@article {115040, title = {Active emulsions in living cell membranes driven by contractile stresses and transbilayer coupling}, journal = {Proc Natl Acad Sci U S A.}, volume = {119}, year = {2022}, month = {07/2022}, pages = {e2123056119}, abstract = {

The spatiotemporal organization of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting, and endocytosis. Earlier studies have shown that multiple types of membrane proteins, including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered glycosylphosphatidylinositol-anchored proteins (GPI-APs), form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drives the creation of active emulsions, mesoscale liquid order (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid phase segregation. To test these ideas, we use spatial imaging of molecular clustering combined with local membrane order, and we demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions and exhibit significant lipid order, consistent with predictions of the active composite model.

}, doi = {10.1073/pnas.2123056119}, author = {Saha, Suvrajit and Das, A and Patra, C and Anilkumar, AA and Sil, Parijat and Mayor, Satyajit and Rao, Madan} } @article {25251, title = {Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions.}, journal = {Proc Natl Acad Sci U S A}, volume = {114}, year = {2017}, month = {2017 Oct 03}, pages = {10648-10653}, abstract = {

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.

}, issn = {1091-6490}, doi = {10.1073/pnas.1701136114}, author = {Swaminathan, Vinay and Kalappurakkal, Joseph Mathew and Mehta, Shalin B and Nordenfelt, Pontus and Moore, Travis I and Koga, Nobuyasu and Baker, David A and Oldenbourg, Rudolf and Tani, Tomomi and Mayor, Satyajit and Springer, Timothy A and Waterman, Clare M} } @article {3546, title = {Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer.}, journal = {Proc Natl Acad Sci U S A}, volume = {113}, year = {2016}, month = {2016 Mar 22}, pages = {E1645-54}, abstract = {

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.

}, issn = {1091-6490}, doi = {10.1073/pnas.1514030113}, author = {K{\"o}ster, Darius Vasco and Husain, Kabir and Iljazi, Elda and Bhat, Abrar and Bieling, Peter and Mullins, R Dyche and Rao, Madan and Mayor, Satyajit} }