However, a mutant form of PACSIN2 lacking one of the Coiled-coil domains (residues 185C218; Fig

However, a mutant form of PACSIN2 lacking one of the Coiled-coil domains (residues 185C218; Fig. (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, AM 2201 and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin 1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated 51, integrin and cytoskeleton strength during cell movement. CDC15 Homology) AM 2201 family of proteins that play a role in remodeling the actin cytoskeleton (Chitu and Stanley, 2007). These adaptor proteins of 50 to 70 kDa contain (1) a Fer/CIP Homology (FCH) domain name, (2) a CDC15 N-terminal homology (CDC15-NT) domain name made up of a Coiled coil (CC) domain name involved in the binding of Filamin AM 2201 (Nikki et al., 2002a), (3) a linker domain name made up of NPF motifs or a Proline rich motif (PACSIN3), and (4) an SH3 domain name. Many proteins are known to bind these different domains (Chitu and Stanley, 2007; Kessels and Qualmann, 2004). The PACSIN proteins homo and hetero-oligomerize through their FCH domain name and possibly CDC15 NT domains (Halbach et al., 2007; Kessels and Qualmann, 2006; Nikki et al., 2002b) (Cousin and Alfandari, unpublished results). This oligomerization is usually thought to be important for the capacity of PACSINs to act as adaptor proteins. The over-expression of either the full length PACSINs or just the SH3 domains has been shown to inhibit endocytosis of the transferin receptor, suggesting a role for PACSINs in endocytosis (Modregger et al., 2000; Qualmann and Kelly, 2000; Simpson et al., 1999). Over-expression of PACSIN was also shown to induce actin-rich filopodia (Nikki et al., 2002a; Qualmann and Kelly, 2000; Sumoy et al., 2001), that could be rescued by co expressing the C-terminal region of N-WASP, an activator of the Arp2/3 complex (Qualmann and Kelly, 2000). Based on these results, it has been proposed that PACSINs are directly involved in endocytosis by linking the actin cytoskeleton to the endocytic machinery (Kessels and Qualmann, 2004; Qualmann and Kelly, 2000). Other groups have shown that PACSINs can also interact with proteins involved in signaling such as the death receptor ligand CD95L (Ghadimi et al., 2002) and AM 2201 the metalloprotease ADAM12 (Mori et al., 2003). In the case of ADAM12, PACSIN3 was shown to bind and activate the ADAM12-mediated shedding of HB-EGF in response to phorbol ester or angiotensin II treatment. We have reported that PACSIN2 is able to bind and inhibit the metalloprotease ADAM13, which is usually involved in the migration of cranial neural crest cell migration in Xenopus (Alfandari et al., 2001; Cousin et al., 2000). This inhibition is not linked to a decrease in cell surface expression of ADAM13 (Cousin et al., 2000). While the regulation of cell migration by a PACSIN family members is a new concept, the PCH family member CD2BP1/PST PIP1 have been implicated in the control of cell-cell adhesion (Badour et al., 2003; Li et al., 1998). Considering the ubiquitous expression of PACSIN2 in Xenopus embryos, we have used gastrulation, a stage where integrin function and regulation has been extensively studied, to further investigate the function of PACSIN2 in modulating cell migration. In this report, HYAL1 we show that PACSIN2 over-expression inhibits gastrulation by down regulating the 51 integrin activity but not AM 2201 cell surface expression. We further show that PACSIN2 over-expression prevents the recruitment of integrin 51 to focal adhesions (FA) but not the recruitment of v integrin. We also show that this membrane localization of PACSIN2, driven by a region near the Coiled-coil domain name, is necessary for this function. Based on that observation and the fact that PACSIN2 forms multimers, we have used a mitochondrial anchored PACSIN2 (PACSIN2-MA) to remove endogenous PACSIN2 from the membrane. PACSIN2-MA blocks gastrulation but does not prevent 51 integrin activation or localization to FA. The over-expression of PACSIN2 and PACSIN2-MA has opposite effects around the cellular localization of filamin. Materials and Methods Eggs and embryos eggs were obtained fertilized, cultured and staged as described previously (Cousin et al., 2000). Constructs The wild type PACSIN2 (wt) and ?CC mutant were cloned into pCS2+ and pCS2-MT (Turner and Weintraub, 1994).