Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion

Franceschi, Nicola De; Alqabandi, Maryam; Weissenhorn, Winfríed; Bassereau, Patricia

doi:10.21769/bioprotoc.3294

Cited by 14 publications

(12 citation statements)

References 12 publications

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“…The fusion procedure was performed as previously described 28 (see ref. 69 for a detailed protocol). Briefly, for each experiment, two types of GUVs extracted from each PVA slide were mixed with the relative external buffer matching the osmolarity and centrifuged for 10 min at 1000g.…”

Section: Methodsmentioning

confidence: 99%

“…The tube pulling experiments and analysis have been performed as described in detail in refs. 47,69 . Briefly, for experiments probing the affinity of the proteins for positive curvature, the tube was formed by bringing briefly the GUV coated with proteins in contact with a streptavidin-coated bead trapped with the optical tweezer and moved away.…”

Section: Methodsmentioning

confidence: 99%

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Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation

et al. 2020

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Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrewlike" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation

et al. 2020

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“…According to current models, the ESCRT-III and VPS4 complexes constitute the core components of the ESCRT machine that execute membrane fission (26)(27)(28)(29). They do so by assembling ESCRT-III monomers into helical filaments and remodeling these filaments by VPS4.…”

Section: Resultsmentioning

confidence: 99%

Asgard ESCRT-III and VPS4 reveal evolutionary conserved chromatin binding properties of the ESCRT machinery

Nachmias

Melnikov

Zorea

et al. 2021

Preprint

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The ESCRT machinery drive membrane remodeling in numerous processes in eukaryotes. Genes encoding for ESCRT proteins have been identified in Asgard archaea, a newly discovered superphylum, currently recognized as the ancestor of all eukaryotes. This begs the question of the functional evolutionary origin of this machinery and its conservation across lineages. Here, we find that Asgard-ESCRT’s exhibit conserved DNA-binding properties, which is derived from recruitment of specific members. We show that Asgard-ESCRT-III/VPS4 homologs interact with one another inside mammalian cells, associate with chromatin, and recruit their counterparts to organize in discrete foci in the mammalian nucleus. This is congruent with human-ESCRT-III homologs. We find that human- and Asgard-ESCRT-IIIs associate with chromatin via the same N terminal domain, and that human-ESCRT-III can recruit Asgard-VPS4 to the nucleus to form foci. Therefore, ESCRTs possess chromatin binding properties that were preserved through the billion years of evolution that separate Asgard and human cells.

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“…This procedure was performed as previously described 32 (see 77 for a detailed protocol). Briefly, for each experiment two types of GUVs extracted from each PVA slide were mixed with the relative external buffer matching the osmolarity and centrifuged for 10 min at 1000 g. GUVs taken from the bottom of the Eppendorf were incubated with gold nanorods 20 min at room temperature and then added to the imaging chamber.…”

Section: Guv Growth Washing and Incubation With Nanorods For Tube Pumentioning

confidence: 99%

“…The tube pulling experiments and analysis have been performed as previously described in 77 and 56 . For experiments probing the affinity of the proteins for positive curvature, the tube was formed by bringing briefly the GUV coated with proteins in contact with a streptavidincoated bead trapped with the optical tweezer and moved away.…”

Section: Tube Experimentsmentioning

confidence: 99%

Human ESCRT-III Polymers Assemble on Positively Curved Membranes and Induce Helical Membrane Tube Formation

Bertin

Franceschi

Mora

et al. 2019

Preprint

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Endosomal sorting complexes required for transport-III (ESCRT-III) are thought to assemble in vivo inside membrane structures with a negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and conversely how ESCRT-III polymers shape membranes is still unclear. Here, we used human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron microscopy, cryo-electron tomography and highspeed AFM. We show that CHMP4B filaments bind preferentially to flat membranes or to membrane tubes with a positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes, the latter winding around the tubes. Although combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes, they also reshape large unilamellar vesicles into helical membrane tubes with a pipe surface shape. Sub-tomogram averaging reveals that the filaments assemble parallel to the tube axis with some local perpendicular connections, highlighting the particular mechanical stresses imposed by ESCRT-III to stabilize the corkscrew-like membrane architecture. Our results thus underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature of ESCRT-III required for all or selected cellular membrane remodeling processes.

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Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion

Cited by 14 publications

References 12 publications

Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation

Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation

Asgard ESCRT-III and VPS4 reveal evolutionary conserved chromatin binding properties of the ESCRT machinery

Human ESCRT-III Polymers Assemble on Positively Curved Membranes and Induce Helical Membrane Tube Formation

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