Date of Award
Doctor of Philosophy (PhD)
Endosomes function to sort, recycle, and degrade a wide range of substances from within and outside the cell. Materials such as incoming pathogens, membrane-destabilizing molecules, or particulates can damage endosomal vesicles. While terminally damaged compartments are degraded by autophagy, pathways that repair salvageable organelles are poorly understood. Here we propose that the ESCRT (Endosomal Sorting Complex Required for Transport) protein machinery, which mediates budding and fission events during endosomal trafficking, also plays an essential role in endosomal repair. ESCRTs rapidly acumulated on endosomes following acute injury by lysosomotropic peptides via a pathway that was independent of autophagy. We used live-cell imaging to demonstrate that ESCRTs responded to small disruptions in endolysosomal membranes and enabled compartments to recover from limited damage. ESCRT recruitment was also triggered by engulfed silica crystals, transfection reagents, osmotic rupture, and oxidative damage, and was especially pronounced in astrocytes and phagocytic cells. ESCRTs might thus provide a defense against endosomal damage likely to be relevant in various physiological and pathological contexts associated with endosomal leakage.
The mechanism of ESCRT-facilitated repair and the architecture of the repair complex are unknown. A major challenge derives from the transient and multifactorial nature of ESCRT structures, which assemble from multiple subunits and are disassembled by the AAA-ATPase VPS4 (Vacuolar Protein Sorting 4). A VPS4 inhibitor would thus be of interest for blocking disassembly and stabilizing ESCRT complexes. We show that the quinazoline derivatives ML240 and DBeQ can inhibit in vitro the ATPase activity of human VPS4A. ML240 additionally caused ESCRTs to accumulate on endosomes and might therefore also inhibit VPS4 in cells. Both compounds are well-known inhibitors of the related AAA-ATPase VCP (Valosin Containing Protein)/p97, but the cellular effects of ML240 on ESCRTs were unrelated to inhibition of this enzyme. We thus propose that VPS4 can be targeted by quinazoline-based molecules, and anticipate that ML240 can provide a starting point for generating more selective VPS4 inhibitors. We note that ML240 also caused extensive tubulation of early endosomes and perturbed endosomal recycling pathways, independently of its effects on VPS4 and VCP. We speculate that the compound might additionally inhibit the microtubule-severing AAA-ATPase Spastin that is homologous to VPS4 and has been suspected to promote recycling tubule fission. ML240 might thus also be exploited to clarify the role of this enzyme in endosomal recycling.
Despite its microtubule-severing activity, Spastin also binds the ESCRT-III proteins CHMP1B and IST1 and evidence supports an ill-defined role for all three factors in endosomal tubule fission. By examining structures formed by CHMP1B and Spastin's effect on their stability, we show that CHMP1B polymers form helical spirals that extrude membrane tubules into the cytoplasm, and that Spastin can resolubilize these polymers. ESCRTs and Spastin might thus comprise a distinct membrane fission machinery. We also demonstrate that CHMP1B polymers are phosphorylated and suggest a role for this modification in ESCRT complex formation.
Chair and Committee
Phyllis I. Hanson
Kendall J. Blumer, Paul H. Schlesinger, Robert P. Mecham, Conrad C. Weihl,
Skowyra, Michal Leszek, "Roles of ESCRT machinery in endosomal repair and recycling" (2018). Arts & Sciences Electronic Theses and Dissertations. 1696.
Available for download on Tuesday, September 10, 2120