Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation

Abstract

Autophagy is a lysosome-based degradation pathway. During autophagy, lysosomes fuse with autophagosomes to form autolysosomes. Following starvation-induced autophagy, nascent lysosomes are formed from autolysosomal membranes through an evolutionarily conserved cellular process, autophagic lysosome reformation (ALR), which is critical for maintaining lysosome homeostasis. Here we report that clathrin and phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) regulate ALR. Combining a screen of candidates identified through proteomic analysis of purified ALR tubules, and large-scale RNAi knockdown, we unveiled a tightly regulated molecular pathway that controls lysosome homeostasis, in which clathrin and PtdIns(4,5)P(2) are the central components. Our functional study demonstrates the central role of clathrin and its associated proteins in cargo sorting, phospholipid conversion, initiation of autolysosome tubulation, and proto-lysosome budding during ALR. Our data not only uncover a molecular pathway by which lysosome homeostasis is maintained through the ALR process, but also reveal unexpected functions of clathrin and PtdIns(4,5)P(2) in lysosome homeostasis.

Interaction between autophagic vesicles and the Coxiella-containing vacuole requires CLTC (clathrin heavy chain)

“…CLTC is delivered to the CCV through autophagosomal fusion, as is it not localized to the CCV membrane without autophagy functionality. This fits with the observation that CLTC localizes to lysosomes upon autophagy induction [21,22]. We propose a model in which autophagy is induced, directly by the bacteria or as a host response to the CCV, and the resulting autophagosomes acquire CLTC, with the aid of Coxiella effectors such as Cig57.…”

“…Recent studies have identified CLTC as playing a role in autophagy, showing that it participates in the regulation of autophagic lysosomal reformation [21]. CLTC is also present on mature autophagosomes, colocalizing with LC3B during initiation of autophagy [22].…”

“…There is no precedence for CLTC to be involved in a membrane fusion event, so it is more plausible that CLTC is not mediating this process directly. As examined in a recent review [33], CLTC recruits downstream effectors on the lysosomal membrane during autophagic lysosome reformation to alter the phosphoinositide signature of the membrane [21]. It is intriguing to consider a similar role for CLTC at the autophagosomal membrane to facilitate interaction with lysosomes.…”

“…A link between CLTC and autophagy has recently been discovered with the observation that CLTC is required both for autophagosome formation and in the later stages for regulation of autophagic lysosomal reformation [20,21]. In addition, it has been reported that CLTC colocalizes with LC3B upon induction of autophagy [22].…”

Early lipofuscin accumulation in frontal lobe epilepsy

“…In particular, clathrin, dynamin-1, adaptor protein-2 (AP2), synapsin, syntaxin, synaptotagmin, and vesicle-associated membrane proteins are involved in synaptic clathrin-mediated vesicle pathways (p 5 0.004; Fig 3D), which have roles in autophagy, phagosome, and lysosomal pathways. 24,25 In contrast, these vesicleassociated proteins showed either no changes or fold decrease in FCDIIB compared to pathology-negative cases.Further immunohistochemistry was carried out for clathrin and dynamin-1 in all cases with FNL, and pathology-negative (E7-E9) and FCDIIB cases (E13 and E17-E18; Fig 4A-I). Using anti-clathrin antibodies specific for the c-terminus of the heavy chain (Fig 4), strong granular immunopositivity was observed in the cytoplasm and axon hillocks of the majority of DN/L in cases with FNL, with a similar distribution to PAS (Fig 4A).…”