LIMD1 phase separation contributes to cellular mechanics and durotaxis by regulating focal adhesion dynamics in response to force

Abstract

Abstract：The mechanical environement affects cell morphology, differentiation and motility. The ability of cells to follow gradients of extracellular matrix stiffness-durotaxis has been implicated in development, fibrosis, and cancer. Cells sense and respond to extra-cellular mechanical cues through cell-matrix adhesions. Interestingly, the maturation of focal adhesions (FAs) is reciprocally force-dependent. How biomechanical cues dictate the status of cell motility and how FAs coordinate force sensing and self-organization remain enigmatic. LIMD1, a member of the LIM domain proteins, localizes to the FAs and has been reported to negatively regulate the Hippo-YAP pathway in response to tension. Here we identify the force sensitive recruitment of LIMD1 to the FAs. We discover that LIMD1 regulates cell spreading, maintains FA dynamics and cellular force, and is critical for durotaxis. Intriguingly, LIMD1 selectively recruits late but not early FA proteins through phase separation at the FAs under force. We suggest a model in which localization of LIMD1 to the FAs, triggered by mechanical force, serves as a phase separation hub for assembling and organizing late FA proteins, allowing for effective FA maturation and efficient cellular mechano-transduction.Force transmission from the extracellular environment to the interior of a cell has been a fascinating subject for decades. Integrin mediated adhesion sites bind to extracellular molecules and initiate a cascade of molecular assembly underneath the plasma membrane, constructing a highly organized structure that is capable of transducing mechanical force 1 . These adhesion sites have been extensively investigated for their crucial roles in mediating both inside-out and outside-in signaling which are essential for cell survival and motility 1-5 . Focal adhesions (FAs) are cell-matrix contacts that mature from nascent focal complexes containing integrins and a few other molecules such as kindlin, talin, FAK and paxillin 6-8 . Only a portion of the nascent focal complexes survives the maturation process and during that they grow by rapidly recruiting late FA proteins to assemble plaque like structures and clutching to the actin stress fibers. Matured FAs exhibited ordered ultrastructure with the force transmission layer sandwiched between the integrin layer and the actin association layer 9 . Specific protein-protein interactions have been identified to participate in FA maturation 10-12 . However, how the army of FA proteins achieves spatial-temporally controlled recruitment and assembly remains unveiled.Efficient force transmission through the FAs is essential for numerous cellular processes including cell migration. Durotaxis is a type of directional cell migration in which cells respond to a gradient of extracellular stiffness 13,14 . Mechanisms underlying durotaxis of mesenchymal cells are proposed to include contractile mechanosensation, probing of the local substrate by actin-based protrusions, and focal adhesion signaling [15][16][17][18][19][20][21] . Force flu...

Force redistribution in clathrin-mediated endocytosis revealed by coiled-coil force sensors

“…Force produced by the cytoskeleton or motor proteins may change the folding of protein domains to promote or inhibit protein phase separation. The phase separation of LIMD1 recruits focal adhesion proteins in a force dependent manner, and CPEB is predicted to be a mechanical prion to facilitate long term memory in synapses 46,47 . The prevalence of low-affinity interactions in the endocytic coat suggests the potential involvement of protein phase separation in normal CME 48,49 , which might be more transient in the natural state but seems to be enhanced by the insertion of the coiled-coil force sensors within End4p.…”