, Congying and Haynes, Elizabeth M. and Suarez, Cristian and Winkelman, Jonathan D. and Johnson, Heath E. and Haugh, Jason M. and Kovar, David R. and Bear, James E.}, year={2015}, month={Jan}, pages={54–67} }
@article{vernekar_wallace_wu_chao_shannon k. o'connor_raleigh_liu_haugh_reichert_2014, title={Bi-ligand surfaces with oriented and patterned protein for real-time tracking of cell migration}, volume={123}, ISSN={["1873-4367"]}, DOI={10.1016/j.colsurfb.2014.09.020}, abstractNote={A bioactive platform for the quantitative observation of cell migration is presented by (1) presenting migration factors in a well-defined manner on 2-D substrates, and (2) enabling continuous cell tracking. Well-defined substrate presentation is achieved by correctly orienting immobilized proteins (chemokines and cell adhesion molecules), such that the active site is accessible to cell surface receptors. A thiol-terminated self-assembled monolayer on a silica slide was used as a base substrate for subsequent chemistry. The thiol-terminated surface was converted to an immobilized metal ion surface using a maleimido-nitrilotriacetic acid (NTA) cross-linker that bound Histidine-tagged recombinant proteins on the surface with uniform distribution and specific orientation. This platform was used to study the influence of surface-immobilized chemokine SDF-1α and cell adhesion molecule ICAM-1 on murine splenic B lymphocyte migration. While soluble SDF-1α induced trans-migration in a Boyden Chamber type chemotaxis assay, immobilized SDF-1α alone did not elicit significant surface-migration on our test-platform surface. Surface-immobilized cell adhesion protein, ICAM-1, in conjunction with activation enabled migration of this cell type on our surface. Controlled exposure to UV light was used to produce stable linear gradients of His-tagged recombinant SDF-1α co-immobilized with ICAM-1 following our surface chemistry approach. XPS and antibody staining showed defined gradients of outwardly oriented SDF-1α active sites. This test platform can be especially valuable for investigators interested in studying the influence of surface-immobilized factors on cell behavior and may also be used as a cell migration enabling platform for testing the effects of various diffusible agents.}, journal={COLLOIDS AND SURFACES B-BIOINTERFACES}, publisher={Elsevier BV}, author={Vernekar, Varadraj N. and Wallace, Charles S. and Wu, Mina and Chao, Joshua T. and Shannon K. O'Connor and Raleigh, Aimee and Liu, Xiaji and Haugh, Jason M. and Reichert, William M.}, year={2014}, month={Nov}, pages={225–235} }
@article{ahmed_grant_edwards_rahman_cirit_goshe_haugh_2014, title={Data-driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states}, volume={10}, ISSN={["1744-4292"]}, DOI={10.1002/msb.134708}, abstractNote={Abstract}, number={1}, journal={MOLECULAR SYSTEMS BIOLOGY}, publisher={Wiley-Blackwell}, author={Ahmed, Shoeb and Grant, Kyle G. and Edwards, Laura E. and Rahman, Anisur and Cirit, Murat and Goshe, Michael B. and Haugh, Jason M.}, year={2014}, month={Jan} }
@article{rahman_haugh_2014, title={Deactivation of a Negative Regulator: A Distinct Signal Transduction Mechanism, Pronounced in Akt Signaling}, volume={107}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2014.10.003}, abstractNote={Kinase cascades, in which enzymes are sequentially activated by phosphorylation, are quintessential signaling pathways. Signal transduction is not always achieved by direct activation, however. Often, kinases activate pathways by deactivation of a negative regulator; this indirect mechanism, pervasive in Akt signaling, has yet to be systematically explored. Here, we show that the indirect mechanism has properties that are distinct from direct activation. With comparable parameters, the indirect mechanism yields a broader range of sensitivity to the input, beyond saturation of regulator phosphorylation, and kinetics that become progressively slower, not faster, with increasing input strength. These properties can be integrated in network motifs to produce desired responses, as in the case of feedforward loops.}, number={10}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Rahman, Anisur and Haugh, Jason M.}, year={2014}, month={Nov}, pages={L29–L32} }
@article{bear_haugh_2014, title={Directed migration of mesenchymal cells: where signaling and the cytoskeleton meet}, volume={30}, ISSN={["1879-0410"]}, DOI={10.1016/j.ceb.2014.06.005}, abstractNote={Cell migration directed by spatial cues, or taxis, is a primary mechanism for orchestrating concerted and collective cell movements during development, wound repair, and immune responses. Compared with the classic example of amoeboid chemotaxis, in which fast-moving cells such as neutrophils are directed by gradients of soluble factors, directed migration of slow-moving mesenchymal cells such as fibroblasts is poorly understood. Mesenchymal cells possess a distinctive organization of the actin cytoskeleton and associated adhesion complexes as its primary mechanical system, generating the asymmetric forces required for locomotion without strong polarization. The emerging hypothesis is that the molecular underpinnings of mesenchymal taxis involve distinct signaling pathways and diverse requirements for regulation.}, journal={CURRENT OPINION IN CELL BIOLOGY}, publisher={Elsevier BV}, author={Bear, James E. and Haugh, Jason M.}, year={2014}, month={Oct}, pages={74–82} }
@article{asokan_johnson_rahman_king_rotty_lebedeva_haugh_bear_2014, title={Mesenchymal Chemotaxis Requires Selective Inactivation of Myosin II at the Leading Edge via a Noncanonical PLC gamma/PKC alpha Pathway}, volume={31}, ISSN={["1878-1551"]}, DOI={10.1016/j.devcel.2014.10.024}, abstractNote={Chemotaxis, migration toward soluble chemical cues, is critical for processes such as wound healing and immune surveillance and is exhibited by various cell types, from rapidly migrating leukocytes to slow-moving mesenchymal cells. To study mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of platelet-derived growth factor (PDGF). Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mammalian target of rapamycin signaling, are dispensable for PDGF chemotaxis. Instead, we find that local inactivation of Myosin IIA, through a noncanonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of live imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge are required for mesenchymal chemotaxis.}, number={6}, journal={DEVELOPMENTAL CELL}, publisher={Elsevier BV}, author={Asokan, Sreeja B. and Johnson, Heath E. and Rahman, Anisur and King, Samantha J. and Rotty, Jeremy D. and Lebedeva, Irina P. and Haugh, Jason M. and Bear, James E.}, year={2014}, month={Dec}, pages={747–760} }
@article{welf_johnson_haugh_2013, title={Bidirectional coupling between integrin-mediated signaling and actomyosin mechanics explains matrix-dependent intermittency of leading-edge motility}, volume={24}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.e13-06-0311}, abstractNote={ Animal cell migration is a complex process characterized by the coupling of adhesion, cytoskeletal, and signaling dynamics. Here we model local protrusion of the cell edge as a function of the load-bearing properties of integrin-based adhesions, actin polymerization fostered by adhesion-mediated signaling, and mechanosensitive activation of RhoA that promotes myosin II–generated stress on the lamellipodial F-actin network. Analysis of stochastic model simulations illustrates how these pleiotropic functions of nascent adhesions may be integrated to govern temporal persistence and frequency of protrusions. The simulations give mechanistic insight into the documented effects of extracellular matrix density and myosin abundance, and they show characteristic, nonnormal distributions of protrusion duration times that are similar to those extracted from live-cell imaging experiments. Analysis of the model further predicts relationships between measurable quantities that reflect the partitioning of stress between tension on F-actin–bound adhesions, which act as a molecular clutch, and dissipation by retrograde F-actin flow. }, number={24}, journal={MOLECULAR BIOLOGY OF THE CELL}, publisher={American Society for Cell Biology (ASCB)}, author={Welf, Erik S. and Johnson, Heath E. and Haugh, Jason M.}, year={2013}, month={Dec}, pages={3945–3955} }
@article{ott_sung_melvin_sheats_haugh_adler_jones_2013, title={Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein}, volume={8}, ISSN={["1932-6203"]}, url={http://europepmc.org/abstract/med/23840497}, DOI={10.1371/journal.pone.0066512}, abstractNote={Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.}, number={6}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Ott, Laura E. and Sung, Eui Jae and Melvin, Adam T. and Sheats, Mary K. and Haugh, Jason M. and Adler, Kenneth B. and Jones, Samuel L.}, editor={Aspenstrom, PontusEditor}, year={2013}, month={Jun} }
@article{johnson_haugh_2013, title={Quantitative Analysis of Phosphoinositide 3-Kinase (PI3K) Signaling Using Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy}, DOI={10.1002/0471143030.cb1414s61}, abstractNote={Abstract}, journal={Current Protocols in Cell Biology}, author={Johnson, Heath E. and Haugh, Jason M.}, year={2013} }
@article{haugh_2012, title={Cell regulation: A time to signal, a time to respond (Comment on DOI 10.1002/bies.201100172)}, volume={34}, ISSN={["0265-9247"]}, DOI={10.1002/bies.201200077}, abstractNote={The behaviours of living cells are normally controlled by growth factors, cytokines, and other molecular cues in vivo, affecting cell division, migration, differentiation, and survival. Specific receptors on the cell surface recognize these cues and mobilize signal transduction networks, which constitute the intracellular machinery responsible for actuating and regulating functional responses. In cancer, certain proteins (oncogenes) are mutated so as to render the cell autonomous from external cues. Consequently, intracellular signalling is robust and uncontrolled, and thus so are cell proliferation, survival, and movement. It is therefore of paramount importance to understand the intricate mechanisms by which signal transduction networks are governed, but the problem is the daunting complexity at the molecular level. Even when considering a single pathway, one must wade through a morass of protein components and post-translational modifications to figure out how it is regulated.
The encouraging news is that considerable progress over the past twenty years or so has resulted in a mature understanding of many signalling pathways. At least conceptually, this simplifies the problem by allowing us to treat pathways as modules, each responsible for the activation of a critical node in the network. Such ‘master regulators’ would include mitogen-activated protein kinases (MAPKs) and other important protein kinases such as Akt. Hence, the notion is that we can reduce the complexity of the problem by splitting it into two parts: one, the convergence of receptor-mediated pathways resulting in activation of a handful of master regulators, and two, the influences of those regulators on cell behaviour. With this paradigm, we can tackle each of the two parts independently and develop quantitative frameworks in which measurements are compared with mathematical models 1. Thus, it is envisioned that we will be able to predict the effects of molecular interventions in both normal and transformed cells, a prospect that is not lost on forward-thinking drug companies 2.
In this issue of BioEssays, Schilling et al. 3 highlight an important fundamental gap in the paradigm outlined above: at some point, one must make the jump from molecules to phenotype. Whereas the molecular world can be described purely mechanistically, i.e., according to physicochemical principles, it is prohibitively difficult to go from signalling to response that way. For example, a mechanistic model of cell growth would need to wholly incorporate gene expression and cell metabolism. The only recourse is a correlative approach 4. Signalling readouts (focused on presumed master regulators) are measured along with cell responses, and then a mathematical relationship between the two may be constructed. But what relationship do we choose? A linear regression is arguably the simplest approach; is that good enough? To a certain extent, the answer has proven to be yes, although the relationships that have been identified incorporate multiple signalling readouts 5; as one might expect, measurement of a single pathway does not adequately predict the outcome across all stimulation/intervention conditions.
Schilling et al. 3 add a new wrinkle to this discussion, which is the temporal aspect of cell signalling. Activation of a signalling network is dynamic, subject to receptor downregulation and other forms of negative feedback adaptation. Thus, the magnitude of pathway activation typically peaks early before reaching a quasi-steady plateau. Negative feedback in signalling networks has been sucessfully characterized through quantitative measurements and models 6, but then linking the temporal output of such models to cell responses requires hard assumptions about how the cell makes decisions. As articulated by Marshall 7, is it the steady state that matters most, or the peak? If the entire time course is important, how should one weight the signalling magnitudes at different times? Schilling et al. 3 discuss the merits of mathematically representing the kinetics as a time integral, calculated numerically. They explain that a primary challenge with this approach is that one must assume when the integration should be truncated, i.e. when the cell's decision is final. This sort of conjecture highlights the fundamental difficulties we face when trying to simplify complex biology using mathematics.}, number={7}, journal={BIOESSAYS}, publisher={Wiley-Blackwell}, author={Haugh, Jason M.}, year={2012}, month={Jul}, pages={528–529} }
@article{cirit_haugh_2012, title={Data-driven modelling of receptor tyrosine kinase signalling networks quantifies receptor-specific potencies of PI3K-and Ras-dependent ERK activation}, volume={441}, ISSN={["0264-6021"]}, DOI={10.1042/bj20110833}, abstractNote={Signal transduction networks in mammalian cells, comprising a limited set of interacting biochemical pathways, are accessed by various growth factor and cytokine receptors to elicit distinct cell responses. This raises the question as to how specificity of the stimulus–response relationship is encoded at the molecular level. It has been proposed that specificity arises not only from the activation of unique signalling pathways, but also from quantitative differences in the activation and regulation of shared receptor-proximal signalling proteins. To address such hypotheses, data sets with greater precision and coverage of experimental conditions will need to be acquired, and rigorous frameworks that codify and parameterize the inherently non-linear relationships among signalling activities will need to be developed. In the present study we apply a systematic approach combining quantitative measurements and mathematical modelling to compare the signalling networks accessed by FGF (fibroblast growth factor) and PDGF (platelet-derived growth factor) receptors in mouse fibroblasts, in which the ERK (extracellular-signal-regulated kinase) cascade is activated by Ras- and PI3K (phosphoinositide 3-kinase)-dependent pathways. We show that, whereas the FGF stimulation of PI3K signalling is relatively weak, this deficiency is compensated for by a more potent Ras-dependent activation of ERK. Thus, as the modelling would predict, the ERK pathway is activated to a greater extent in cells co-stimulated with FGF and PDGF, relative to the saturated levels achieved with either ligand alone. It is envisaged that similar approaches will prove valuable in the elucidation of quantitative differences among other closely related receptor signalling networks.}, number={1}, journal={BIOCHEMICAL JOURNAL}, publisher={Portland Press Ltd.}, author={Cirit, Murat and Haugh, Jason M.}, year={2012}, month={Jan}, pages={77–85} }
@article{welf_haugh_2012, title={Erratum to: Stochastic Dynamics of Membrane Protrusion Mediated by the DOCK180/Rac Pathway in Migrating Cells}, volume={5}, ISSN={1865-5025 1865-5033}, url={http://dx.doi.org/10.1007/S12195-012-0252-9}, DOI={10.1007/S12195-012-0252-9}, abstractNote={In our previously published article, we described a computational model and analysis investigating the stochastic dynamics of cell protrusion during cell migration. In that work, we presented results of stochastic simulations plotted in two-dimensional parameter space. To supplement this analysis, the associated region of model bistability was identified by analysis of the deterministic model equations, and this region was plotted on the same graphs (panels a & b in each of Figs. 2–6). Since publication of our article, we became aware that certain parameter combinations thought to lie in the bistable region are, in fact, monostable. We wish to thank Dr. Michael Savageau (Biomedical Engineering, UC Davis), who studied our model during the preparation of his review article, for bringing the matter to our attention. We traced the error to the use of incorrect values of certain parameters in the bistability analysis. Whereas the values reported in the paper are correct for the stochastic model results, the bistability analysis was performed using three incorrect parameter values as shown in Table C1. Shown below as Fig. C1 are the corrected figure panels, which now show the bistability regions using the correct parameter values as used in the stochastic simulations. One of our previous conclusions concerned the lack of a definitive relationship between phenotypic switching in the stochastic simulations and model bistability. This conclusion was based, in part, on stochastic simulations for parameter sets that were supposed to lie in regions of bistability (Fig. 2e and Fig. 5e of the paper). Since the corrected results show that the parameter sets in question do not lie in the bistable regime, we have performed additional simulations to confirm that the original conclusions are valid (Figure C2). In these simulations, the analysis shown in Fig. 5 of the original paper was repeated with a larger value of the Cs parameter in order to expand the region of bistability. Comparison of stochastic runs on either side of the bistability region, as originally intended, confirms that although switching between protrusion and adhesion phenotypes is likely to occur in regions of parameter space that are close to the region of bistability, model bistability is not required for this behavior. As explained in the original article, bistable regions of parameter space usually lie between those regions that give monostable low and monostable high protrusion, and in the vicinity of the interface between the two, the stochastic model readily produces transient departures from the stable state.}, number={4}, journal={Cellular and Molecular Bioengineering}, publisher={Springer Science and Business Media LLC}, author={Welf, Erik S. and Haugh, Jason M.}, year={2012}, month={Oct}, pages={514–516} }
@article{haugh_2012, title={Live-Cell Fluorescence Microscopy with Molecular Biosensors: What Are We Really Measuring?}, volume={102}, ISSN={["0006-3495"]}, DOI={10.1016/j.bpj.2012.03.055}, abstractNote={Engineered protein biosensors, such as those based on Förster resonance energy transfer, membrane translocation, or solvatochromic shift, are being used in combination with live-cell fluorescence microscopy to reveal kinetics and spatial localization of intracellular processes as they occur. Progress in the application of this approach has been steady, yet its general suitability for quantitative measurements remains unclear. To address the pitfalls of the biosensor approach in quantitative terms, simple reaction-diffusion models were analyzed. The analysis shows that although high-affinity molecular recognition allows robust detection of the fluorescence readout, either of two detrimental effects is fostered. Binding of an intramolecular biosensor or of a relatively abundant intermolecular biosensor introduces observer effects in which the dynamics of the target molecule under study are significantly perturbed, whereas binding of a sparingly expressed intermolecular biosensor is subject to a saturation effect, where the pool of unbound biosensor is significantly depleted. The analysis explores how these effects are manifest in the kinetics and spatial gradients of the biosensor-target complex. A sobering insight emerges: the observer or saturation effect is always significant; the question is whether or not it can be tolerated or accounted for. The challenge in managing the adverse effects is that specification of the biosensor-target affinity to within a certain order of magnitude is required.}, number={9}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Haugh, Jason M.}, year={2012}, month={May}, pages={2003–2011} }
@article{welf_ahmed_johnson_melvin_haugh_2012, title={Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism}, volume={197}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.201108152}, abstractNote={Mesenchymal cell migration as exhibited by fibroblasts is distinct from amoeboid cell migration and is characterized by dynamic competition among multiple protrusions, which determines directional persistence and responses to spatial cues. Localization of phosphoinositide 3-kinase (PI3K) signaling is thought to play a broadly important role in cell motility, yet the context-dependent functions of this pathway have not been adequately elucidated. By mapping the spatiotemporal dynamics of cell protrusion/retraction and PI3K signaling monitored by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K-dependent branching and pivoting of protrusions. PI3K inhibition did not affect the initiation of newly branched protrusions, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the onset of local protrusion and was required for the lateral spreading and stabilization of nascent branches. During chemotaxis, the branch experiencing the higher chemoattractant concentration was favored, and, thus, the cell reoriented so as to align with the external gradient.}, number={1}, journal={JOURNAL OF CELL BIOLOGY}, publisher={Rockefeller University Press}, author={Welf, Erik S. and Ahmed, Shoeb and Johnson, Heath E. and Melvin, Adam T. and Haugh, Jason M.}, year={2012}, month={Apr}, pages={105–114} }
@article{welf_haugh_2012, title={Stochastic Models of Cell Protrusion Arising From Spatiotemporal Signaling and Adhesion Dynamics}, DOI={10.1016/b978-0-12-388403-9.00009-6}, abstractNote={During cell migration, local protrusion events are regulated by biochemical and physical processes that are in turn coordinated with the dynamic properties of cell-substratum adhesion structures. In this chapter, we present a modeling approach for integrating the apparent stochasticity and spatial dependence of signal transduction pathways that promote protrusion in tandem with adhesion dynamics. We describe our modeling framework, as well as its abstraction, parameterization, and validation against experimental data. Analytical techniques for identifying and evaluating the effects of model bistability on simulation simulation results are shown, and implications of this analysis for understanding cell protrusion behavior are offered.}, journal={Computational Methods in Cell Biology}, publisher={Elsevier BV}, author={Welf, Erik S. and Haugh, Jason M.}, year={2012}, pages={223–241} }
@article{welf_haugh_2012, title={Stochastic dynamics of membrane protrusion mediated by the DOCK180/Rac pathway in migrating cells (vol 3, pg 30, 2010)}, volume={5}, number={4}, journal={Cellular and Molecular Bioengineering}, author={Welf, E. S. and Haugh, J. M.}, year={2012}, pages={514–516} }
@inproceedings{welf_haugh_2012, title={Stochastic models of cell protrusion arising from spatiotemporal signaling and adhesion dynamics}, volume={110}, booktitle={Methods in cell biology, vol 110: computational methods in cell biology}, author={Welf, E. S. and Haugh, J. M.}, year={2012}, pages={223–241} }
@article{cirit_grant_haugh_2012, title={Systemic Perturbation of the ERK Signaling Pathway by the Proteasome Inhibitor, MG132}, volume={7}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0050975}, abstractNote={Inhibition of the ubiquitin-proteasome protein degradation pathway has been identified as a viable strategy for anti-tumor therapy based on its broad effects on cell proliferation. By the same token, the variety of elicited effects confounds the interpretation of cell-based experiments using proteasome inhibitors such as MG132. It has been proposed that MG132 treatment reduces growth factor-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), at least in part through upregulation of dual specificity phosphatases (DUSPs). Here, we show that the effects of MG132 treatment on ERK signaling are more widespread, leading to a reduction in activation of the upstream kinase MEK. This suggests that MG132 systemically perturbs the intracellular phosphoproteome, impacting ERK signaling by reducing phosphorylation status at multiple levels of the kinase cascade.}, number={11}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Cirit, Murat and Grant, Kyle G. and Haugh, Jason M.}, editor={Buday, LaszloEditor}, year={2012}, month={Nov} }
@article{buhrman_kumar_cirit_haugh_mattos_2011, title={Allosteric Modulation of Ras-GTP Is Linked to Signal Transduction through RAF Kinase}, volume={286}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m110.193854}, abstractNote={Ras is a key signal transduction protein in the cell. Mutants of Gly12 and Gln61 impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln61 in the active site. We show that the “on” and “off” conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex.}, number={5}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Buhrman, Greg and Kumar, V. S. Senthil and Cirit, Murat and Haugh, Jason M. and Mattos, Carla}, year={2011}, month={Feb}, pages={3323–3331} }
@article{haugh_2011, title={Cells see the light to bring signaling under control}, volume={8}, DOI={10.1038/nmeth.1708}, number={10}, journal={Nature Methods}, publisher={Springer Nature}, author={Haugh, Jason M}, year={2011}, month={Sep}, pages={808–809} }
@article{chylek_hu_blinov_emonet_faeder_goldstein_gutenkunst_haugh_lipniacki_posner_et al._2011, title={Guidelines for visualizing and annotating rule-based models}, volume={7}, ISSN={["1742-2051"]}, DOI={10.1039/c1mb05077j}, abstractNote={Rule-based modeling provides a means to represent cell signaling systems in a way that captures site-specific details of molecular interactions. For rule-based models to be more widely understood and (re)used, conventions for model visualization and annotation are needed. We have developed the concepts of an extended contact map and a model guide for illustrating and annotating rule-based models. An extended contact map represents the scope of a model by providing an illustration of each molecule, molecular component, direct physical interaction, post-translational modification, and enzyme-substrate relationship considered in a model. A map can also illustrate allosteric effects, structural relationships among molecular components, and compartmental locations of molecules. A model guide associates elements of a contact map with annotation and elements of an underlying model, which may be fully or partially specified. A guide can also serve to document the biological knowledge upon which a model is based. We provide examples of a map and guide for a published rule-based model that characterizes early events in IgE receptor (FcεRI) signaling. We also provide examples of how to visualize a variety of processes that are common in cell signaling systems but not considered in the example model, such as ubiquitination. An extended contact map and an associated guide can document knowledge of a cell signaling system in a form that is visual as well as executable. As a tool for model annotation, a map and guide can communicate the content of a model clearly and with precision, even for large models.}, number={10}, journal={MOLECULAR BIOSYSTEMS}, publisher={Royal Society of Chemistry (RSC)}, author={Chylek, Lily A. and Hu, Bin and Blinov, Michael L. and Emonet, Thierry and Faeder, James R. and Goldstein, Byron and Gutenkunst, Ryan N. and Haugh, Jason M. and Lipniacki, Tomasz and Posner, Richard G. and et al.}, year={2011}, pages={2779–2795} }
@article{melvin_welf_wang_irvine_haugh_2011, title={In Chemotaxing Fibroblasts, Both High-Fidelity and Weakly Biased Cell Movements Track the Localization of PI3K Signaling}, volume={100}, ISSN={["0006-3495"]}, DOI={10.1016/j.bpj.2011.02.047}, abstractNote={Cell movement biased by a chemical gradient, or chemotaxis, coordinates the recruitment of cells and collective migration of cell populations. During wound healing, chemotaxis of fibroblasts is stimulated by platelet-derived growth factor (PDGF) and certain other chemoattractants. Whereas the immediate PDGF gradient sensing response has been characterized previously at the level of phosphoinositide 3-kinase (PI3K) signaling, the sensitivity of the response at the level of cell migration bias has not yet been studied quantitatively. In this work, we used live-cell total internal reflection fluorescence microscopy to monitor PI3K signaling dynamics and cell movements for extended periods. We show that persistent and properly aligned (i.e., high-fidelity) fibroblast migration does indeed correlate with polarized PI3K signaling; accordingly, this behavior is seen only under conditions of high gradient steepness (>10% across a typical cell length of 50 μm) and a certain range of PDGF concentrations. Under suboptimal conditions, cells execute a random or biased random walk, but nonetheless move in a predictable fashion according to the changing pattern of PI3K signaling. Inhibition of PI3K during chemotaxis is accompanied by loss of both cell-substratum contact and morphological polarity, but after a recovery period, PI3K-inhibited fibroblasts often regain the ability to orient toward the PDGF gradient.}, number={8}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Melvin, Adam T. and Welf, Erik S. and Wang, Yana and Irvine, Darrell J. and Haugh, Jason M.}, year={2011}, month={Apr}, pages={1893–1901} }
@article{wang_cirit_haugh_2011, title={PI3K-dependent cross-talk interactions converge with Ras as quantifiable inputs integrated by Erk (vol 5, pg 246, 2009)}, volume={7}, ISSN={["1744-4292"]}, DOI={10.1038/msb.2011.61}, number={1}, journal={MOLECULAR SYSTEMS BIOLOGY}, publisher={Wiley-Blackwell}, author={Wang, Chun-Chao and Cirit, Murat and Haugh, Jason M.}, year={2011}, month={Aug} }
@article{ahmed_yang_ozcam_efimenko_weiger_genzer_haugh_2011, title={Poly(vinylmethylsiloxane) Elastomer Networks as Functional Materials for Cell Adhesion and Migration Studies}, volume={12}, ISSN={["1526-4602"]}, DOI={10.1021/bm101549y}, abstractNote={Cell migration is central to physiological responses to injury and infection and in the design of biomaterial implants. The ability to tune the properties of adhesive materials and relate those properties in a quantitative way to the dynamics of intracellular processes remains a definite challenge in the manipulation of cell migration. Here, we propose the use of poly(vinylmethylsiloxane) (PVMS) networks as novel substrata for cell adhesion and migration. These materials offer the ability to tune independently chemical functionality and elastic modulus. Importantly, PVMS networks are compatible with total internal reflection fluorescence (TIRF) microscopy, which is ideal for interrogating the cell-substratum interface; this latter characteristic presents a distinct advantage over polyacrylamide gels and other materials that swell with water. To demonstrate these capabilities, adhesive peptides containing the arginyl-glycyl-aspartic acid (RGD) tripeptide motif were successfully grafted to the surface of PVMS network using a carboxyl-terminated thiol as a linker. Peptide-specific adhesion, spreading, and random migration of NIH 3T3 mouse fibroblasts were characterized. These experiments show that a peptide containing the synergy sequence of fibronectin (PHSRN) in addition to RGD promotes more productive cell migration without markedly enhancing cell adhesion strength. Using TIRF microscopy, the dynamics of signal transduction through the phosphoinositide 3-kinase pathway were monitored in cells as they migrated on peptide-grafted PVMS surfaces. This approach offers a promising avenue for studies of directed migration and mechanotransduction at the level of intracellular processes.}, number={4}, journal={BIOMACROMOLECULES}, publisher={American Chemical Society (ACS)}, author={Ahmed, Shoeb and Yang, Hyun-kwan and Ozcam, Ali E. and Efimenko, Kirill and Weiger, Michael C. and Genzer, Jan and Haugh, Jason M.}, year={2011}, month={Apr}, pages={1265–1271} }
@article{cirit_haugh_2011, title={Quantitative models of signal transduction networks}, volume={4}, DOI={10.4161/cib.4.3.15149}, abstractNote={Receptor-mediated signal transduction networks, comprised of multiple biochemical pathways, control cell responses and are therefore central to normal and aberrant physiological processes. An appreciation for the inherent complexities of these networks has matured in recent years, to the point where it is now apparent that experimental measurements will need to be combined with computational modeling and analysis to best interpret and predict how individual mechanisms (protein-protein interactions, enzymatic reactions, etc.) are integrated at the network level. To progress along these lines, there is a major barrier to overcome: although a deep mechanistic understanding of signal transduction has been achieved, data sets of a suitably quantitative nature are still lacking. Based on our efforts to systematically acquire and analyze such measurements, we contend that the level of detail in models of signaling networks ought to be limited by the availability of quantitative data, rather than by the much greater availability of qualitative information about signaling interactions. Although this approach is sensible from a data-driven modeling perspective, it is controversial because it gives the false impression that molecular-level details are being ignored.}, number={3}, journal={Communicative & Integrative Biology}, publisher={Informa UK Limited}, author={Cirit, Murat and Haugh, Jason M.}, year={2011}, month={May}, pages={353–356} }
@article{welf_haugh_2011, title={Signaling pathways that control cell migration: models and analysis}, volume={3}, ISSN={["1939-005X"]}, DOI={10.1002/wsbm.110}, abstractNote={Abstract}, number={2}, journal={WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE}, publisher={Wiley-Blackwell}, author={Welf, Erik S. and Haugh, Jason M.}, year={2011}, pages={231–240} }
@article{weiger_ahmed_welf_haugh_2010, title={Directional Persistence of Cell Migration Coincides with Stability of Asymmetric Intracellular Signaling}, volume={98}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2009.09.051}, abstractNote={It has long been appreciated that spatiotemporal dynamics of cell migration are under the control of intracellular signaling pathways, which are mediated by adhesion receptors and other transducers of extracellular cues. Further, there is ample evidence that aspects of cell migration are stochastic: how else could it exhibit directional persistence over timescales much longer than typical signal transduction processes, punctuated by abrupt changes in direction? Yet the mechanisms by which signaling processes affect those behaviors remain unclear. We have developed analytical methods for relating parallel live-cell microscopy measurements of cell migration dynamics to the intracellular signaling processes that govern them. In this analysis of phosphoinositide 3-kinase signaling in randomly migrating fibroblasts, we observe that hot spots of intense signaling coincide with localized cell protrusion and endure with characteristic lifetimes that correspond to those of cell migration persistence. We further show that distant hot spots are dynamically and stochastically coupled. These results are indicative of a mechanism by which changes in a cell's direction of migration are determined by a fragile balance of relatively rapid intracellular signaling processes.}, number={1}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Weiger, Michael C. and Ahmed, Shoeb and Welf, Erik S. and Haugh, Jason M.}, year={2010}, month={Jan}, pages={67–75} }
@article{welf_haugh_2010, title={Stochastic Dynamics of Membrane Protrusion Mediated by the DOCK180/Rac Pathway in Migrating Cells}, volume={3}, ISSN={["1865-5033"]}, DOI={10.1007/s12195-010-0100-8}, abstractNote={Cell migration is regulated by processes that control adhesion to extracellular matrix (ECM) and force generation. While our fundamental understanding of how these control mechanisms are actuated at the molecular level (signal transduction) has been refined over many years, appreciation of their dynamics has grown more recently. Here, we formulate and analyze by stochastic simulation a quantitative model of signaling mediated by the integrin family of adhesion receptors. Nascent adhesions foster the activation of the small GTPase Rac by at least two distinct signaling pathways, one of which involves tyrosine phosphorylation of the scaffold protein paxillin and formation of multiprotein complexes containing the guanine nucleotide exchange factor DOCK180. Active Rac promotes protrusion of the cell’s leading edge, which in turn enhances the rate of nascent adhesion nucleation; we call this feedback mechanism the core protrusion cycle. Protrusion is antagonized by stable adhesions, which form by myosin-dependent maturation of nascent adhesions, and we propose here a feedforward mechanism mediated by the tyrosine kinase c-Src by which this antagonism is regulated so as to allow transient protrusion at higher densities of ECM. We show that this “buffering of inhibition” mechanism, coupled with the core protrusion cycle, is capable of tuning the frequencies of protrusion and adhesion maturation events.}, number={1}, journal={CELLULAR AND MOLECULAR BIOENGINEERING}, publisher={Springer Nature}, author={Welf, Erik S. and Haugh, Jason M.}, year={2010}, month={Mar}, pages={30–39} }
@article{cirit_krajcovic_choi_welf_horwitz_haugh_2010, title={Stochastic Model of Integrin-Mediated Signaling and Adhesion Dynamics at the Leading Edges of Migrating Cells}, volume={6}, ISSN={["1553-7358"]}, DOI={10.1371/journal.pcbi.1000688}, abstractNote={Productive cell migration requires the spatiotemporal coordination of cell adhesion, membrane protrusion, and actomyosin-mediated contraction. Integrins, engaged by the extracellular matrix (ECM), nucleate the formation of adhesive contacts at the cell's leading edge(s), and maturation of nascent adhesions to form stable focal adhesions constitutes a functional switch between protrusive and contractile activities. To shed additional light on the coupling between integrin-mediated adhesion and membrane protrusion, we have formulated a quantitative model of leading edge dynamics combining mechanistic and phenomenological elements and studied its features through classical bifurcation analysis and stochastic simulation. The model describes in mathematical terms the feedback loops driving, on the one hand, Rac-mediated membrane protrusion and rapid turnover of nascent adhesions, and on the other, myosin-dependent maturation of adhesions that inhibit protrusion at high ECM density. Our results show that the qualitative behavior of the model is most sensitive to parameters characterizing the influence of stable adhesions and myosin. The major predictions of the model, which we subsequently confirmed, are that persistent leading edge protrusion is optimal at an intermediate ECM density, whereas depletion of myosin IIA relieves the repression of protrusion at higher ECM density.}, number={2}, journal={PLOS COMPUTATIONAL BIOLOGY}, publisher={Public Library of Science (PLoS)}, author={Cirit, Murat and Krajcovic, Matej and Choi, Colin K. and Welf, Erik S. and Horwitz, Alan F. and Haugh, Jason M.}, editor={Asthagiri, Anand R.Editor}, year={2010}, month={Feb} }
@article{cirit_wang_haugh_2010, title={Systematic Quantification of Negative Feedback Mechanisms in the Extracellular Signal-regulated Kinase (ERK) Signaling Network}, volume={285}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m110.148759}, abstractNote={Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.}, number={47}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Cirit, Murat and Wang, Chun-Chao and Haugh, Jason M.}, year={2010}, month={Nov}, pages={36736–36744} }
@article{barua_faeder_haugh_2009, title={A Bipolar Clamp Mechanism for Activation of Jak-Family Protein Tyrosine Kinases}, volume={5}, ISSN={["1553-7358"]}, DOI={10.1371/journal.pcbi.1000364}, abstractNote={Most cell surface receptors for growth factors and cytokines dimerize in order to mediate signal transduction. For many such receptors, the Janus kinase (Jak) family of non-receptor protein tyrosine kinases are recruited in pairs and juxtaposed by dimerized receptor complexes in order to activate one another by trans-phosphorylation. An alternative mechanism for Jak trans-phosphorylation has been proposed in which the phosphorylated kinase interacts with the Src homology 2 (SH2) domain of SH2-B, a unique adaptor protein with the capacity to homo-dimerize. Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH) receptor/Jak2/SH2-Bβ system. The modeling results suggest that, whereas Jak2-(SH2-Bβ)2-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bβ and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling. Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar ‘clamp’ that stabilizes the active configuration of two Jak2 molecules in the same macro-complex.}, number={4}, journal={PLOS COMPUTATIONAL BIOLOGY}, publisher={Public Library of Science (PLoS)}, author={Barua, Dipak and Faeder, James R. and Haugh, Jason M.}, editor={Kannan, NatarajanEditor}, year={2009}, month={Apr} }
@article{haugh_2009, title={Analysis of Reaction-Diffusion Systems with Anomalous Subdiffusion}, volume={97}, ISSN={["0006-3495"]}, DOI={10.1016/j.bpj.2009.05.014}, abstractNote={Abstract
Reaction-diffusion equations are the cornerstone of modeling biochemical systems with spatial gradients, which are relevant to biological processes such as signal transduction. Implicit in the formulation of these equations is the assumption of Fick's law, which states that the local diffusive flux of species i is proportional to its concentration gradient; however, in the context of complex fluids such as cytoplasm and cell membranes, the use of Fick's law is based on empiricism, whereas evidence has been mounting that such media foster anomalous subdiffusion (with mean-squared displacement increasing less than linearly with time) over certain length scales. Particularly when modeling diffusion-controlled reactions and other systems where the spatial domain is considered semi-infinite, assuming Fickian diffusion might not be appropriate. In this article, two simple, conceptually extreme models of anomalous subdiffusion are used in the framework of Green's functions to demonstrate the solution of four reaction-diffusion problems that are well known in the biophysical context of signal transduction: fluorescence recovery after photobleaching, the Smolochowski limit for diffusion-controlled reactions in solution, the spatial range of a diffusing molecule with finite lifetime, and the collision coupling mechanism of diffusion-controlled reactions in two dimensions. In each case, there are only subtle differences between the two subdiffusion models, suggesting how measurements of mean-squared displacement versus time might generally inform models of reactive systems with partial diffusion control.}, number={2}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Haugh, Jason M.}, year={2009}, month={Jul}, pages={435–442} }
@article{wang_cirit_haugh_2009, title={PI3K‐dependent cross‐talk interactions converge with Ras as quantifiable inputs integrated by Erk}, volume={5}, ISSN={1744-4292 1744-4292}, url={http://dx.doi.org/10.1038/msb.2009.4}, DOI={10.1038/msb.2009.4}, abstractNote={Although it is appreciated that canonical signal-transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross-talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross-talk mechanisms in the platelet-derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3-kinase (PI3K) and Ras/extracellular signal-regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross-talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K-dependent and -independent modes of Ras/Erk activation.}, number={1}, journal={Molecular Systems Biology}, publisher={EMBO}, author={Wang, Chun‐Chao and Cirit, Murat and Haugh, Jason M}, year={2009}, month={Jan}, pages={246} }
@article{weiger_wang_krajcovic_melvin_rhoden_haugh_2009, title={Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts}, volume={122}, ISSN={["1477-9137"]}, DOI={10.1242/jcs.037564}, abstractNote={During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3′-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3′-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts.}, number={3}, journal={JOURNAL OF CELL SCIENCE}, publisher={The Company of Biologists}, author={Weiger, Michael C. and Wang, Chun-Chao and Krajcovic, Matej and Melvin, Adam T. and Rhoden, John J. and Haugh, Jason M.}, year={2009}, month={Feb}, pages={313–323} }
@article{haugh_2008, title={Biophysics - Cells get in shape for a crawl}, volume={453}, ISSN={["0028-0836"]}, DOI={10.1038/453461a}, abstractNote={A cell's shape changes as it moves along a surface. The forward-thinking cytoskeletal elements are all for progress, but the conservative cell membrane keeps them under control by physically opposing their movement. Cell shape is determined by the interaction of many elements such as the cytoskeleton, cell membrane and interaction of cells with their substrate. Keren et al. have analysed the natural cell-to-cell variability in a large population of motile cells (keratocytes) from fish skin to reveal mechanisms of shape determination. They develop a model that accurately predicts both cell shape and speed based on a physically realistic, molecularly detailed model of an actin network treadmilling in an inextensible membrane bag.}, number={7194}, journal={NATURE}, publisher={Springer Nature}, author={Haugh, Jason M.}, year={2008}, month={May}, pages={461–462} }
@article{monine_haugh_2008, title={Cell population-based model of dermal wound invasion with heterogeneous intracellular signaling properties}, volume={2}, DOI={10.4161/cam.2.2.6511}, abstractNote={A deterministic model of dermal wound invasion, which accounts for the platelet-derived growth factor (PDGF) gradient sensing mechanism in fibroblasts mediated by cell surface receptors and the phosphoinositide 3-kinase (PI3K) signal transduction pathway, was previously described (Biophys J 2006; 90:2297-2308). Here, we extend that work and implement a hybrid modeling strategy that treats fibroblasts as discrete entities endowed with heterogeneous properties, namely receptor, PI3K and 3’ phosphoinositide phosphatase expression levels. Analysis of the model suggests that the wound environment fosters the advancement of cells within the population that are better fit to migrate and/or proliferate in response to PDGF stimulation. Thus, cell-to-cell variability results in a significantly higher rate of wound invasion as compared with the deterministic model, in a manner that depends on the way in which individual cell properties are sampled or inherited upon cell division.}, number={2}, journal={Cell Adhesion & Migration}, publisher={Informa UK Limited}, author={Monine, Michael I. and Haugh, Jason M.}, year={2008}, month={Apr}, pages={137–145} }
@article{comfort_haugh_2008, title={Combinatorial Signal Transduction Responses Mediated by Interleukin-2 and-4 Receptors in a Helper T-H2 Cell Line}, volume={1}, ISSN={["1865-5033"]}, DOI={10.1007/s12195-008-0015-9}, abstractNote={The cytokines interleukin (IL)-2 and IL-4 are important regulators of the adaptive immune response, due in part to their effects on clonal expansion and differentiation of T cells. When IL-2 and IL-4 are administered together, both antagonistic and synergistic effects have been reported, but little is known in general concerning the mechanisms underlying such combinatorial effects. We found evidence for both effects in the proliferation responses of the IL-2 and IL-4 responsive T cell line, HT-2; IL-4 delays the onset of cell growth yet ultimately allows a higher cell density to be achieved in static culture. At the level of signal transduction pathways, we found that IL-4 partially inhibits IL-2 receptor-mediated pathways (PI3K/Akt, Ras/Erk, and STAT5a/b) and does not prolong their transient kinetics. This mode of antagonism, but not the effects on cell proliferation, is overcome at higher concentrations of IL-2 that are sufficient to saturate the signaling responses. By comparison, IL-4-stimulated activation of STAT6 is unaffected by IL-2 and shows sustained kinetics, and we speculate that this or another IL-4 receptor-specific pathway is responsible for the effects of IL-4 on IL-2-stimulated proliferation. A possibly related observation is that IL-4 induces a dramatic cell adhesion phenotype.}, number={2-3}, journal={CELLULAR AND MOLECULAR BIOENGINEERING}, publisher={Springer Nature}, author={Comfort, Kristen K. and Haugh, Jason M.}, year={2008}, month={Sep}, pages={163–172} }
@article{barua_faeder_haugh_2008, title={Computational models of tandem Src homology 2 domain interactions and application to phosphoinositide 3-kinase}, volume={283}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M708359200}, abstractNote={Intracellular signal transduction proteins typically utilize multiple interaction domains for proper targeting, and thus a broad diversity of distinct signaling complexes may be assembled. Considering the coordination of only two such domains, as in tandem Src homology 2 (SH2) domain constructs, gives rise to a kinetic scheme that is not adequately described by simple models used routinely to interpret in vitro binding measurements. To analyze the interactions between tandem SH2 domains and bisphosphorylated peptides, we formulated detailed kinetic models and applied them to the phosphoinositide 3-kinase p85 regulatory subunit/platelet-derived growth factor β-receptor system. Data for this system from different in vitro assay platforms, including surface plasmon resonance, competition binding, and isothermal titration calorimetry, were reconciled to estimate the magnitude of the cooperativity characterizing the sequential binding of the high and low affinity SH2 domains (C-SH2 and N-SH2, respectively). Compared with values based on an effective volume approximation, the estimated cooperativity is 3 orders of magnitude lower, indicative of significant structural constraints. Homodimerization of full-length p85 was found to be an alternative mechanism for high avidity binding to phosphorylated platelet-derived growth factor receptors, which would render the N-SH2 domain dispensable for receptor binding.}, number={12}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Barua, Dipak and Faeder, James R. and Haugh, Jason M.}, year={2008}, month={Mar}, pages={7338–7345} }
@article{delisa_haugh_2008, title={First International Conference on Biomolecular Engineering (ICBE)}, volume={24}, number={1}, journal={Biotechnology Progress}, author={DeLisa, M. and Haugh, J.}, year={2008}, pages={1–1} }
@article{haugh_2008, title={Mathematical Modelling of Biological Signaling Networks}, DOI={10.1002/9780470048672.wecb646}, abstractNote={Abstract}, journal={Wiley Encyclopedia of Chemical Biology}, author={Haugh, Jason M.}, year={2008}, month={May} }
@article{monine_haugh_2008, title={Signal transduction at point-blank range: Analysis of a spatial coupling mechanism for pathway crosstalk}, volume={95}, ISSN={["0006-3495"]}, DOI={10.1529/biophysj.108.128892}, abstractNote={The plasma membrane provides a physical platform for the orchestration of molecular interactions and biochemical conversions involved in the early stages of receptor-mediated signal transduction in living cells. In that context, we introduce here the concept of spatial coupling, wherein simultaneous recruitment of different enzymes to the same receptor scaffold facilitates crosstalk between different signaling pathways through the local release and capture of activated signaling molecules. To study the spatiotemporal dynamics of this mechanism, we have developed a Brownian dynamics modeling approach and applied it to the receptor-mediated activation of Ras and the cooperative recruitment of phosphoinositide 3-kinase (PI3K) by activated receptors and Ras. Various analyses of the model simulations show that cooperative assembly of multimolecular complexes nucleated by activated receptors is facilitated by the local release and capture of membrane-anchored signaling molecules (such as active Ras) from/by receptor-bound signaling proteins. In the case of Ras/PI3K crosstalk, the model predicts that PI3K is more likely to be recruited by activated receptors bound or recently visited by the enzyme that activates Ras. By this mechanism, receptor-bound PI3K is stabilized through short-range, diffusion-controlled capture of active Ras and Ras/PI3K complexes released from the receptor complex. We contend that this mechanism is a means by which signaling pathways are propagated and spatially coordinated for efficient crosstalk between them.}, number={5}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Monine, Michael I. and Haugh, Jason M.}, year={2008}, month={Sep}, pages={2172–2182} }
@misc{haugh_2007, title={Membrane-binding/modification model of signaling protein activation and analysis of its control by cell morphology}, volume={92}, ISSN={["0006-3495"]}, DOI={10.1529/biophysj.107.105213}, abstractNote={A mechanism for cell shape control of intracellular signal transduction, whereby the average concentration of activated proteins in the cytosol increases as the height of the cell decreases, has been described recently. An important modification of this analysis is offered, recognizing that signaling proteins are not only activated at the plasma membrane but must first form complexes with signaling molecules that reside there, such as receptors and lipids. With these more realistic boundary conditions, it is shown that the region of parameter space where cell shape amplifies the average cytosolic activity is greatly expanded. Moreover, this model allows for amplification of the activated protein bound at the membrane, which is considered more relevant for certain, spatially driven signaling processes in cell migration.}, number={11}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Haugh, Jason M.}, year={2007}, month={Jun}, pages={L93–L95} }
@article{barua_faeder_haugh_2007, title={Structure-based kinetic models of modular signaling protein function: Focus on Shp2}, volume={92}, ISSN={["0006-3495"]}, DOI={10.1529/biophysj.106.093484}, abstractNote={We present here a computational, rule-based model to study the function of the SH2 domain-containing protein tyrosine phosphatase, Shp2, in intracellular signal transduction. The two SH2 domains of Shp2 differentially regulate the enzymatic activity by a well-characterized mechanism, but they also affect the targeting of Shp2 to signaling receptors in cells. Our kinetic model integrates these potentially competing effects by considering the intra- and intermolecular interactions of the Shp2 SH2 domains and catalytic site as well as the effect of Shp2 phosphorylation. Even for the isolated Shp2/receptor system, which may seem simple by certain standards, we find that the network of possible binding and phosphorylation states is composed of over 1000 members. To our knowledge, this is the first kinetic model to fully consider the modular, multifunctional structure of a signaling protein, and the computational approach should be generally applicable to other complex intermolecular interactions.}, number={7}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Barua, Dipak and Faeder, James R. and Haugh, Jason M.}, year={2007}, month={Apr}, pages={2290–2300} }
@article{haugh_2006, title={Deterministic model of dermal wound invasion incorporating receptor-mediated signal transduction and spatial gradient sensing}, volume={90}, ISSN={["1542-0086"]}, DOI={10.1529/biophysj.105.077610}, abstractNote={During dermal wound healing, platelet-derived growth factor (PDGF) serves as both a chemoattractant and mitogen for fibroblasts, potently stimulating their invasion of the fibrin clot over a period of several days. A mathematical model of this process is presented, which accurately accounts for the sensitivity of PDGF gradient sensing through PDGF receptor/phosphoinositide 3-kinase-mediated signal transduction. Analysis of the model suggests that PDGF receptor-mediated endocytosis and degradation of PDGF allows a constant PDGF concentration profile to be maintained at the leading front of the fibroblast density profile as it propagates, at a constant rate, into the clot. Thus, the constant PDGF gradient can span the optimal concentration range for asymmetric phosphoinositide 3-kinase signaling and fibroblast chemotaxis, with near-maximal invasion rates elicited over a relatively broad range of PDGF secretion rates. A somewhat surprising finding was that extremely sharp PDGF gradients do not necessarily stimulate faster progression through the clot, because maintaining such a gradient through PDGF consumption is a potentially rate-limiting process.}, number={7}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Haugh, JM}, year={2006}, month={Apr}, pages={2297–2308} }
@article{haugh_schneider_2006, title={Effectiveness factor for spatial gradient sensing in living cells}, volume={61}, ISSN={["1873-4405"]}, DOI={10.1016/j.ces.2006.04.041}, abstractNote={We consider the steady-state pattern of messenger molecules produced in the membrane of a cell perceiving and responding to an extracellular gradient of chemoattractant, which directs cell movement towards the chemoattractant source. Specifically, we analyze the undesirable effect of lateral diffusion in blurring the intracellular messenger profile. The concept of an effectiveness factor, akin to the analysis of reactions in porous catalysts, is applied to the spatial gradient sensing problem, with the distinction that slow, not fast, diffusion is required for effective gradient sensing. Analytical effectiveness factor expressions are derived for ideal geometries and then generalized to arbitrary cell shapes. In the case of mouse fibroblasts responding to gradients of platelet-derived growth factor, we conclude that the cell morphology and orientation with respect to the gradient can dictate whether messenger diffusion obliterates gradient sensing or has very little effect. The analysis outlined here allows the effect of intracellular messenger diffusion on spatial gradient sensing to be quantified for individual cells.}, number={17}, journal={CHEMICAL ENGINEERING SCIENCE}, publisher={Elsevier BV}, author={Haugh, Jason M. and Schneider, Ian C.}, year={2006}, month={Sep}, pages={5603–5611} }
@article{bryan_haugh_mccune_2006, title={Fast imaging of partially conductive linear cracks using impedance data}, volume={22}, ISSN={["0266-5611"]}, DOI={10.1088/0266-5611/22/4/013}, abstractNote={We develop two closely-related fast and simple numerical algorithms to address the inverse problem of identifying a collection of disjoint linear cracks in a two-dimensional homogeneous electrical conductor from exterior boundary voltage/current measurements. We allow the possibility that the cracks are partially conductive. Our approach also allows us to determine the actual number of cracks present, as well as make use of one or multiple input fluxes. We illustrate our algorithms with a variety of computational examples.}, number={4}, journal={INVERSE PROBLEMS}, author={Bryan, Kurt and Haugh, Janine and McCune, David}, year={2006}, month={Aug}, pages={1337–1358} }
@article{schneider_haugh_2006, title={Mechanisms of gradient sensing and chemotaxis - Conserved pathways, diverse regulation}, volume={5}, ISSN={["1551-4005"]}, DOI={10.4161/cc.5.11.2770}, abstractNote={Directed cell migration is critical for normal development, immune responses, and wound healing and plays a prominent role in tumor metastasis. In eukaryotes, cell orientation is biased by an external chemoattractant gradient through a spatial contrast in chemoattractant receptor-mediated signal transduction processes that differentially affect cytoskeletal dynamics at the cell front and rear. Mechanisms of spatial gradient sensing and chemotaxis have been studied extensively in the social amoeba Dictyostelium discoideum and mammalian leukocytes (neutrophils), which are similar in their remarkable sensitivity to shallow gradients and robustness of response over a broad range of chemoattractant concentration. Recently, we have quantitatively characterized a different gradient sensing system, that of platelet-derived growth factor-stimulated fibroblasts, an important component of dermal wound healing. The marked differences between this system and the others have led us to speculate on the diversity of gradient sensing mechanisms and their biological implications.}, number={11}, journal={CELL CYCLE}, publisher={Informa UK Limited}, author={Schneider, Ian C. and Haugh, Jason M.}, year={2006}, month={Jun}, pages={1130–1134} }
@article{kaur_park_lewis_haugh_2006, title={Quantitative model of Ras-phosphoinositide 3-kinase signalling cross-talk based on co-operative molecular assembly}, volume={393}, ISSN={["1470-8728"]}, DOI={10.1042/bj20051022}, abstractNote={In growth-factor-stimulated signal transduction, cell-surface receptors recruit PI3Ks (phosphoinositide 3-kinases) and Ras-specific GEFs (guanine nucleotide-exchange factors) to the plasma membrane, where they produce 3′-phosphorylated phosphoinositide lipids and Ras-GTP respectively. As a direct example of pathway networking, Ras-GTP also recruits and activates PI3Ks. To refine the mechanism of Ras–PI3K cross-talk and analyse its quantitative implications, we offer a theoretical model describing the assembly of complexes involving receptors, PI3K and Ras-GTP. While the model poses the possibility that a ternary receptor–PI3K–Ras complex forms in two steps, it also encompasses the possibility that receptor–PI3K and Ras–PI3K interactions are competitive. In support of this analysis, experiments with platelet-derived growth factor-stimulated fibroblasts revealed that Ras apparently enhances the affinity of PI3K for receptors; in the context of the model, this suggests that a ternary complex does indeed form, with the second step greatly enhanced through membrane localization and possibly allosteric effects. The apparent contribution of Ras to PI3K activation depends strongly on the quantities and binding affinities of the interacting molecules, which vary across different cell types and stimuli, and thus the model could be used to predict conditions under which PI3K signalling is sensitive to interventions targeting Ras.}, number={1}, journal={BIOCHEMICAL JOURNAL}, publisher={Portland Press Ltd.}, author={Kaur, H and Park, CS and Lewis, JM and Haugh, JM}, year={2006}, month={Jan}, pages={235–243} }
@article{schneider_haugh_2005, title={Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts}, volume={171}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.200509028}, abstractNote={Migration of eukaryotic cells toward a chemoattractant often relies on their ability to distinguish receptor-mediated signaling at different subcellular locations, a phenomenon known as spatial sensing. A prominent example that is seen during wound healing is fibroblast migration in platelet-derived growth factor (PDGF) gradients. As in the well-characterized chemotactic cells Dictyostelium discoideum and neutrophils, signaling to the cytoskeleton via the phosphoinositide 3-kinase pathway in fibroblasts is spatially polarized by a PDGF gradient; however, the sensitivity of this process and how it is regulated are unknown. Through a quantitative analysis of mathematical models and live cell total internal reflection fluorescence microscopy experiments, we demonstrate that PDGF detection is governed by mechanisms that are fundamentally different from those in D. discoideum and neutrophils. Robust PDGF sensing requires steeper gradients and a much narrower range of absolute chemoattractant concentration, which is consistent with a simpler system lacking the feedback loops that yield signal amplification and adaptation in amoeboid cells.}, number={5}, journal={JOURNAL OF CELL BIOLOGY}, publisher={Rockefeller University Press}, author={Schneider, IC and Haugh, JM}, year={2005}, month={Dec}, pages={883–892} }
@article{monine_haugh_2005, title={Reactions on cell membranes: Comparison of continuum theory and Brownian dynamics simulations}, volume={123}, ISSN={["1089-7690"]}, DOI={10.1063/1.2000236}, abstractNote={Biochemical transduction of signals received by living cells typically involves molecular interactions and enzyme-mediated reactions at the cell membrane, a problem that is analogous to reacting species on a catalyst surface or interface. We have developed an efficient Brownian dynamics algorithm that is especially suited for such systems and have compared the simulation results with various continuum theories through prediction of effective enzymatic rate constant values. We specifically consider reaction versus diffusion limitation, the effect of increasing enzyme density, and the spontaneous membrane association/dissociation of enzyme molecules. In all cases, we find the theory and simulations to be in quantitative agreement. This algorithm may be readily adapted for the stochastic simulation of more complex cell signaling systems.}, number={7}, journal={JOURNAL OF CHEMICAL PHYSICS}, publisher={AIP Publishing}, author={Monine, MI and Haugh, JM}, year={2005}, month={Aug} }
@article{schneider_parrish_haugh_2005, title={Spatial analysis of 3 ' phosphoinositide signaling in living fibroblasts, III: Influence of cell morphology and morphological polarity}, volume={89}, ISSN={["1542-0086"]}, DOI={10.1529/biophysj.105.061218}, abstractNote={Activation of phosphoinositide (PI) 3-kinase is a required signaling pathway in fibroblast migration directed by platelet-derived growth factor. The pattern of 3' PI lipids in the plasma membrane, integrating local PI 3-kinase activity as well as 3' PI diffusion and turnover, influences the spatiotemporal regulation of the cytoskeleton. In fibroblasts stimulated uniformly with platelet-derived growth factor, visualized using total internal reflection fluorescence microscopy, we consistently observed localized regions with significantly higher or lower 3' PI levels than adjacent regions (hot and cold spots, respectively). A typical cell contained multiple hot spots, coinciding with apparent leading edge structures, and at most one cold spot at the rear. Using a framework for finite-element modeling with actual cell contact area geometries, we find that although the 3' PI pattern is affected by irregular contact area shape, cell morphology alone cannot explain the presence of hot or cold spots. Our results and analysis instead suggest that these regions reflect different local 3' PI dynamics, specifically through a combination of mechanisms: enhanced PI 3-kinase activity, reduced 3' PI turnover, and possibly slow/constrained 3' PI diffusion. The morphological polarity of the cell may thus bias 3' PI signaling to promote persistent migration in fibroblasts.}, number={2}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Schneider, IC and Parrish, EM and Haugh, JM}, year={2005}, month={Aug}, pages={1420–1430} }
@article{haugh_2004, title={Mathematical model of human growth hormone (hGH)-stimulated cell proliferation explains the efficacy of hGH variants as receptor agonists or antagonists}, volume={20}, ISSN={["8756-7938"]}, DOI={10.1021/bp0499101}, abstractNote={Human growth hormone (hGH) is a therapeutically important endocrine factor that signals various cell types. Structurally and functionally, the interactions of hGH with its receptor have been resolved in fine detail, such that hGH and hGH receptor variants can be practically engineered by either random or rational approaches to achieve significant changes in the free energies of binding. A somewhat unique feature of hGH action is its homodimerization of two hGH receptors, which is required for intracellular signaling and stimulation of cell proliferation, yet the potencies of hGH mutants in cell‐based assays rarely correlate with their overall receptor‐binding avidities. Here, a mathematical model of hGH‐stimulated cell signaling is posed, accounting not only for binding interactions at the cell surface but induction of receptor endocytosis and downregulation as well. Receptor internalization affects ligand potency by imposing a limit on the lifetime of an active receptor complex, irrespective of ligand‐receptor binding properties. The model thus explains, in quantitative terms, the numerous published observations regarding hGH receptor agonism and antagonism and challenges the interpretations of previous studies that have not considered receptor trafficking as a central regulatory mechanism in hGH signaling.}, number={5}, journal={BIOTECHNOLOGY PROGRESS}, publisher={Wiley-Blackwell}, author={Haugh, JM}, year={2004}, pages={1337–1344} }
@article{haugh_schneider_lewis_2004, title={On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling}, volume={230}, ISSN={["1095-8541"]}, DOI={10.1016/j.jtbi.2004.04.023}, abstractNote={Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.}, number={1}, journal={JOURNAL OF THEORETICAL BIOLOGY}, publisher={Elsevier BV}, author={Haugh, JM and Schneider, IC and Lewis, JM}, year={2004}, month={Sep}, pages={119–132} }
@article{haugh_schneider_2004, title={Spatial analysis of 3 ' phosphoinositide signaling in living fibroblasts: I. Uniform stimulation model and bounds on dimensionless groups}, volume={86}, ISSN={["1542-0086"]}, DOI={10.1016/S0006-3495(04)74137-5}, abstractNote={Fluorescent protein probes now permit spatial distributions of specific intracellular signaling molecules to be observed in real time. Mathematical models have been used to simulate molecular gradients and other spatial patterns within cells, and the output of such models may be compared directly with experiments if the binding of the fluorescent probe and the physics of the imaging technique are each incorporated. Here we present a comprehensive model describing the dynamics of 3' phosphoinositides (PIs), lipid second messengers produced in the plasma membrane in response to stimulation of the PI 3-kinase signaling pathway, as monitored in the cell-substratum contact area using total internal reflection fluorescence microscopy. With this technique it was previously shown that uniform stimulation of fibroblasts with platelet-derived growth factor elicits the formation of axisymmetric 3' PI gradients, which we now characterize in the context of our model. We find that upper and lower bounds on the relevant dimensionless model parameter values for an individual cell can be calculated from four well-defined fluorescence measurements. Based on our analysis, we expect that the key dimensionless group, the ratio of 3' PI turnover and diffusion rates, can be estimated within approximately 20% or less.}, number={1}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Haugh, JM and Schneider, IC}, year={2004}, month={Jan}, pages={589–598} }
@article{schneider_haugh_2004, title={Spatial analysis of 3 ' phosphoinositide signaling in living fibroblasts: II. Parameter estimates for individual cells from experiments}, volume={86}, ISSN={["1542-0086"]}, DOI={10.1016/S0006-3495(04)74138-7}, abstractNote={Fibroblast migration is directed by gradients of platelet-derived growth factor (PDGF) during wound healing. As in other chemotactic systems, it has been shown recently that localized stimulation of intracellular phosphoinositide (PI) 3-kinase activity and production of 3' PI lipids in the plasma membrane are important events in the signaling of spatially biased motility processes. In turn, 3' PI localization depends on the effective diffusion coefficient, D, and turnover rate constant, k, of these lipids. Here we present a systematic and direct comparison of mathematical model calculations and experimental measurements to estimate the values of the effective 3' PI diffusion coefficient, D, turnover rate constant, k, and other parameters in individual fibroblasts stimulated uniformly with PDGF. In the context of our uniform stimulation model, the values of D and k in each cell were typically estimated within 10-20% or less, and the mean values across all of the cells analyzed were D = 0.37 +/- 0.25 microm2/s and k = 1.18 +/- 0.54 min(-1). In addition, we report that 3' PI turnover is not affected by PDGF receptor signaling in our cells, allowing us to focus our attention on the regulation of 3' PI production as this system is studied further.}, number={1}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Schneider, IC and Haugh, JM}, year={2004}, month={Jan}, pages={599–608} }
@article{park_schneider_haugh_2003, title={Kinetic analysis of platelet-derived growth factor receptor/phosphoinositide 3-kinase/Akt signaling in fibroblasts}, volume={278}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.M304968200}, abstractNote={Isoforms of the serine-threonine kinase Akt coordinate multiple cell survival pathways in response to stimuli such as platelet-derived growth factor (PDGF). Activation of Akt is a multistep process, which relies on the production of 3′-phosphorylated phosphoinositide (PI) lipids by PI 3-kinases. To quantitatively assess the kinetics of PDGF receptor/PI 3-kinase/Akt signaling in fibroblasts, a systematic study of this pathway was performed, and a mechanistic mathematical model that describes its operation was formulated. We find that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, and its kinetics are quantitatively consistent with a mechanism in which receptor dimerization is initially mediated by the association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high concentrations of PDGF, consistent with the loss of activated receptors upon endocytosis. By comparison, Akt activation responds to lower PDGF concentrations and exhibits more sustained kinetics. Further analysis and modeling suggest that the pathway is saturated at the level of PI 3-kinase activation, and that the p110α catalytic subunit of PI 3-kinase contributes most to PDGF-stimulated 3′-PI production. Thus, at high concentrations of PDGF the kinetics of 3′-PI production are limited by the turnover rate of these lipids, while the Akt response is additionally influenced by the rate of Akt deactivation.}, number={39}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Park, CS and Schneider, IC and Haugh, JM}, year={2003}, month={Sep}, pages={37064–37072} }
@article{haugh_2002, title={A unified model for signal transduction reactions in cellular membranes}, volume={82}, ISSN={["0006-3495"]}, DOI={10.1016/S0006-3495(02)75424-6}, abstractNote={Abstract
An analytical solution is obtained for the steady-state reaction rate of an intracellular enzyme, recruited to the plasma membrane by active receptors, acting upon a membrane-associated substrate. Influenced by physical and chemical effects, such interactions are encountered in numerous signal-transduction pathways. The generalized modeling framework is the first to combine reaction and diffusion limitations in enzyme action, the finite mean lifetime of receptor–enzyme complexes, reactions in the bulk membrane, and constitutive and receptor-mediated substrate insertion. The theory is compared with other analytical and numerical approaches, and it is used to model two different signaling pathway types. For two-state mechanisms, such as activation of the Ras GTPase, the diffusion-limited activation rate constant increases with enhanced substrate inactivation, dissociation of receptor–enzyme complexes, or crowding of neighboring complexes. The latter effect is only significant when nearly all of the substrate is in the activated state. For regulated supply and turnover pathways, such as phospholipase C-mediated lipid hydrolysis, an additional influence is receptor-mediated substrate delivery. When substrate consumption is rapid, this process significantly enhances the effective enzymatic rate constant, regardless of whether enzyme action is diffusion limited. Under these conditions, however, enhanced substrate delivery can result in a decrease in the average substrate concentration.}, number={2}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Haugh, JM}, year={2002}, month={Feb}, pages={591–604} }
@article{active egf receptors have limited access to ptdins(4,5)p2 in endosomes: implications for phospholipase c and pi 3-kinase signaling_2002, journal={Journal of Cell Science}, year={2002}, month={Jan} }
@article{haugh_meyer_2002, title={Active EGF receptors have limited access to Ptdlns(4,5)P-2 in endosomes: implications for phospholipase C and PI 3-kinase signaling}, volume={115}, number={2}, journal={Journal of Cell Science}, author={Haugh, J. M. and Meyer, T.}, year={2002}, pages={303–310} }
@article{haugh_2002, title={Localization of Receptor-Mediated Signal Transduction Pathways: The Inside Story}, volume={2}, DOI={10.1124/mi.2.5.292}, abstractNote={Receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) elicit proliferation, migration, and differentiation in a wide spectrum of cell types through various signal transduction pathways. These activities are attenuated by receptor internalization, intracellular trafficking through endosomes, and degradation in lysosomes, resulting in decreased receptor expression. However, there is now considerable evidence that EGFRs continue to signal in endosomes, forcing us to reevaluate the outcomes of receptor trafficking. An exciting revelation is that internalized receptors extend some signaling activities but not others, suggesting that certain responses, such as cell motility, must be mediated at the cell surface. Still, only when the effects of decreased receptor populations and signaling compartmentalization are integrated can we hope to understand and manipulate receptor function at the molecular level.}, number={5}, journal={Molecular Interventions}, publisher={CrossRef Test Account}, author={Haugh, J. M.}, year={2002}, month={Sep}, pages={292–307} }
@article{haugh_wells_lauffenburger_2000, title={Mathematical modeling of epidermal growth factor receptor signaling through the phospholipase C pathway: Mechanistic insights and predictions for molecular interventions}, volume={70}, ISSN={["1097-0290"]}, DOI={10.1002/1097-0290(20001020)70:2<225::AID-BIT12>3.0.CO;2-S}, abstractNote={Combining engineering analyses and mathematical modeling with intervention and detection methodologies at the molecular level will allow manipulation of intracellular signal transduction pathways, and therefore rational control of functional processes central to medicine and biotechnology. We have formulated a simple mathematical model of a key signaling pathway required for regulated migration of fibroblasts and other cell types: activation of the intracellular enzyme phospholipase C (PLC) mediated by epidermal growth factor receptor (EGFR) and a multitude of other transmembrane receptors. One of the interesting features of this pathway is that the substrate of PLC, the lipid phosphatidylinositol (4,5)-bisphosphate (PIP(2)), is turned over quite rapidly and must be constantly resupplied to the plasma membrane by a known transfer mechanism. The model, which accounts for regulation of PIP(2) concentration, is sufficiently detailed to explain unique quantitative features of recent experimental data. We find that competitive pathways that deplete PIP(2) from the membrane, as well as receptor-mediated enhancement of PIP(2) supply, must be significant for agreement between model and experiment. Importantly, the mechanistic nature of the model also allowed us to predict the efficacy of various molecular intervention strategies, including overexpression of wild-type and variant proteins in the pathway as well as treatment with specific drug inhibitors. For many parameter conditions the intuitive strategy of targeting the enzyme itself is actually predicted to be relatively inefficient, with a novel and potentially useful alternative being disruption of the reactant supply mechanism.}, number={2}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Haugh, JM and Wells, A and Lauffenburger, DA}, year={2000}, month={Oct}, pages={225–238} }
@article{haugh_wells_lauffenburger_2000, title={Mathematical modeling of epidermal growth factor receptor signaling through the phospholipase C pathway: Mechanistic insights and predictions for molecular interventions}, volume={70}, DOI={10.1002/1097-0290(20001020)70:2<225::aid-bit12>3.3.co;2-j}, abstractNote={Combining engineering analyses and mathematical modeling with intervention and detection methodologies at the molecular level will allow manipulation of intracellular signal transduction pathways, and therefore rational control of functional processes central to medicine and biotechnology. We have formulated a simple mathematical model of a key signaling pathway required for regulated migration of fibroblasts and other cell types: activation of the intracellular enzyme phospholipase C (PLC) mediated by epidermal growth factor receptor (EGFR) and a multitude of other transmembrane receptors. One of the interesting features of this pathway is that the substrate of PLC, the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2), is turned over quite rapidly and must be constantly resupplied to the plasma membrane by a known transfer mechanism. The model, which accounts for regulation of PIP2 concentration, is sufficiently detailed to explain unique quantitative features of recent experimental data. We find that competitive pathways that deplete PIP2 from the membrane, as well as receptor-mediated enhancement of PIP2 supply, must be significant for agreement between model and experiment. Importantly, the mechanistic nature of the model also allowed us to predict the efficacy of various molecular intervention strategies, including overexpression of wild-type and variant proteins in the pathway as well as treatment with specific drug inhibitors. For many parameter conditions the intuitive strategy of targeting the enzyme itself is actually predicted to be relatively inefficient, with a novel and potentially useful alternative being disruption of the reactant supply mechanism. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 225–238, 2000.}, number={2}, journal={Biotechnology and Bioengineering}, publisher={Wiley-Blackwell}, author={Haugh, Jason M. and Wells, Alan and Lauffenburger, Douglas A.}, year={2000}, month={Oct}, pages={225–238} }
@article{haugh_codazzi_teruel_meyer_2000, title={Spatial sensing in fibroblasts mediated by 3 ' phosphoinositides}, volume={151}, ISSN={["1540-8140"]}, DOI={10.1083/jcb.151.6.1269}, abstractNote={The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3′ PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein–tagged Akt pleckstrin homology domain (GFP–AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3′ PI lipids in the adhesion zone of fibroblasts. Polar GFP–AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3′ PI production and rapid membrane spreading implicates 3′ PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3′ PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 μm2/s) and short lifetime of 3′ PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3′ PI second messengers.}, number={6}, journal={JOURNAL OF CELL BIOLOGY}, publisher={Rockefeller University Press}, author={Haugh, JM and Codazzi, F and Teruel, M and Meyer, T}, year={2000}, month={Dec}, pages={1269–1279} }
@article{haugh_schooler_wells_wiley_lauffenburger_1999, title={Effect of Epidermal Growth Factor Receptor Internalization on Regulation of the Phospholipase C- 1 Signaling Pathway}, volume={274}, DOI={10.1074/jbc.274.13.8958}, abstractNote={The epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-α (TGFα) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGFα-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-γ1 (PLC-γ1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGFα stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP2) hydrolysis over those stimulated by TGFα. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP2 hydrolysis, PLC-γ1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-γ1 phosphorylation and PIP2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-γ1 to its substrate in endocytic trafficking organelles.}, number={13}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Haugh, J. M. and Schooler, K. and Wells, A. and Wiley, H. S. and Lauffenburger, D. A.}, year={1999}, month={Mar}, pages={8958–8965} }
@article{haugh_huang_wiley_wells_lauffenburger_1999, title={Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21ras in Fibroblasts}, volume={274}, DOI={10.1074/jbc.274.48.34350}, abstractNote={Regulated activation of the highly conserved Ras GTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc·Grb2·Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor down-regulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-α, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.}, number={48}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Haugh, J. M. and Huang, A. C. and Wiley, H. S. and Wells, A. and Lauffenburger, D. A.}, year={1999}, month={Nov}, pages={34350–34360} }
@article{haugh_lauffenburger_1998, title={Analysis of Receptor Internalization as a Mechanism for Modulating Signal Transduction}, volume={195}, DOI={10.1006/jtbi.1998.0791}, abstractNote={The past decade has witnessed a profound explosion of knowledge in the field of signal transduction mediated by receptor tyrosine kinases. Upon binding of cognate extracellular ligands, these receptors interact with various enzymes and other signaling molecules intracellularly. These protein substrates, which are generally freely diffusing residents of the cytoplasm, as well as the predominantly membrane-associated downstream targets that they activate, are now fairly well characterized molecules. Despite this surge in signaling research, the mechanisms that regulate signaling interactions in a dynamic fashion remain poorly understood, particulary in quantitative terms. We have developed a generalized mathematical model describing the relationships among receptor, substrate, and target molecules with the aim of gaining fundamental insights into one suggested control mechanism: endocytic trafficking-the rapid and induced internalization of ligated receptors and subsequent relocation to intracellular trafficking organelles. This model is very well-suited, in particular, for the epidermal growth factor receptor. Our major conclusion is that when downstream signaling does not rely on phosphorylation of the substrate by the receptor, or when the substrate is phosphorylated to the same extent regardless of receptor location, receptor internalization can still affect signaling if the membrane-associated target of the substrate is differentially partitioned between surface and internal membrane environments. Specificity of this target "availability" effect can derive from molecular specificity of the retention mechanisms that drive this partitioning. A second conclusion is then that differences in substrate or receptor phosphorylation stoichiometries, perhaps due to partitioning of other membrane kinases or phosphatases, can provide additional influence on signaling. Whatever the mechanism, the total observed activity-i.e. the rate of activated target molecules produced per unit time- can be correlated with receptor/ligand binding and trafficking data to determine whether internalization abrogates or amplifies signaling.}, number={2}, journal={Journal of Theoretical Biology}, publisher={Elsevier BV}, author={Haugh, Jason M. and Lauffenburger, Douglas A.}, year={1998}, month={Nov}, pages={187–218} }
@article{lauffenburger_fallon_haugh_1998, title={Scratching the (cell) surface: cytokine engineering for improved ligand/receptor trafficking dynamics}, volume={5}, DOI={10.1016/s1074-5521(98)90110-7}, abstractNote={Cytokines can be engineered for greater potency in stimulating cellular functions. An obvious test criterion for an improved cytokine is receptor-binding affinity, but this does not always correlate with improved biological response. By combining protein-engineering techniques with studies of receptor trafficking and signaling, it might be possible to identify the ligand receptor-binding properties that should be sought.}, number={10}, journal={Chemistry & Biology}, publisher={Elsevier BV}, author={Lauffenburger, Douglas A. and Fallon, Eric M. and Haugh, Jason M.}, year={1998}, month={Oct}, pages={R257–R263} }
@article{haugh_lauffenburger_1997, title={Physical modulation of intracellular signaling processes by locational regulation}, volume={72}, DOI={10.1016/s0006-3495(97)78846-5}, abstractNote={Recent observations in the field of signal transduction suggest that where a protein is located within a cell can be as important as its activity measured in solution for activation of its downstream pathway. The physical organization of the cell can provide an additional layer of control upon the chemical reaction networks that govern ultimately perceived signals. Using the cytosol and plasma membrane as relevant compartmental distinctions, we analyze the effect of relocation on the rate of association with a membrane-associated target. We quantify this effect as an enhancement factor E in terms of measurable parameters such as the number of available targets, molecular diffusivities, and intrinsic reaction rate constants. We then employ two simple yet relevant example models to illustrate how relocation can affect the dynamics of signal transduction pathways. The temporal profiles and phase behavior of these models are investigated. We also relate experimentally observable aspects of signal transduction such as peak activation and the relative time scales of stimulus and response to quantitative aspects of the relocation mechanisms in our models. In our example schemes, nearly complete relocation of the cytosolic species in the signaling pair is required to generate meaningful activation of the model pathways when the association rate enhancement factor E is as low as 10; when E is 100 or greater, only a small fraction of the protein must be relocated.}, number={5}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Haugh, J.M. and Lauffenburger, D.A.}, year={1997}, month={May}, pages={2014–2031} }
@article{haugh_weiger, title={Modeling Intracellular Signal Transduction Processes}, DOI={10.1002/9783527619375.ch17b}, abstractNote={The prelims comprise: Introduction Receptor-Binding and Regulation Mechanisms Receptor-mediated Covalent Modifications and Molecular Interactions Spatial Organization and Gradients on Cellular and Subcellular Length Scales Downstream Signaling Cascades and Networks Prospects and Challenges Concluding Remarks}, journal={Chemical Biology}, publisher={Wiley-Blackwell}, author={Haugh, Jason M. and Weiger, Michael C.}, pages={1061–1081} }
@misc{welf_ahmed_johnson_melvin_haugh, title={PI3K's Pivotal Role in Cell Migration}, DOI={10.4016/47795.01}, journal={SciVee}, publisher={SciVee, Inc}, author={Welf, E. and Ahmed, S. and Johnson, H.E. and Melvin, A.T. and Haugh, J.M.} }