KD of integrin 1 attenuated the localization of both Abi1 and Pfn-1 at the leading edge (Fig
KD of integrin 1 attenuated the localization of both Abi1 and Pfn-1 at the leading edge (Fig.?6C,D). Open in a separate window Figure 6 Integrin 1 controls the positioning of Abi1 and Pfn-1 at tip of protrusion. lamellipodia and its protrusion coordinated with F-actin at the leading cell edge of live cells. In addition, Rabbit Polyclonal to C1QB we identified profilin-1 (Pfn-1), a G-actin transporter, as a new partner for Abi1. Abi1 knockdown reduced the recruitment of Pfn-1 to the leading cell edge. Moreover, Abi1 knockdown reduced the localization of the actin-regulatory proteins c-Abl (Abelson tyrosine kinase) and N-WASP (neuronal WiskottCAldrich Syndrome Protein) at the cell edge without affecting other migration-related proteins including pVASP (phosphorylated vasodilator stimulated phosphoprotein), cortactin and vinculin. Furthermore, we found that c-Abl and integrin 1 regulated the positioning of Abi1 at the leading edge. Taken together, the results suggest that Abi1 regulates cell migration by affecting Pfn-1 and N-WASP, but not pVASP, cortactin and focal adhesions. Integrin 1 and c-Abl are important for the recruitment of Abi1 to the leading edge. test was used for statistical analysis. Proline-rich domain of Abi1 is important for cell migration Because Abi1?PP mutant did not bind to Pfn-1 (Fig.?2B), we evaluated whether Abi1?PP mutant affects the distribution of Pfn-1 in cells. We found that wild type (WT) Abi1, but not Abi1?PP mutant, localized at the tip of lamellipodia (Fig.?3C). Furthermore, the expression of Abi1?PP mutant attenuated the distribution of Pfn-1 at the cell edge (Fig.?3C,D). Next, we determined the effects of Abi1?PP mutant on cell migration by using time-lapse microscopy. Abi1?PP mutant inhibited accumulated distance, Euclidean distance GSK2795039 and speed of cell migration (Fig.?3ECG). Abi1 differentially affects localization of c-Abl, N-WASP, cortactin and vinculin in cells Because c-Abl, N-WASP, cortactin and vinculin are important for the regulation of cell migration, we used immunofluorescent microscopy to determine whether Abi1 regulates distribution of these proteins. Abi1 KD reduced the localization of c-Abl and pN-WASP (Y256) (indication of N-WASP activation)16 at GSK2795039 the leading cell edge (Fig.?4A,B). Furthermore, Abi1 KD diminished F-actin distribution at the tip of protrusion (Fig.?4A,B). However, cortactin localization at the leading edge was not affected by Abi1 KD (Fig.?4A,B). Moreover, Abi1 KD did not impact distribution of vinculin, a focal adhesion marker (Fig.?4A,B). Open in a separate window Number 4 Differential part of Abi1 in spatial localization of migration-associated proteins. (A) Abi1 KD attenuated localization of c-Abl, pN-WASP and F-actin at the leading edge without influencing cortactin positioning. In addition, Abi1 KD did not impact vinculin relative intensity and area. Scale pub, 20?m. White colored arrows point to the leading edges. Red arrows point to focal adhesions. (B) Data are mean ideals of experiments from at least 32 cells for each group. Error bars show SD. **test was used for statistical analysis. c-Abl tyrosine kinase modulates localization of Abi1 and Pfn-1 at the tip of protrusion c-Abl tyrosine kinase has a part in controlling cell migration23,27. We found that c-Abl was concentrated at the leading cell border of motile cells (Fig.?5A), which is supported by earlier studies23. Thus, we evaluated whether c-Abl regulates the recruitment of Abi1 and Pfn-1. KD of c-Abl reduced the recruitment of Abi1 and Pfn-1 to the leading edge of motile cells (Fig.?5B,C). Open in a separate windowpane Number 5 c-Abl regulates the recruitment of Abi1 and Pfn-1 to the leading edge. (A) c-Abl is definitely localized at the tip of lamellipodia. Level pub, 10?m. (B) Immunoblot analysis of stable c-Abl knockdown cells and control cells. Data are mean ideals of experiments from five batches of cell tradition. Error bars show SD. (C) KD of c-Abl reduced the localization of Abi1 and Pfn-1 at the leading edge. Scale pub, 10?m. Data are mean ideals of experiments from at least 30 cells for each group. Error bars show SD. **test was used for statistical analysis. Integrin 1 GSK2795039 regulates localization of Abi1 and Pfn-1 at the leading edge Integrin 1 is definitely.