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1.  Epitaxially Grown Collagen Fibrils Reveal Diversity in Contact Guidance Behavior among Cancer Cells 
Langmuir  2014;31(1):307-314.
Invasion of cancer cells into the surrounding tissue is an important step during cancer progression and is driven by cell migration. Cell migration can be random, but often it is directed by various cues such as aligned fibers composed of extracellular matrix (ECM), a process called contact guidance. During contact guidance, aligned fibers bias migration along the long axis of the fibers. These aligned fibers of ECM are commonly composed of type I collagen, an abundant structural protein around tumors. In this paper, we epitaxially grew several different patterns of organized type I collagen on mica and compared the morphology and contact guidance behavior of two invasive breast cancer cell lines (MDA-MB-231 and MTLn3 cells). Others have shown that these cells randomly migrate in qualitatively different ways. MDA-MB-231 cells exert large traction forces, tightly adhere to the ECM, and migrate with spindle-shaped morphology and thus adopt a mesenchymal mode of migration. MTLn3 cells exert small traction forces, loosely adhere to the ECM, and migrate with a more rounded morphology and thus adopt an amoeboid mode of migration. As the degree of alignment of type I collagen fibrils increases, cells become more elongated and engage in more directed contact guidance. MDA-MB-231 cells perceive the directional signal of highly aligned type I collagen fibrils with high fidelity, elongating to large extents and migrating directionally. Interestingly, behavior in MTLn3 cells differs. While highly aligned type I collagen fibril patterns facilitate spreading and random migration of MTLn3 cells, they do not support elongation or directed migration. Thus, different contact guidance cues bias cell migration differently and the fidelity of contact guidance is cell type dependent, suggesting that ECM alignment is a permissive cue for contact guidance, but requires a cell to have certain properties to interpret that cue.
PMCID: PMC4295811  PMID: 25531276
2.  Mechanical Integration of Actin and Adhesion Dynamics in Cell Migration 
Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.
PMCID: PMC4437624  PMID: 19575647
F-actin cytoskeleton; adhesion maturation; traction force; adhesion strength; focal adhesion
3.  Epidermal Growth Factor–induced Contraction Regulates Paxillin Phosphorylation to Temporally Separate Traction Generation from De-adhesion 
Molecular Biology of the Cell  2009;20(13):3155-3167.
Directed cell migration is mediated by cycles of protrusion, adhesion, traction generation on the extracellular matrix and retraction. However, how the events after protrusion are timed, and what dictates their temporal order is completely unknown. We used acute epidermal growth factor (EGF) stimulation of epidermal keratinocytes to initiate the cell migration cycle to study the mechanism of the timing of adhesion, traction generation, and de-adhesion. Using microscopic and biochemical assays, we surprisingly found that at ∼2 min after EGF stimulation protrusion, activation of myosin-II, traction generation, adhesion assembly, and paxillin phosphorylation occurred nearly simultaneously, followed by a 10-min delay during which paxillin became dephosphorylated before cell retraction. Inhibition of myosin-II blocked both the EGF-stimulated paxillin phosphorylation and cell retraction, and a paxillin phosphomimic blocked retraction. These results suggest that EGF-mediated activation of myosin-II acts as a mechanical signal to promote a cycle of paxillin phosphorylation/dephosphorylation that mediates a cycle of adhesion strengthening and weakening that delays cell retraction. Thus, we reveal for the first time a mechanism by which cells may temporally segregate protrusion, adhesion, and traction generation from retraction during EGF-stimulated cell migration.
PMCID: PMC2704165  PMID: 19403690
4.  Differences in adhesion and protrusion properties correlate with differences in migration speed under EGF stimulation 
BMC Biophysics  2012;5:8.
Cell migration plays an essential role in many biological processes, such as cancer metastasis, wound healing and immune response. Cell migration is mediated through protrusion and focal adhesion (FA) assembly, maturation and disassembly. Epidermal growth factor (EGF) is known to enhance migration rate in many cell types; however it is not known how FA maturation, FA dynamics and protrusion dynamics are regulated during EGF-induced migration. Here we use total internal reflection fluorescence (TIRF) microscopy and image analysis to quantify FA properties and protrusion dynamics under different doses of EGF stimulation.
EGF was found to broaden the distribution of cell migration rates, generating more fast and slow cells. Furthermore, groups based on EGF stimulation condition or cell migration speed were marked by characteristic signatures. When data was binned based on EGF stimulation conditions, FA intensity and FA number per cell showed the largest difference among stimulation groups. FA intensity decreased with increasing EGF concentration and FA number per cell was highest under intermediate stimulation conditions. No difference in protrusion behavior was observed. However, when data was binned based on cell migration speed, FA intensity and not FA number per cell showed the largest difference among groups. FA intensity was lower for fast migrating cells. Additionally, waves of protrusion tended to correlate with fast migrating cells.
Only a portion of the FA properties and protrusion dynamics that correlate with migration speed, correlate with EGF stimulation condition. Those that do not correlate with EGF stimulation condition constitute the most sensitive output for identifying why cells respond differently to EGF. The idea that EGF can both increase and decrease the migration speed of individual cells in a population has particular relevance to cancer metastasis where the microenvironment can select subpopulations based on some adhesion and protrusion characteristics, leading to a more invasive phenotype as would be seen if all cells responded like an “average” cell.
PMCID: PMC3414788  PMID: 22577847
5.  Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation 
The Journal of Cell Biology  2010;188(6):877-890.
FAK-mediated myosin-dependent paxillin phosphorylation is necessary to bring vinculin to maturing focal adhesions, reinforcing the link between the cytoskeleton and the ECM.
Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II–mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II–dependent manner and to examine the mechanism for their myosin II–sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II–mediated FAK phosphorylation of paxillin that promotes vinculin FA recruitment to reinforce the cytoskeletal ECM linkage and drive FA maturation.
PMCID: PMC2845065  PMID: 20308429
6.  Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts 
The Journal of Cell Biology  2005;171(5):883-892.
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.
PMCID: PMC2171296  PMID: 16314431

Results 1-6 (6)