Studies On Mechanisms Of F-actin Regulating The Formation Of Endocytic Vesicles And SNX-3 Mediating Endosomal Sorting

Endocytosis is required for general cellular functions,including uptake of membrane receptors,signals and nutrients.There are several endocytic pathways that utilize different mechanisms to internalize cargoes.The best studied endocytosis pathway is the clathrin-dependent endocytosis(CDE).However,studies on clathrin-independent endocytosis(CIE)are relatively slow and inconsistent due to the mechanisms and cargo destinations vary in different cell types.After endocytosis,the internalized cargo proteins are transported to early endosomes(EEs)from which they can be recycled back to the plasma membrane,transported to the trans Golgi network via retrograde recycling,or sorted to lysosomes for degradation.Researchers found that CDE cargo proteins are usually transported through vesicles in cells,while several CIE cargoes transport via tubular carriers.Endocytosis and membrane transport defects can lead to major diseases such as nerves and immune system disorders.Therefore,it is helpful for us to understand cell biology and the pathogenesis of related diseases by exploring the mechanisms of various endocytosis pathways and membrane transport.Here,by using in vivo time-lapse microscopy and taking advantage of the powerful genetic tools available for Caenorhabditis elegans(C.elegans),we conducted a small-scale screen for potential regulators,and identified the interaction interfaces underlying endocytosis and endosomal sorting.The main contents include:1.Studies on mechanisms of cytoskeleton regulating endocytic vesicle formation in CDE pathway.In yeast,endocytosis involving clathrin is critically dependent on actin assembly,which facilitates membrane invagination and vesicle scission.In mammals,Dynamin family are the undisputed critical component for the scission of clathrin-coated pits(CCPs)from the plasma membrane.However,the extent to which F-actin participates in CCPs scission,and the recruitment,regulation and functional interdependence of F-actin and Dynamin during this process remain only partly understood.Here,we found that loss of Wiskott-Aldrich syndrome protein(WASP)-interacting protein(WIP-1)impaired CDE in a manner that is independent of the C.elegans homolog of WASP/N-WASP(WSP-1)and is mediated by direct binding to G-actin.Moreover,the cortactin-binding domain of WIP-1 serves as the binding interface for DBN-1(also known in other organisms as Abp1),another actin-binding protein.We further identified a novel role for WIP-1 and DBN-1 in regulating F-actin assembly at the endocytic sites and cooperating with DYN-1 by identifying their mutual interaction interfaces.Firstly,we found loss of either WIP-1 or DYN-1 resulted in defective scission of CCPs on the basolateral plasma membrane of intestines.WIP-1 and DBN-1 are involved in stabilizing F-actin polymerization at the CCP sites,which contributes to the recruitment of DYN-1.Additionally,DBN-1 binds to the assembled actin through its N-terminal ADF-H and 3CC domains,which is a prerequisite for DYN-1 recruitment to CCPs.2.Explore the regulatory role of Sorting nexin on the endosomal sorting of CIE cargo proteins.Among several CIE pathways,research on the Arf6-associated CIE is more extensive.However,the regulatory mechanisms of sorting of Arf6-associated CIE cargoes on EEs remain unclear.Based on the presence of a phox-homology(PX)domain,Sorting nexins(SNXs)loaclize on EEs and function in endosomal sorting of various CDE cargo proteins.Here,we screened the SNXs expressed in C.elegans,and for the first time revealed that loss of SNX-3 which only contains a PX domain results in missorting of CIE cargoes to lysosome for degradation.Accumulating evidence has indicated that retrograde cargo proteins are missorted and degraded in lysosomes in response to loss of SNX3.While,how snx-3 affects endosomal sorting still only partly understood.By examining the intracellular distribution of regulators responsible for degradation pathways on EEs,we found the distribution and patterns of ESCRT component HGRS-1(Hrs homologue)were not affected by snx-3 mutation.However,lack of SNX-3 function resulted in EEA-1(EEA1 homologue)being more localized on EEs and colocalized well with CIE cargo proteins.EEA-1 and SNX-3 have the possibility of competitive binding to EEs.In addition,more EEA-1 is recruited onto EEs in snx-3 mutant intestines may disturb the sorting mechanisms.VPS complex is not involved in SNX-3 recognition of Arf6-associated CIE cargo proteins.Loss of function of VPS complex resulted in missorting of CDE cargoes.By contrast,the distribution of CIE cargoes such as hTAC and DAF-4 were not significantly affected by snx-3 mutation.VPS-35 was less colocalized with hTAC,while SNX-3 colocalized with hTAC in wild-type as well as in vps-35 mutant intestines.SNX-3 may recognize CIE cargo proteins either directly or by interacting with other regulator proteins.