Investigating the regulation of WIPI2b function at the phagophore by phosphorylation in starvation-induced autophagy

Macroautophagy, here referred to as autophagy, is an intracellular degradation pathway cells use to maintain their homeostasis. Autophagy is also required for cell survival during nutrient deprivation, as well as development and immunity in higher eukaryotes. Aberrations in autophagy can lead to pathologies including cancer, neurodegeneration and diabetes. Autophagy is characterised by the formation of a double membrane phagophore, which sequesters cytosolic cargo and forms a vesicle termed an autophagosome. The autophagosome eventually fuses with the lysosome, resulting in the degradation of the cytosolic cargo. Although autophagosome formation is orchestrated by the sequential action of the core autophagy proteins, a key question remains – what gives rise to a double membrane phagophore? The key event in phagophore biogenesis is the production of PI3P at the phagophore formation sites. WIPI2b, a PI3P effector protein, directly interacts with ATG16L1 and is recruited to the omegasomes, which is the basis for LC3 recruitment to the forming phagophore. To address how the function of WIPI2b at the phagophore is regulated, I focused on phosphorylation, as there have been reports about potential phosphorylation sites on WIPI2b. I confirmed an interactive relationship between WIPI2b and ULK1 that was reported previously and identified a number of phosphorylation sites on WIPI2b upon overexpression of ULK1 kinase. I found that phospho-mutants of WIPI2b S68 exhibit reduced interaction with ATG16L1 and WIPI4. I generated and characterised a WIPI2 CRISPR knockout cell line and found that WIPI2b S68 phospho-mutants are unable to rescue LC3 lipidation in WIPI2 CRISPR knockout cells. I also found that WIPI2b S284 phosphorylation is important for the regulation of WIPI2b association with membranes. I propose WIPI2b phosphorylation by ULK1 provides a feedback loop during autophagy to control the amount of functional WIPI2b at phagophores and therefore allows phagophore elongation.Open Acces

The endolysosomal adaptor PLEKHM1 is a direct target for both mTOR and MAPK pathways

The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 co-precipitates with, and is directly phosphorylated by, mTOR. Using a phospho-specific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism

A guide to the regulation of selective autophagy receptors

Autophagy is a highly conserved catabolic process cells use to maintain their homeostasis by degrading misfolded, damaged and excessive proteins, nonfunctional organelles, foreign pathogens and other cellular components. Hence, autophagy can be nonselective, where bulky portions of the cytoplasm are degraded upon stress, or a highly selective process, where preselected cellular components are degraded. To distinguish between different cellular components, autophagy employs selective autophagy receptors, which will link the cargo to the autophagy machinery, thereby sequestering it in the autophagosome for its subsequent degradation in the lysosome. Autophagy receptors undergo post-translational and structural modifications to fulfil their role in autophagy, or upon executing their role, for their own degradation. We highlight the four most prominent protein modifications – phosphorylation, ubiquitination, acetylation and oligomerisation – that are essential for autophagy receptor recruitment, function and turnover. Understanding the regulation of selective autophagy receptors will provide deeper insights into the pathway and open up potential therapeutic avenues

ER remodeling via ER-phagy

The endoplasmic reticulum (ER) is a hotspot for many essential cellular functions. The ER membrane is highly dynamic, which affects many cellular processes that take place within the ER. One such process is ER-phagy, a selective degradation of ER fragments (including membranes and luminal content), which serves to preserve the size of ER while adapting its morphology under basal and stress conditions. In order to be degraded, the ER undergoes selective fragmentation facilitated by specialized ER-shaping proteins that also act as ER-phagy receptors. Their ability to sense and induce membrane curvature, as well as to bridge the ER with autophagy machinery, allows for a successful ER fragmentation and delivery of these fragments to the lysosome for degradation and recycling. In this review, we provide insights into ER-phagy from the perspective of membrane remodeling. We highlight the importance of ER membrane dynamics during ER-phagy and emphasize how its dysregulation reflects on human physiology and pathology

Minimized combinatorial CRISPR screens identify genetic interactions in autophagy

Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale

Suppression of autophagy during mitosis via CUL4-RING ubiquitin ligases-mediated WIPI2 polyubiquitination and proteasomal degradation

10.1080/15548627.2019.1596484Autophagy15111917-193

The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension

Abstract Human WIPI β-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan