IFT88 Associated With Secretory Pathway And Mediated The Genesis And Maintenance Of Primary Cilia

Primary cilia are conserved organelles that project from cell surface and exist ubiquitously in vertebrate cells.A growing number of studies revealed that this structure is a hub of numerous important signaling pathways,such as hedgehog signaling pathway.As an independent cellular compartment,cilium possesses special protein and membrane composition.Since lack of protein synthesis mechanism,proteins for maintaining the genesis and function of cilia are synthesized in cell body and then transported to cilia.There are hypotheses suggest that the travels of both membrane and soluble proteins are mediated by the intracellular vesicle transport:not only those membrane proteins which are trafficking along with vesicles during their synthesis and sorting,but also the soluble proteins which are synthesized on free polysomes are peripherally-associated with vesicles targeting to cilia.The intraflagellar transport(IFT)machinery mediates the transport along ciliary axoneme,however,recent studies have found that except for localizing at cilia and cilia base to mediate intraflagellar transport,some IFT proteins also associated with secretory pathway and mediate the transport of those intracellular vesicles targeting to cilia.IFT88 is a core member of IFT-B complex,whose knockout leads to cilia deficiency and embryonic lethal.In addition to mediating intraflagellar process,IFT88 also has been reported to form a complex at ER with Cdh23,Harmonin and Myo7aa to mediate the trafficking of USH proteins from ER to ERGIC.Although there are clues suggesting that IFT88 might be involved in intracellular vesicle transport system,no clear statement has been proposed to illustrate this hypothesis.In our study,we demonstrated in NIH3T3 cells that IFT88 could localize on ER,associate with ER-derived COP?-coated vesicles,and interact with coat protein SEC 13.An IFT88 binding partner DGK? colocalized with IFT88 on ER,especially on ER export site(ERES).The absence of DGK? blocked the release of IFT88-containing COP?-coated vesicles,suggesting that DGK? might facilitate the formation of these vesicles.The decrease of IFT88-containing COP?-coated vesicles formation lead to reduction of both IFT88 accumulation at primary and cilia length,and blocked the transport of a membrane protein Periphrin/Prd to primary cilia shipped from ER as well.These results suggested that IFT88 involved in targeting ER-derived secretory vesicles to primary cilia in a golgi bypass way.As a particular region on plasma membrane,ciliary membrane possesses special lipid composition:the major phosphoinositide of the primary cilia membrane is phosphatidylinositol 4-phosphate(PI4P),while phosphatidylinositol 4,5 bis-phosphate(PI(4,5)P2)enriched at ciliary base.As a member of diacylglycerol kinases family,the function of DGK? is phosphorylating DAG to produce PA.This reaction is the initial step of phosphatidylinositol cycle and starts the resynthesis of PI(4,5)P2.And we found that IFT88-associated vesicles also enriched with PI(4,5)P2,suggesting that this might be the source of the enrichment of PI(4,5)P2 at ciliary base.Knockdown of DGK? could inhibit Hedgehog signaling activity and GCP proliferation.Meanwhile,DGK inhibitors could reduce Smo accumulation at cilia and inhibit Hedgehog signaling activity,suggesting that DGK?could involve in hedgehog signaling transduction through mediating vesicle targeting to primary cilia.Based on our results,we considered that IFT88 could associate with COP?coated vesicle which released from ER and carried specific proteins and lipids,and mediate its targeting to primary cilia.IFT88 binding protein DGK? could trigger IFT8 8-containing vesicles release from ER and modify the lipid composition of these vesicles,and then facilitate the genesis and function of primary and the transduction of hedgehog signaling.