ZF21 PROTEIN REGULATES CELL ADHESION AND MOTILITY

Immunoprecipitation assay for endogenous ZF21 in MDA-MB231 cells. The lysates of MDA-MB231 cells were incubated with chicken anti-ZF21 IgY. The chicken anti-ZF21 IgY was precipited with chicken IgY precipitating agarose beads (Millipore). Proteins bound to ZF21 were analyzed by Western blotting using each antibody.

. The formation of adhesion structures during the spreading and migration of cultured cells on ECM layers has been studied extensively (8,9). During cell migration, 1 primitive adhesion structures can be observed at the migration front as relatively small focal complexes along the periphery of ruffling membranes (10). Most of these structures are short lived, but some grow to become more stable adhesion links to the actin cytoskeleton so as to permit the generation of force for cell movement via actin contraction (10,11).  Immunofluorescence microscopy-Cells were fixed in -20°C methanol for 10 min and permeabilized using 0.1% Triton-X100 in PBS for 10 min. After the samples were blocked in PBS containing 5% goat serum and 3% BSA, they were incubated with primary antibodies.
All primary antibodies were visualized with either an Alexa 488-or 568-conjugated goat anti-mouse or anti-rabbit antibody (Invitrogen).

Images of cells were captured with Leica
ASMDW with CCD camera (Leica).

RESULTS
The  In parallel with the decrease in the number of FAs, the area covered by the FAs also decreased (Fig. 5N, shLacZ). In contrast, the decrease in the number of FAs (Fig. 5, G and M) and in the area covered by the FAs (Fig.   5N) was significantly delayed in cells in which expression of ZF21 was knocked down. Thus, ZF21 appears to play a role during microtubule-induced disassembly of FAs.

ZF21 is a component of focal adhesions-
Since ZF21 regulates turnover of FAs, we localized activation with a laser beam. We made use of mCherry-tagged Zyxin as a marker for FAs (Fig. 6B, red), while ZF21 was monitored by the fluorescence of the m1Venus tag (Fig. 6B, green) GST-ZF21 as bait (Fig. 6D). An N-terminal fragment that lacks the kinase domain (KD) and FAT domain failed to bind ZF21 (Fig. 6D, FERM). In contrast, the KD of FAK alone was sufficient to bind ZF21 (Fig. 6D, KD).
EEA1, an endosomal protein, was largely absent from the residual membrane fragments after hypo-osmotic treatment of the cells compared to the untreated cells (Fig. 6F, lanes   4). In contrast, FAK and integrin-1 were detected in the residual membrane fragments even after hypo-osmotic treatment of the cells (Fig. 6F, lane4). Similarly to FAK and integrin-1, endogenous ZF21 protein was detected in the residual fraction adhering to the dishes (Fig. 6F, lane 4). vesicles, ZF21 protein also localizes to FAs, as we observed using TIRF microscopy to visualize proteins in close proximity to cell attachment sites on the slide glasses (Fig. 6B).
We used a pull-down assay to show that ZF21 can bind FAK, but not paxillin or zyxin (Fig.   6C). The FYVE domain of ZF21 is important not only for the localization to endosomes but also for the binding to FAK (Fig. 6E).
Enrichment of ZF21 at sites of cell attachment was further confirmed by analyzing membrane fragments attached to the culture dish after removal of most cellular material following 12 exposure of the cells to hypo-osmotic conditions (Fig. 6F). Enrichment of ZF21 to these sites of attachment was dependent on the presence of FAK.
Extension of microtubules to FAs induces disassembly of their structure (23,24).
Presumably, microtubules convey critical protein components that trigger disassembly of