Project 4: FMN2 makes perinuclear actin to protect nuclei from damage during confined cell migration and promote metastasis Colleen T. Skau Cell migration in confined 3D tissue microenvironments is critical for development, immune function, and the dissemination of tumor cells in metastasis. How cells squeeze their large and stiff nuclei through such environments is unclear. We discovered a cytoskeletal structure that prevents damage to the nucleus and genetic material during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is compositionally and functionally distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In cells migrating in confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is highly upregulated in a range of human melanomas, and show that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Collectively our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration, and thus promote cancer metastasis. A paper resulting from these studies is in press at Cell Project 3: Colleen Skau. INF2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly Actin filaments and integrin-based focal adhesions (FA) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator INF2 localizes specifically to FA and dorsal stress fibers (SF) in fibroblasts. High resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF-FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FA during maturation rather than during their initial generation, and once there acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately three dimensional matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SF and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM. A paper resulting from these studies was published in PNAS and a review was published in Ann Rev Biophys Project 4: Vinay Swaminathan The FAK-Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin. Cell migration is initiated in response to biochemical or physical cues in the environment that promote actin-mediated lamellipodial protrusion followed by the formation of nascent integrin adhesions (NA) within the protrusion to drive leading edge advance. Although the focal adhesion-associated non-receptor tyrosine kinase FAK is known to be required for cell migration through effects on mature focal adhesions, its role in NA formation and lamellipodial dynamics is not clear. Live-cell microscopy of FAK -/- cells with expression of phosphorylation or a FERM domain mutant deficient in Arp2/3 binding revealed a requirement for FAK in promoting the dense formation, transient stabilization and timely turnover of NA within lamellipodia to couple actin-driven protrusion to adhesion and advance of the leading edge. We show that phosphorylation on Y397 of FAK promotes dense NA formation, but is dispensible for transient NA stabilization and leading edge advance. In contrast, transient NA stabilization and advance of the cell edge requires FAK-Arp2/3 interaction, which promotes Arp2/3 localization to NA and reduces FAK activity. Haptosensing of ECM concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading. Together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration. A paper resulting from these studies was published in MBOC and a review was published

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Luo, Ruibai; Chen, Pei-Wen; Kuo, Jean-Cheng et al. (2018) ARAP2 inhibits Akt independently of its effects on focal adhesions. Biol Cell 110:257-270
Wu, Zhanghan; Plotnikov, Sergey V; Moalim, Abdiwahab Y et al. (2017) Two Distinct Actin Networks Mediate Traction Oscillations to Confer Focal Adhesion Mechanosensing. Biophys J 112:780-794
Swaminathan, Vinay; Alushin, Gregory M; Waterman, Clare M (2017) Mechanosensation: A Catch Bond That Only Hooks One Way. Curr Biol 27:R1158-R1160
Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Mehta, Shalin B et al. (2017) Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions. Proc Natl Acad Sci U S A 114:10648-10653
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Baird, Michelle A; Billington, Neil; Wang, Aibing et al. (2017) Local pulsatile contractions are an intrinsic property of the myosin 2A motor in the cortical cytoskeleton of adherent cells. Mol Biol Cell 28:240-251
Swaminathan, Vinay; Waterman, Clare M (2016) The molecular clutch model for mechanotransduction evolves. Nat Cell Biol 18:459-61
Skau, Colleen T; Fischer, Robert S; Gurel, Pinar et al. (2016) FMN2 Makes Perinuclear Actin to Protect Nuclei during Confined Migration and Promote Metastasis. Cell 167:1571-1585.e18

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