Regulation of the chaperone‐mediated autophagy receptor LAMP2A by DYNC1LI2 in cystinosis

Abstract

The dynein motor protein complex is required for retrograde transport but the specific functions of the intermediate and light chains that form the cargo‐binding complex are not elucidated and the importance of individual subunits in the maintenance of cellular homeostasis is unknown. Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Increased levels of intra‐lysosomal cystine lead to cell malfunction, which is especially manifested in the kidney epithelial proximal tubule cells (PTCs). Many cellular defects in cystinosis are independent of lysosomal overload, suggesting that cystinosin regulates cellular processes independently of its role as a cystine transporter. We showed that cystinotic cells (Ctns−/−) are characterized by vesicular transport defects, increased endoplasmic reticulum (ER) stress and susceptibility to oxidative stress. Here, using a mRNA array and immunoblot assays, we show that the dynein subunit, intermediate chain 2 (DYNC1LI2) is downregulated in cystinotic cells. Reconstitution of the expression of DYNC1LI2 in Ctns−/− fibroblasts re‐established endolysosomal dynamics quantified by Total Internal Reflection Fluorescence microscopy (TIRFM). Rescue of vesicular trafficking by DYNC1LI2 correlated with decreased ER stress manifested as decreased levels of the chaperone Grp78 in cystinotic cells. Mitochondrial fragmentation in cystinotic fibroblasts was also reduced by DYNC1LI2 expression. Survival of cystinotic cells to oxidative stress insult was increased by DYNC1LI2 but not by its paralog DYNC1LI1, which also failed to reestablish endolysosomal trafficking, or to decrease ER stress levels and mitochondrial fragmentation. Cystinotic (CTNS‐KO) PTCs, generated using CRISPR‐Cas9, showed defective endolysosomal trafficking and decreased expression of the Rab‐GTPase Rab11 carrier of the chaperone‐mediated autophagy receptor LAMP2A and the endocytic receptor megalin, both mislocalized in cystinosis. Restoring DYNC1LI2 expression rescued the localization of LAMP2A and restored the expression of megalin at the plasma membrane of CTNS‐KO cells. Expression of GFP‐DYNC1LI2 failed to rescue phenotypes in cystinotic cells (Ctns−/−) when co‐expressed with dominant negative (DN) RAB7/RAB11 but not DN‐Rab5 and in correlation with this finding, TR‐FRET assays show that DYNC1LI2 associates with RILP, a RAB7 effector and Rab11‐FIP4, a Rab11 effector. The rescue of the increased ER stress phenotype by DYNC1LI2 in cystinotic cells was blocked by downregulation of LAMP2A expression. Our results suggest that DYNC1LI2 reconstitution improves cellular homeostasis in a LAMP2A‐dependent manner highlighting DYNC1LI2 as a possible target molecule to improve cellular function in cystinosis.Support or Funding InformationNIHCRF