Septin‐based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles

Beber, Alexandre; Alqabandi, Maryam; Prévost, Coline; Viars, Fanny; Lévy, Daniel; Bassereau, Patricia; Bertin, Aurélie; Mangenot, Stéphanie

doi:10.1002/cm.21480

Cited by 33 publications

(58 citation statements)

References 61 publications

(76 reference statements)

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“…To investigate how membrane-binding affects septin polymerization, we deposited the purified fly septin hexamers in polymerization buffer on glass-supported lipid bilayers (SLBs) composed of net-neutral PC lipids doped with anionic lipids (Figure 2A). To test whether fly septins bind PI(4,5)P2, as reported for yeast septins [57,58], we doped the PC membranes with different mole percentages of PI(4,5)P2 ranging from 1 to 8%. Already at 1% PI(4,5)P2, septins formed immobile bundles adhering to the membrane ( Figure 2B).…”

Section: Lipid Membranes Recruit Septins and Promote Their Assemblymentioning

confidence: 99%

“…The cell membrane in the fly has been reported to contain 3-10% PS, but the composition varies among tissues and developmental stages [54][55][56]. PI (4,5)P2 is an anionic signaling lipid previously shown to interact with yeast and mammalian septins [37,57,58] and assumed to be important for septin-membrane interactions in the fly [59,60]. We show by fluorescence imaging that fly septin hexamers have a high affinity for membranes containing PS and/or PI (4,5)P2, and form dense membrane-associated meshworks.…”

Section: Introductionmentioning

confidence: 99%

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Membrane binding controls ordered self-assembly of animal septins

Szuba

Bano

François

et al. 2020

Preprint

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Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here we show by cell-free reconstitution that membrane binding requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septins form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12 to 18 nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4 nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

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Section: Lipid Membranes Recruit Septins and Promote Their Assemblymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Membrane binding controls ordered self-assembly of animal septins

Szuba

Bano

François

et al. 2020

Preprint

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“…Finally, we used cryo-EM to study the organization of CHMP4B on tubes. Our LUV preparation was generated after a crude re-suspension of a dried lipid film, which preserves PI(4,5)P2 lipids within the lipid bilayer 57 . This methodology generates a rather heterogeneous suspension of vesicles both in size and in geometry.…”

Section: Chmp4b Polymers Do Not Deform Membranes and Have No Particulmentioning

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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“…For all GUV compositions used, a buffer control experiment was performed to ensure that it had no effect on the vesicles. POPC was initially chosen in order to probe the interaction with the major phospholipid component in the bilayer, mimicking consistent with previous reports on the importance of phosphatidylinositol derived lipids for septin association [38,39]. A network of filamentous structures can be seen over the membrane surface on introducing the septin complex ( Fig.…”

Section: Sept5-sept6-sept7 Complex Interacts With a Biomimetic Membramentioning

confidence: 99%

Repositioning septins within the core particle

Mendonça

Macêdo

Itri³

et al. 2019

Preprint

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Abbreviations (Define non-standard or uncommon abbreviations). AbstractSeptins are GTP binding proteins considered to be a novel component of the cytoskeleton. They polymerize into filaments based on hetero-oligomeric core particles which, in humans, are either hexamers or octamers composed of two copies each of either three or four different septins from the 13 available. Not all combinations are possible as it is believed that these must obey substitution rules which determine that different septins must be derived from four distinct and well-established sub-groups. Here, we have purified and characterized one such combinations, SEPT5-SEPT6-SEPT7, and used TEM to derive the first structural information concerning its assembly. The full complex was purified using an affinity tag attached to only one of its components (SEPT7) and was able to bind to and perturb lipid bilayers. Although the complex assembled into elongated hexameric particles, the position of SEPT5 was incompatible with that predicted by the reported structure of SEPT2-SEPT6-SEPT7 based on the substitution rules. MBP-fusion constructs for SEPT5 and SEPT2 and immuno-staining clearly show that these septins occupy the terminal positions of the SEPT5-SEPT6-SEPT7 and SEPT2-SEPT6-SEPT7 hexamers, respectively. In so doing they expose a so-called NC interface which we show to be more susceptible to perturbation at high salt concentrations. Our results show that the true structure of the hexamer is inverted with respect to that described previously and, as such, is more compatible with that reported for yeast. Taken together, our results suggest that the mechanisms involved in spontaneous self-assembly of septin core particles and their filaments deserve further reflection.

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Septin‐based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles

Cited by 33 publications

References 61 publications

Membrane binding controls ordered self-assembly of animal septins

Membrane binding controls ordered self-assembly of animal septins

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

Repositioning septins within the core particle

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