To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered lysosome-associated membrane protein (LAMP)-carrier vesicles around multivesicular bodies, as well as the appearance of ‘hourglass’ profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of charged multi-vesicular body protein 6 (CHMP6), and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.
Bibliographical noteFunding Information:
This work was supported by the Medical Research Council (MRC) [research grants MR/M010007/1 and MR/R0009015/1 to J.P.L. and N.A.B., research studentship to M.D.J.P.]; the Biotechnology and Biological Sciences Research Council (BBSRC) [research grant BB/L002841/1 to A.A.P.] and the BBSRC with GSK Research and Development Ltd [industrial CASE studentship to L.J.D. grant no. 1574760]. Open access funding provided by University of Cambridge. Deposited in PMC for immediate release.
© 2021. Published by The Company of Biologists Ltd.
- Membrane fusion