Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions

Dar, Srishti; Kamerkar, Sukrut C.; Pucadyil, Thomas J.

doi:10.1038/nprot.2016.173

Cited by 27 publications

(37 citation statements)

References 34 publications

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“…These templates are formed by flow-induced extrusion of a membrane reservoir (Fig. 3a ) 27 , 28 , and display a planar supported lipid bilayer (SLB) that is connected to an array of unilamellar membrane tubes. Templates contained an environment- and membrane curvature-insensitive fluorescent lipid, p -Texas Red DHPE for visualizing the membrane and estimating tube dimensions according to methods reported earlier 28 .…”

Section: Resultsmentioning

confidence: 99%

“…The membrane curvature- and protein-binding-insensitive lipid probe p -Texas red DHPE was separated from the mixed isomers of Texas Red DHPE (Invitrogen) using thin-layer chromatography on silica gel plates (Sigma) 28 . DOPS, DOPC, and p -Texas Red DHPE were reconstituted in a 40:59:1 molar ratio in chloroform and brought to a final concentration of 1 mM total lipid.…”

Section: Methodsmentioning

confidence: 99%

“…Lipid stocks were stored at −40 °C and brought to room temperature before use. Membrane templates were formed by spreading 2 μl of the lipid stock on glass coverslips passivated by the covalent attachment of PEG8000 28 . The coverslip was left in vacuum and assembled in a flow cell (FCS2, Bipotechs, PA).…”

Section: Methodsmentioning

confidence: 99%

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ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

et al. 2018

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Endocytic and recycling pathways generate cargo-laden transport carriers by membrane fission. Classical dynamins, which generate transport carriers during endocytosis, constrict and cause fission of membrane tubes in response to GTP hydrolysis. Relatively, less is known about the ATP-binding Eps15-homology domain-containing protein1 (EHD1), a dynamin family member that functions at the endocytic-recycling compartment. Here, we show using cross complementation assays in C. elegans that EHD1’s membrane binding and ATP hydrolysis activities are necessary for endocytic recycling. Further, we show that ATP-bound EHD1 forms membrane-active scaffolds that bulge tubular model membranes. ATP hydrolysis promotes scaffold self-assembly, causing the bulge to extend and thin down intermediate regions on the tube. On tubes below 25 nm in radius, such thinning leads to scission. Molecular dynamics simulations corroborate this scission pathway. Deletion of N-terminal residues causes defects in stable scaffolding, scission and endocytic recycling. Thus, ATP hydrolysis-dependent membrane remodeling links EHD1 functions to endocytic recycling.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

et al. 2018

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“…The decrease in lipid mobility in a membrane tube in the presence of an actin sleeve is confirmed by fluorescence recovery after photobleaching (FRAP) experiments [ fig. S6, Supplementary Materials, and (28)]. By balancing the membrane forces driving this flow with the dissipation due to the friction of a membrane of viscosity  against a density  of actin attachment points, we compute the force relaxation dynamics to lowest order in W/ as…”

Section: Relaxation Of Elongated Maasmentioning

confidence: 99%

Actin modulates shape and mechanics of tubular membranes

Allard

Bouzid

Betz³

et al. 2020

Sci. Adv.

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The actin cytoskeleton shapes cells and also organizes internal membranous compartments. In particular, it interacts with membranes for intracellular transport of material in mammalian cells, yeast, or plant cells. Tubular membrane intermediates, pulled along microtubule tracks, are formed during this process and destabilize into vesicles. While the role of actin in tubule destabilization through scission is suggested, literature also provides examples of actin-mediated stabilization of membranous structures. To directly address this apparent contradiction, we mimic the geometry of tubular intermediates with preformed membrane tubes. The growth of an actin sleeve at the tube surface is monitored spatiotemporally. Depending on network cohesiveness, actin is able to entirely stabilize or locally maintain membrane tubes under pulling. On a single tube, thicker portions correlate with the presence of actin. These structures relax over several minutes and may provide enough time and curvature geometries for other proteins to act on tube stability.

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“…These supported membranes have been successfully used to reveal the physical basis of membrane remodelling promoted by protein crowding 97 or performed by endocytic [98][99][100][101] and autophagic proteins 102 . Similarly, the supported membrane tubes (SMrT) 103 are synthetic membranes mimicking membrane tethers pulled by proteins 104,105 that can be used to study the impact of local curvature on protein-membrane interactions 101 .…”

Section: Engineering Curved Membranes To Understand the Mechanisms Ofmentioning

confidence: 99%

Topography design in model membranes: Where biology meets physics

Chand

Beales

Claeyssens

et al. 2018

Exp Biol Med (Maywood)

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Phospholipid membranes are necessary for the compartmentalisation of chemistries within biological cells and for initiation and propagation of cell signalling. The morphological and chemical complexity of cellular membranes represent a challenge for dissecting the biochemical processes occurring at these interfaces. Therefore, investigations of the biological events occurring at the membrane require suitable models able to reproduce the complexities of this surface. Solid-supported lipid bilayers (SLB) are simplified physical replicas of biological membranes that allow the bottom-up reconstruction of the molecular mechanisms occurring at cellular interfaces. In this brief review we introduce how the properties of SLBs can be tuned to mimic biological membranes, highlighting the engineering approaches for creating spatially resolved patterns of lipid bilayers and supported membranes with curved geometries. Additionally, we present how SLBs have 2 ! been employed to reconstitute the biophysical basis of molecular mechanisms involved in intercellular signalling and more recently, membrane trafficking. KeywordsSolid-supported lipid bilayers, membrane patterning, membrane curvature, synthetic biology, in vitro reconstitution. Impact StatementArtificial membranes with complex topography aid the understanding of biological processes where membrane geometry plays a key regulatory role. In this review, we highlight how emerging material and engineering technologies have been employed to create minimal models of cell signalling pathways, in vitro. These artificial systems allow life scientists to answer ever more challenging questions in regards to mechanisms in cellular biology. In vitro reconstitution of biology is an area that draws on the expertise and collaboration between biophysicists, material scientists and biologists and has recently generated a number of high impact results, some of which are also discussed in this review.

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Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions

Cited by 27 publications

References 34 publications

ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

Actin modulates shape and mechanics of tubular membranes

Topography design in model membranes: Where biology meets physics

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