Key Points Cyfip1 plays a crucial role for branching of actin filaments and for lamellipodium formation. Lamellipodium formation is not required for the formation of a hemostatic plug or thrombus.

Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrinmediated adhesion Highlights d Loss of WRC in macrophages impairs phagocytosis, spreading, and (de)adhesion d WRC loss of function perturbs integrin activation and FAK/ paxillin phosphorylation d Arp2/3 complex and WRC functions on integrin adhesion are at least partly separable

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# equal contribution One sentence summaryInterference of Hem1 function in mice and cells uncovers a hitherto unrecognized role in integrinmediated cell adhesion that is crucial for macrophage function and connects to recently discovered immunodeficiencies in patients carrying Hem1 mutations. ABSTRACTThe hematopoietic-specific protein 1 (Hem1) comprises an essential subunit of the WAVE Regulatory Complex (WRC) in immune cells. WRC has a fundamental role in Arp2/3 complex activation and the protrusion of branched actin networks in motile cells.Hem1 deficiency leads to suppression of the entire WRC in immune cells. Defective WRC function in macrophages results in loss of lamellipodia and migration defects. Moreover, phagocytosis, commonly accompanied by lamellipodium protrusion during cup formation, is altered in Hem1 null cells concerning frequency and efficacy. When analyzing cell spreading, adhesion and podosome formation, we found that Hem1 null cells are capable, in principle, of podosome formation and consequently, do not show any quantitative differences in extracellular matrix degradation. Their adhesive behavior, however, was significantly altered. Specifically, adhesion as well as de-adhesion of Hem1 null cells was strongly compromised, likely contributing to the observed reduced efficiency of phagocytosis. In line with this, phosphorylation of the prominent adhesion component paxillin was diminished. Nonhematopoietic somatic cells disrupted in expression for both Hem1 and its ubiquitous orthologue Nckassociated protein 1 (Nap1) or the essential WRC components Sra-1/PIR121 did not only confirm defective paxillin phosphorylation, but also revealed that paxillin turnover in focal adhesions is accelerated in the absence of WRC. Finally, adhesion assays using platelets lacking functional WRC as model system unmasked radically decreased IIb 3 integrin activation.Our results thus demonstrate that WRC-driven actin networks impact on integrin-dependent processes controlling formation and dismantling of different types of cell-substratum adhesion.Cell migration follows an orchestrated sequence of events, a cycle of cell protrusion, adhesion to newly occupied space and contraction to drag the cell body forward. Polymerization of actin filaments (Factin) at the cell front pushes the plasma membrane into the direction of migration, which then attaches to the extracellular substratum via members of the integrin transmembrane receptors family.Cells in metazoan organisms are capable of migrating through complex, 3-dimensional environments and their license keys to different organismal compartments constitute combinations of integrins they carry on their surface, allowing them to specifically adhere to given substrata (1, 2). Once engaged, the integrins dynamically couple to the actin cytoskeleton, which in turn can exert pulling forces through myosin II activation and contraction, culminating in locomotion of the cell body (3-5)Integrins are heterodimeric cell surface receptors that are responsible for cell adhesion duri...

Highlights d RhoA/Cdc42 DKO mice display severe macrothrombocytopenia and defective hemostasis d DKO megakaryocytes show defective cytoplasmic maturation but normal endomitosis d Loss of RhoA/Cdc42 signaling abolishes proplatelet formation d RhoA/Cdc42 signaling regulates gene expression during megakaryocyte maturation