Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway

Luther J Davis; Nicholas A Bright; James R Edgar; Michael D J Parkinson; Lena Wartosch; Judith M Mantell; Andrew A Pedan; J Paul Luzio

doi:10.1242/jcs.255463

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway

Luther J Davis, Nicholas A Bright^*, James R Edgar, Michael D J Parkinson, Lena Wartosch, Judith M Mantell, Andrew A Pedan, J Paul Luzio^*

^*Corresponding author for this work

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Abstract

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.

Original language	English
Article number	jcs255463
Number of pages	25
Journal	Journal of Cell Science
Volume	134
Issue number	10
DOIs	https://doi.org/10.1242/jcs.255463
Publication status	Published - 27 May 2021

Bibliographical note

Funding 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.

Publisher Copyright:
© 2021. Published by The Company of Biologists Ltd.

Keywords

Lysosome
Endosome
Membrane fusion

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10.1242/jcs.255463

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title = "Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway",

abstract = "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 {\textquoteleft}hourglass{\textquoteright} 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.",

keywords = "Lysosome, Endosome, Membrane fusion",

author = "Davis, \{Luther J\} and Bright, \{Nicholas A\} and Edgar, \{James R\} and Parkinson, \{Michael D J\} and Lena Wartosch and Mantell, \{Judith M\} and Pedan, \{Andrew A\} and Luzio, \{J Paul\}",

note = "Funding 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. Publisher Copyright: {\textcopyright} 2021. Published by The Company of Biologists Ltd.",

year = "2021",

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language = "English",

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AU - Davis, Luther J

AU - Bright, Nicholas A

AU - Edgar, James R

AU - Parkinson, Michael D J

AU - Wartosch, Lena

AU - Mantell, Judith M

AU - Pedan, Andrew A

AU - Luzio, J Paul

N1 - Funding 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. Publisher Copyright: © 2021. Published by The Company of Biologists Ltd.

PY - 2021/5/27

Y1 - 2021/5/27

N2 - 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.

AB - 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.

KW - Lysosome

KW - Endosome

KW - Membrane fusion

U2 - 10.1242/jcs.255463

DO - 10.1242/jcs.255463

M3 - Article (Academic Journal)

C2 - 34042162

SN - 0021-9533

VL - 134

JO - Journal of Cell Science

JF - Journal of Cell Science

IS - 10

M1 - jcs255463

ER -

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway

Abstract

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Keywords

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