We first developed a poly clonal antibody against SNX16 and this antibody suc cessfully detects the ectopically expressed SNX16 GFP in MCF 7 cells. SNX16 is enriched in brain and muscles in mouse, so we tested whether SNX16 is dis tributed novel to the cell cortex in these tissues. We performed immunofluorensence staining on mouse heart frozen sec tions using our home made antibody. Cell cortex staining Inhibitors,Modulators,Libraries of SNX16 is detected at mouse heart sections but not the same sample pre blocked with the purified SNX16 soluble protein. This result suggests that the staining is specific and we conclude that a fraction of SNX16 is present at cell cortex both in vitro and in vivo. Signals required for the cell cortex distribution of SNX16 SNX23KIF16B is a kinesin family protein that can regu late the microtubule based peripheral transport of early endosomes.

It is reported to co localize with early endo some marker EEA1 at the cell cortex in Hela cells. This distribution pattern of SNX23 is similar to what we observed for SNX16 here, so we compared the subcel lular distribution Inhibitors,Modulators,Libraries patterns of SNX16 and SNX23. We co transfected SNX16 and 23 into the MCF 7 cells and found that they co localize with each other at cell cortex. Since SNX23 Inhibitors,Modulators,Libraries is a motor protein that can regulate the cell peripheral transport of early endosomes, we determined whether the SNX23 transport pathway is required for the cell cortex distribution of SNX16. We knocked down SNX23 by siRNAs then determined the subcellular distribution pattern of SNX16.

Our siRNAs effectively down regulate the mRNA Inhibitors,Modulators,Libraries level of Inhibitors,Modulators,Libraries SNX23 and we found that down regulation of SNX23 abolishes the peripheral distribution of SNX16. In fact, the majority of SNX16 vesicles are now detected at the perinuclear regions. The microtubule filaments are required for the SNX23 mediated cargo transport, so we investigated whether the microtubules are involved in the trafficking of SNX16 vesicles. Pretreatment of MCF 7 cells with colchicine, an inhibitor of microtubule polymerization, disrupts the cortex localization of SNX16 vesicles. On the other hand, inhibition of the actin fila ments by cytochalasin B does not affect the cell cortex distribution of SNX16. So, the SNX23 and microtubule dependent transport route is required for the cell cortex distribution of SNX16 vesicles. The PX domain of SNX16 can bind to PI3P thus the PI3 kinase pathway is able to regulate the early endosome localization of SNX16.

We analyzed whether the PI3 kinase pathway is involved in the cell cortex distribu tion of SNX16 as well. We found that the inhibition of PI3 kinase by small chemical wortmannin PF01367338 abolishes the cell cortex localization of SNX16 vesicles. On the other hand, inhibition of mTOR which is a PI3K related kinase by rapamycin does not induce similar ef fect.