Biology subjects

McEwan, D. G.

Publications and source records attributed to McEwan, D. G..

3 recordsLinked to original sources

bioRxiv2023

Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via CASM

Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired, or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological and genetic approaches, we show that lysosome damage induces rapid Conjugation of ATG8s to Single Membranes (CASM). ATG8 proteins are recruited directly onto damaged membranes, independently of ATG13/WIPI2, and conjugated to PS, as well as PE, a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, and direct recruitment of ATG16L1, dependent on K490 (WD40-domain), and sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with the formation of dynamic LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics.

cell biology↗

bioRxiv2021

kinesin recruitment by adapter SKIP on melanosomes is dynamically controlled by LC3B phosphorylation

Anterograde melanosome transport is essential for adaptive skin tanning response. However, the molecular components involved, their interplay and regulation by external cues in melanosome transport remain under-explored. Silencing of kinesin motors revealed that several members including the established KIF5B and a novel candidate KIF1B, mediate melanosome movement. The camouflage behaviour of zebrafish embryos induced by incident light or -MSH requires kif1b, suggesting a conserved melanosome transport machinery across vertebrates. Interestingly, the peri-nuclear melanosome accumulation upon kinesin knockdown is recapitulated by the silencing of autophagy effector MAP1LC3B (LC3B). Pull-down assays identified KIF1B, but not KIF5B, to be the LC3B-associated kinesin. LC3B binds the adapter SKIP via its LIR docking region that is proximal to Thr12 residue, a site for phosphorylation by Protein Kinase A. We demonstrate that phosphorylation of LC3B at Thr12 is stimulated by -MSH, which potentiates the anterograde melanosome transport. Thereby, our study, identifies a novel kinesin motor KIF1B for melanosome movement and establishes LC3B as the key molecular component that facilitates -MSH responsive mobilization of melanosomes. Key HighlightsO_LIKinesin screen reveals non-redundant use of KIF5B, KIF1B motors for melanosome transport C_LIO_LIkif1b is required for camouflage response in zebrafish and melanosome movement in mammals C_LIO_LIN-terminal region of LC3B interacts with adapter SKIP and couples kinesin KIF1B C_LIO_LI-MSH activates PKA-mediated phosphorylation of LC3B to potentiate anterograde movement C_LI SignificanceMelanosomes are lysosome related organelles containing melanin pigment, that are synthesized in melanocytes and transferred to the recipient keratinocytes of skin. This involves long range melanosome movement within melanocytes to reach cell periphery for the transfer to follow. Physiologically, UV protection involves local secretion of melanocyte stimulating hormone (-MSH) that acts on melanocytes to promote skin tanning response. Herein, we investigate the components involved in this process and establish that the melanosome movement is dynamically controlled by -MSH through phosphorylation of LC3B. These findings establish the mechanism behind the rapid distribution of melanosomes during tanning response and provide opportunity to intervene for sun protection.

cell biology↗

bioRxiv2019

A conserved ATG2-GABARAP interaction is critical for phagophore closure

The intracellular trafficking pathway, macroautophagy, acts as a recycling and disposal service that can be upregulated during periods of stress, to maintain cellular homeostasis. An essential transition point in the pathway is the sealing of the immature phagophore to form an autophagosome, isolating unwanted cargo prior to lysosomal degradation. However, little mechanistic detail is known about phagophore closure. Human ATG2A and ATG2B proteins, through their interaction with WIPI proteins, are thought to be key players during phagophore closure. We have identified a highly-conserved motif driving the interaction between human ATG2 and GABARAP proteins that is in close proximity to the ATG2-WIPI4 interaction site. We show that the ATG2-GABARAP interaction mutants are unable to close phagophores resulting in blocked autophagy, similar to ATG2A/ATG2B double knock-out cells. In contrast, the ATG2-WIPI4 interaction mutant fully restored phagophore closure and autophagy flux, similar to wild type ATG2. Taken together, we provide new mechanistic insights to the requirements for ATG2 function at the phagophore and suggest that an ATG2-GABARAP interaction is essential for phagophore closure, whereas ATG2-WIPI4 interaction is dispensable.

cell biology↗