10.6084/m9.figshare.7959500.v2 Jinzhong Zhang Jinzhong Zhang Jing He Jing He Jennifer L. Johnson Jennifer L. Johnson Gennaro Napolitano Gennaro Napolitano Mahalakshmi Ramadass Mahalakshmi Ramadass Farhana Rahman Farhana Rahman Sergio D. Catz Sergio D. Catz Cross-regulation of defective endolysosome trafficking and enhanced autophagy through TFEB in UNC13D deficiency Taylor & Francis Group 2019 Autophagy endosomal trafficking FHL3 fusion lysosomal signaling secretion TFEB 2019-08-17 07:55:06 Dataset https://tandf.figshare.com/articles/dataset/Cross-regulation_of_defective_endolysosome_trafficking_and_enhanced_autophagy_through_TFEB_in_UNC13D_deficiency/7959500 <p>Several lines of evidence support the occurrence of cross-regulation between the endocytic pathway and autophagy, but the molecular mechanisms regulating this process are not well-understood. Here, we show that the calcium sensor UNC13D regulates the molecular mechanism of late endosomal trafficking and endosomal maturation, and defects in UNC13D lead to macroautophagy upregulation. <i>unc13d</i>-null cells showed impaired endosomal trafficking and defective endocytic flux. The defective phenotypes were rescued by the expression of UNC13D but not by its STX7-binding-deficient mutant. This defective endosomal function in UNC13D-deficient cells resulted in increased autophagic flux, increased long-lived protein degradation, decreased SQSTM1/p62 protein levels and increased autolysosome formation as determined by biochemical, microscopy and structural methods. The autophagic phenotype was not associated with increased recruitment of the UNC13D-binding proteins and autophagy regulators, RAB11 or VAMP8, but was caused, at least in part, by TFEB-mediated upregulation of a subset of autophagic and lysosomal genes, including <i>Atg9b</i>. Downregulation of TFEB decreased <i>Atg9b</i> levels and decreased macroautophagy in <i>unc13d</i>-null cells. UNC13D upregulation corrected the defects in endolysosomal trafficking and decreased the number of accumulated autophagosomes in a cellular model of the lysosomal-storage disorder cystinosis, under both fed and starvation conditions, identifying UNC13D as an important new regulatory molecule of autophagy regulation in cells with lysosomal disorders.</p> <p>ACTB: actin, beta; CTSB: cathepsin B; EEA1: early endosome antigen 1; ESCRT: endosomal sorting complex required for transport; FHL3: familial hemophagocytic; lymphohistiocytosis type 3; HEX: hexosaminidase; HLH: hemophagocytic lymphohistiocytosis; LSD: lysosomal storage disorder; MEF: mouse embryonic fibroblast; SEM: standard errors of the mean; SNARE: soluble n-ethylmaleimide-sensitive-factor attachment receptor; STX: syntaxin; SYT7: synaptotagmin VII; TFE3: transcription factor E3; TFEB: transcription factor EB; TIRF: total internal reflection fluorescence ULK1: unc-51 like kinase 1; UNC13D: unc-13 homolog d; VAMP: vesicle-associate membrane protein; WT: wild-type</p>