The roles of JSAP1 and JIP1 in cell adhesion and spreading were examined using mouse embryonic fibroblasts (MEFs) deficient in JIP1 (JIP1-KO). JSAP1 (JSAP1-KO), and in both JIP1 and JSAP1 (double-KO), and by using their wild type. After being plated oil fibronectin-coated culture plates, wild type MEFs rapidly adhered and differentiated to typical longitudinal fibroblasts in 4h. JSAP1-KO MEFs showed a similar Sequence of adhesion and cell spreading, but their adhesion was weak, and cell spreading sequence proceeded in a delayed manner compared with the wild type. In spreading JSAP1-KO MEFs, adhesion-triggered actin cytoskeleton reorganization and FAK activation proceeded at a slower pace than in wild type MEFs. The cellular properties of double-KO MEFs and JIP1-KO MEFs were similar to those of JSAP1-KO MEFs and wild type MEFs, respectively. These results suggest that JSAP1 plays a role in adhesion and cell spreading by regulating the rapid reorganization of the actin cytoskeleton. (c) 2006 Elsevier Inc, All rights reserved.