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Mechanosensing by the actin cytoskeleton Living cells need to respond to mechanical forces for many essential biological functions. This mechanosensing activity is thought to be a property of the actin cytoskeleton, but no specific mechanisms have yet been identified. In this study, Ehrlicher et al . identify the actin-binding protein filamin A (FLNa) as a central mechanotransduction element. In a minimal reconstituted system, ligand binding to filamin is affected by mechanical forces, causing certain binding partners to dissociate and others to adhere more strongly. This selectivity may provide a direct molecular link between physical forces and biological activity. Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders 1 , 2 , 3 , 4 such as cardiac failure 5 and pulmonary injury 6 . The actin cytoskeleton’s connectivity enables it to transmit forces rapidly over large distances 7 , implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA) 8 , 9 as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of β-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with β-integrin tails connecting to the actin cytoskeleton by binding directly to filamin 4 . FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation 10 . Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching 11 , we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases β-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.

We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditions is a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces.

AGONISTS that stimulate protein kinase C (PKC) induce profound changes in cell morphology correlating with the reorganization of submembranous actin 1,2 , but no direct connection between PKC and actin assembly has been identified 3 . The myristoylated, alanine-rich C kinase substrate (MARCKS) binds calmodulin 4,5 and is a predominant, specific substrate of PKC which is phosphorylated during macrophage and neutrophil activation 6–8 , growth factor-dependent mitogenesis 9,10 and neurosecretion 11,12 ; it is redistributed from plasma membrane to cytoplasm when phosphorylated 13–15 and is involved in leukocyte motility 14,15 . Here we report that MARCKS is a filamentous (F) actin crosslinking protein, with activity that is inhibited by PKC-mediated phosphorylation and by binding to calcium–calmodulin. MARCKS may be a regulated crossbridge between actin and the plasma membrane, and modulation of the actin crosslinking activity of the MARCKS protein by calmodulin and phosphorylation represents a potential convergence of the calcium–calmodulin and PKC signal transduction pathways in the regulation of the actin cytoskeleton.

Key Points Molecules that crosslink actin filaments into particular architectures are important components of cell structure and movement. Filamins are one of the first of such components recognized and are among the most important. Filamins are extended dimers composed of subunits that contain characteristic β-pleated sheet repeats. Vertebrate filamins have amino-terminal actin-binding domains and self-associate at the carboxyl termini of their subunits. The main human filamin (filamin A) is encoded on the X chromosome. A second filamin gene (filamin B) is encoded on chromosome 3 and a muscle-specific filamin gene (filamin C) is encoded on chromosome 7. So far two filamin genes have been recognized in Drosophila . Dictyostelium amoebae have only one filamin species which is truncated compared with vertebrate and Drosophila filamins. Filamins cause actin filaments to branch with high angles leading efficiently to the formation of actin gels in vitro . The filamins reside at branches between orthogonally intersecting filaments in the peripheral cytoplasm of cells. Filamins also bind over 20 diverse cellular proteins, including membrane receptors and intracellular signalling macromolecules. Cells missing the main filamins have defects in surface stability and locomotion and in some of the functions ascribed to the filamin binding partners. A mutation in the filamin A gene is lethal for males and the cause of periventricular heterotopia in females chimeric for the mutation. Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.

Cooling of blood platelets clusters the von Willebrand factor receptor complex. Macrophage$\\alpha_M\\beta_2$integrins bind to the$GPIb\\alpha$subunit of the clustered complex, resulting in rapid clearance of transfused, cooled platelets. This precludes refrigeration of platelets for transfusion, but the current practice of room temperature storage has major drawbacks. We document that$\\alpha_M\\beta_2$is a lectin that recognizes exposed$\\beta-N-acetylglucosamine$residues of N-linked glycans on$GPIb\\alpha$. Enzymatic galactosylation of chilled platelets blocks$\\alpha_M\\beta_2$recognition, prolonging the circulation of functional cooled platelets. Platelet-associated galactosyltransferase produces efficient galactosylation when uridine phosphate-galactose is added, affording a potentially simple method for storing platelets in the cold.

Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott–Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella .

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA's effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.

The Wiskott–Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.