@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. Building better biosensors The accuracy of a biosensor used to measure signaling events in live cells depends on both the specificity of the biosensor for its intended target and the absence of interference with the signaling pathway. Phosphotyrosine-binding SH2 domains have been used as biosensors for receptor tyrosine kinase activation. Tiruthani et al. showed that a paired SH2 domain biosensor for monitoring phosphorylation of the epidermal growth factor receptor (EGFR) at Tyr992 was not specific for EGFR and was not recruited to the membrane in a manner that accurately reflected the kinetics of EGFR signaling. Hence, the authors used two different mutagenesis and screening approaches to engineer new biosensors, mSH2 and SPY992, that exhibited greater specificity for EGFR Tyr992 and more accurately reported EGFR signaling kinetics. These approaches were extended to develop SPY1148, a biosensor for phosphorylation of EGFR at Tyr1148, and could be extended to generate phospho-specific biosensors for various targets. Live-cell fluorescence microscopy is broadly applied to study the dynamics of receptor-mediated cell signaling, but the availability of intracellular biosensors is limited. A biosensor based on the tandem SH2 domains from phospholipase C–γ1 (PLCγ1), tSH2-WT, has been used to measure phosphorylation of the epidermal growth factor receptor (EGFR). Here, we found that tSH2-WT lacked specificity for phosphorylated EGFR, consistent with the known promiscuity of SH2 domains. Further, EGF-stimulated membrane recruitment of tSH2-WT differed qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model suggested, and experiments confirmed, that the high avidity of tSH2-WT resulted in saturation of its target and interference with EGFR endocytosis. To overcome the apparent target specificity and saturation issues, we implemented two protein engineering strategies. In the first approach, we screened a combinatorial library generated by random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1 and isolated a mutant form (mSH2) with enhanced specificity for phosphorylated Tyr992 (pTyr992) of EGFR. A biosensor based on mSH2 closely reported the kinetics of EGFR phosphorylation but retained cross-reactivity similar to tSH2-WT. In the second approach, we isolated a pTyr992-binding protein (SPY992) from a combinatorial library generated by mutagenesis of the Sso7d protein scaffold. Compared to tSH2-WT and mSH2, SPY992 exhibited superior performance as a specific, moderate-affinity biosensor. We extended this approach to isolate a biosensor for EGFR pTyr1148 (SPY1148). This approach of integrating theoretical considerations with protein engineering strategies can be generalized to design and evaluate suitable biosensors for various phospho-specific targets.}, 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{singh_zhang_ahmed_ramakrishnan_ekkad_2019, title={Effect of micro-roughness shapes on jet impingement heat transfer and fin-effectiveness}, volume={132}, ISSN={0017-9310}, url={http://dx.doi.org/10.1016/j.ijheatmasstransfer.2018.11.135}, DOI={10.1016/j.ijheatmasstransfer.2018.11.135}, abstractNote={With recent advancements in the field of additive manufacturing, the design domain for development of complicated cooling configurations has significantly expanded. The motivation of the present study is to develop high performance impingement cooling designs catered towards applications requiring high rates of heat removal, e.g. gas turbine blade leading edge and double-wall cooling, air-cooled electronic devices, etc. In the present study, jet impingement is combined with strategic roughening of the target surface, to achieve high heat removal rates. Steady state experiments have been carried out to calculate the heat transfer coefficient for jet impingement onto different target surface configurations. The jet-to-jet spacing (x/d = y/d) was varied from 2 to 5, and jet-to-target distance (z/d) was varied from 1 to 5. The target surface configurations featured cylindrical, cubic and concentric shaped roughness elements, fabricated through binder jetting process. The baseline case for the roughened target surface was a smooth target. Heat transfer and pressure drop experiments were carried out at Reynolds numbers ranging from 2500 to 10,000. Further, numerical simulations were carried out to model flow and heat transfer for all configurations at a representative Reynolds number. Through our experiments and numerical results, we have demonstrated that the novel “concentric” roughness shape was the best in terms of fin effectiveness and Nusselt numbers levels, amongst the investigated shapes. The concentric-shape roughened target resulted in fin effectiveness up to 1.6, whereas the cubic- and cylindrical-shape roughened targets yielded in fin effectiveness up to 1.4 and 1.3, respectively. Further, it was experimentally found that the addition of micro-roughness elements does not result in a discernable increment in pressure losses, compared to the impingement on the smooth target surface. Hence, the demonstrated configuration with the highest heat transfer coefficient also resulted in highest thermal hydraulic performance.}, journal={International Journal of Heat and Mass Transfer}, publisher={Elsevier BV}, author={Singh, Prashant and Zhang, Mingyang and Ahmed, Shoaib and Ramakrishnan, Kishore R. and Ekkad, Srinath}, year={2019}, month={Apr}, pages={80–95} }
 @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={The extracellular signal-regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live-cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole-cell ERK phosphorylation is a by-product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose-dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.}, 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{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={Migrating fibroblasts reorient directionality by PI3K-dependent branching and pivoting of protrusions, a mechanism that allows fibroblasts to align with an external chemotactic 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{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{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} }