Studies On Mechanisms Of SNX-3-Mediated Tubular Endosomal Recycling And ATP8A1-Regulated Post-endocytic Degradation

Endosomal sorting and trafficking are important for the maintenance of cellular homeostasis,disregulation of which always causes cancer and Alzheimer’s disease.The investigation of the regulatory mechanisms of endosomal sorting and trafficking is helpful to elucidate the pathogenesis of related diseases and provide valuable therapeutic approach.There are two mechanistically and morphologically distinct endocytic pathways,the clathrin-dependent(CDE)and clathrin-independent endocytosis(CIE).The transmembrane proteins(collectively referred to as cargoes)are internalised from the plasma membrane(PM)into early endosomes(EEs).Here,cargo proteins can either be sorted for degradation in lysosomes or be recycled to the PM,or retrieved to the trans-Golgi network(TGN)for reuse.EEs are the sorting hubs for the cargo proteins,wherein many different regulators play important roles for endocytic trafficking,including Rab GTPases,sorting nexins(SNXs),retromer complex and endosomal sorting complexes required for transport(ESCRT)complex.It is widely recognized that SNXs are essential for the formation of membrane transport carriers in the recycling pathway.There is one type of tubular endosomes in cell,which don’t undergo appearant vesiculation and fission,and are responsible for the recycling of ARF6-associated CIE cargoes.However,whether SNXs are involved in regulating tubular endosomal recycling of CIE cargo proteins is poorly understood.In the first part of this thesis,by using in vivo microscopy with high spatio-temporal resolution,RNA interference,and western blot,etc,the molecular mechanism uderlying SNX-3-mediated tubular endosomal recycling of CIE cargos was studied in Caenorhabditis elegans(C.elegans).Firstly,the effect on the ARF-6-associated CIE recycling endosomal tubules was screened for the SNX family members.The simplest member SNX-3 was identified as a novel regulator for the formation of tubular endosomal carriers independent of retromer complex.The loss of SNX-3 results in misrouting of CIE cargo proteins into the lysosome for degradation via ESCRT complex,instead of recycling back to PM.Moreover,it was demonstrated that SNX-3 competes with EEA-1 for binding to phosphatidylinositol-3-phosphate(PI3P)enriching EEs,which facilitates the sorting of CIE cargo into tubular endosomes for recycling,and avoids the sorting of CIE cargo proteins into EEA-1-labeled classical sorting endosomes.In addition,phospholipids are the significant membrane components which also play pivotal roles in endosomal sorting and trafficking.Unlike the above mentioned protein regulators,phospholipids regulate membrane remodeling and transport carrier biogenesis through recruiting and activating effector proteins on the target membranes or directly generating membrane curvature.As the most abundant anionic phospholipid of eukaryotic cells,phosphatidylserine(PS)is restricted to the cytoplasmic leaflet of PM,Golgi,and endosomes.The flippase P4-ATPase regulates the formation of secretory vesicles by mediating the tranport of PS across TGN membrane.However,whether P4-ATPase is involved in mediating the transport of PS across endosomal membrane and regulating of endosomal sorting and trafficking remains to be explored.In the second part of this paper,the multicolor imaging technology,RNA interference,immunofluorescence and western blot were used to investigate the mechanism of ATP8A1,a member of P4-ATPase,regulating trafficking along the endocytic degradative pathway by mediating the transport of endosomal PS across a membrane bilayer.Firstly,it was found that ATP8A1 mainly localizes in the endolysosomal system of Hela cells,which is responsible for maintaining PS on the cytosolic leaflet.Further studies showed that the reduction of ATP8A1 resulted in the aggregation of endosomes in the perinuclear region and accelerated degradation of epidermal growth factor receptor(EGFR).It is suggested that ATP8A1 plays a negative regulatory role in endosomal degradative pathway.Finally,it was demonstrated that ATP8A1 regulates the entry of EGFR into multivesicular bodies(MVBs)by mediating the the tranport of PS across endosomal membrane,which affects the formation of ESCRT degradative microdomain.Taken together,this paper reveals the molecular mechanism of SNX-3-mediated ARF-6-associated CIE tubular endosomal recycling for the first time,which provids new insights into the sorting and trafficking of CIE pathways.In addition,this paper also uncovers and investigates the mechanism of ATP8A1 regulating endosomal degradative pathway for the first time,which provids new evidences and clues for further understanding the role of P4-ATPase in endosomal sorting and trafficking.