WTS-1/Lats Regulates Endocytic Recycling By Restraining F-actin Assembly

Through multiple endocytic pathways,eukaryotic cells uptake plasma membrane components,including lipids(phospholipids and cholesterol)and proteins.Endosomal actin homeostasis is essential for recycling transport.Specifically,endosomal actin structure dynamics,including assembly and disassembly,are involved in recycling regulation.Disturbance of endosomal actin architecture is deleterious to endocytic recycling.However,the underlying homeostatic mechanisms that regulate actin organization during recycling remain unclear.To elucidate the mechanisms of endocytic recycling,we performed a genome-wide RNAi screen and identified WTS-1 as a new recycling regulator in C.elegans.Wts/LATS was initially identified in D.melanogaster as a core kinase of the Hippo pathway.wts-1/Wts/LATS mutant worms,flies,and mice displayed embryonic lethality or larval arrest,indicating that WTS-1/Wts/LATS is required for proper development.Studies of C.elegans demonstrated that WTS-1 is located in the subapical region of the epithelia.The loss of WTS-1 activity disrupted intestinal apicobasal polarity,while this phenotype could be associated with disturbed apical secretion.Further analysis demonstrated that wts-1 mutations caused more actin to be localized to structures near the apical membrane.To explore the mechanism of WTS-1 affecting endocytic recycling and the underlying mechanisms of WTS-1 regulating actin organization during recycling.Deploying experimental techniques of cell biology,genetics,and biochemistry to dissect the function of WTS-1.Loss of WTS-1 impaired the basolateral recycling of clathrin-independent cargo h TAC.WTS-1/LATS resides on the sorting endosomes and colocalizes with the actin polymerization regulator PTRN-1/CAMSAP.Of note,we observed an increase of PTRN-1-labeled structures in WTS-1-deficient cells,suggesting that WTS-1 is capable of limiting the endosomal localization of PTRN-1.Accordingly,the actin overaccumulation phenotype in WTS-1-depleted cells was abolished by the concomitant loss of PTRN-1.We further showed that recycling defects and actin overaccumulation in WTS-1-deficient cells were effectively alleviated by the overexpression of constitutively active UNC-60A/cofilin(S3A),consistent with the identification of LATS as a negative regulator of cofilin activity.Together our data consolidated previous findings,and we proposed an additional model,working in parallel to the UNC-60A/cofilin-mediated actin disassembly,that WTS-1 acts to restrict the assembly of endosomal F-actin via curbing PTRN-1 dwelling on endosomes,ensuring the progress of recycling transport.Our results indicate that WTS-1 synergistically maintains endosomal actin homeostasis by restraining PTRN-1-mediated F-actin assembly and facilitating UNC-60A-mediated F-actin disassembly,thereby sustaining recycling transport.