Fig. 5. Protein recruitment to C-Nap1 patches. (A) U2OS cells were transfected with either myc-C-Nap1 or C-Nap1 and fixed with methanol 24 hours later. In the former case, they were stained with a monoclonal anti-myc antibody (9E10) for C-Nap1 (a) and counterstained with a rabbit anti-Nek2 antibody (b). In the latter case, C-Nap1 was stained with C-Ab (c), and -tubulin was visualized with a monoclonal antibody (d). No signal was observed in cells stained with either anti-rabbit or anti-mouse secondary antibodies alone (data not shown). Bar, 10 µm (d). (B) MT re-growth assay. Following overexpression of C-Nap1, MTs were depolymerized by cold treatment, and MT re-polymerization was induced for 60 seconds by placing cells into pre-warmed medium (37°C). Cells were then fixed with methanol and stained with C-Ab (a) and a mouse anti--tubulin antibody (b). The arrowhead points to a transfected cell harboring a C-Nap1 patch; note that MT nucleation is severely suppressed in this cell. Bar, 10 µm (b). (C) Mutational domain analysis of C-Nap1. On the right, the C-Nap1 domain structure and the ability of different C-Nap1 deletion mutants to form patches is summarized schematically. The dark boxes designate the predicted coiled-coil domains in C-Nap1; wt (wild-type) denotes the full-length C-Nap1 protein. On the left, the phenotype observed after overexpression of the T4 C-Nap1 deletion mutant is illustrated. U2OS cells were analyzed by immunofluorescence microscopy, using anti-myc (9E10) antibodies for staining of T4. Bar, 10 µm.