21 Thus, Cas has a modular structure, and the individual module transmits signals by interacting with selected intracellular molecules. We previously reported that Cas-deficient (Cas−/−) mice died in utero at 12.5 days post coitum (dpc) and showed retarded cardiac development AZD6244 in vivo with disorganized myofibrils and disrupted Z-disks.22 We also observed that Cas−/− fibroblasts had impaired actin stress fiber formation and profound defects in cell motility, migration, and spreading.22, 23 These results demonstrated that Cas functions as an actin-assembling molecule and plays vital roles in cell dynamics and organ development.

To further understand the roles of Cas in organogenesis, we generated mice with a hypomorphic Cas allele devoid of the exon 2–derived region (CasΔex2/Δex2) encoding the entire SH3 domain. Interestingly, although CasΔex2/Δex2 mice also died as embryos, they had no cardiovascular system defects but instead showed progressive liver degeneration with hepatocyte apoptosis. Because Cas expression in the liver was not found in hepatocytes but was detected in SECs, it is likely that exon 2–deleted Cas (Cas Δex2) indirectly affects hepatocyte survival by altering SEC function. By employing an SEC line as an in vitro model system, we demonstrated that MG-132 manufacturer Cas lacking SH3, which possesses biochemical properties similar to those of Cas Δex2, resulted in impaired actin

stress fiber formation and loss of fenestrae in SECs. These results indicated that Cas plays pivotal roles in liver development through the regulation of SEC fenestration. Cas, p130 Crk-associated learn more substrate; Cas Δex2, exon 2–deleted p130 Crk-associated substrate; Cas ΔSH3, p130 Crk-associated substrate mutant lacking

Src homology domain 3; Cas FL, full-length p130 Crk-associated substrate; cDNA, complementary DNA; Cre, cyclization recombination; dpc, days post coitum; FN, fibronectin; HA, hemagglutinin; HE, hematoxylin and eosin; loxP, locus of X-over P1; MEF, mouse embryonic fibroblast; Neo, neomycin resistance; NS, not significant; PCR, polymerase chain reaction; SBD, Src-binding domain; SD, substrate domain; SEC, sinusoidal endothelial cell; SH2, Src homology domain 2; SH3, Src homology domain 3; Stab2, stabilin 2; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; WT, wild-type; YxxP, Tyr-x-x-Pro. The construction of the targeting vector and the generation of CasΔex2/Δex2 mice are described in detail in the supporting information. Western blotting and immunoprecipitation were performed as described.22 The procedure and antibodies used for the experiments are described in detail in the supporting information. Histological, immunohistochemical, immunocytochemical, and immunofluorescent analyses were performed as described.22 The procedure and antibodies used for the experiments are described in detail in the supporting information.