@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{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{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{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{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} }