Curvature- and Phase-Induced Protein Sorting Quantified in Transfected Cell-Derived Giant Vesicles

Moreno-Pescador, Guillermo; Florentsen, Christoffer D.; Östbye, Henrik; Sønder, Stine Lauritzen; Boye, Theresa Louise; Veje, Emilie L.; Sonne, Alexander K.; Semsey, Szabolcs; Nylandsted, Jesper; Daniels, Robert; Bendix, Poul Martin

doi:10.1021/acsnano.9b01052

Cited by 41 publications

(65 citation statements)

References 71 publications

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“…concentration whereas ANXA2 did not show this behavior as reported in Ref. 13 Since the ANXA4 tested here forms trimers after binding to the membrane 6,26 we proceeded to investigate the effect of trimerization on the sorting of ANXA4. We introduced a modified version of the mutant protein, engineered not to form trimers (ANXA4-Mutant) in both MCF7 cells and in HEK293T cells.…”

Section: Anxa4 Monomers Do Not Sense Membrane Curvaturementioning

confidence: 63%

“…The GPMVs are devoid of internal membrane structures like cytoskeleton and nucleus, 17 but they do contain proteins from the cytosol and transmembrane proteins from the plasma membrane, including proteins produced from transient transfection. 13 These vesicles are therefore well suited for the purpose of observing protein-lipid interactions in a complex plasma membrane environment. Human embryonic kidney (HEK293T) and breast carcinoma (MCF-7) cell lines were used as donors for the GPMVs and ANXA4-GFP proteins were transiently expressed in these cell lines.…”

Section: Resultsmentioning

confidence: 99%

“…In Ref. 13 it was observed that ANXA5, but not ANXA2, senses negative membrane curvature. Moreover, ANXA5 is known to form trimers in environments with high Ca 2+…”

Section: Anxa4 Monomers Do Not Sense Membrane Curvaturementioning

confidence: 99%

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Annexin A4 trimers are recruited by high membrane curvatures in Giant Plasma Membrane Vesicles

Florentsen

Kamp-Sonne

Moreno-Pescador

et al. 2020

Preprint

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The plasma membrane of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the plasma membrane (PM) is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including plasma membrane repair. Its binding to membranes is activated by Ca 2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca 2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant plasma membrane vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.

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Section: Anxa4 Monomers Do Not Sense Membrane Curvaturementioning

confidence: 63%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Annexin A4 trimers are recruited by high membrane curvatures in Giant Plasma Membrane Vesicles

Florentsen

Kamp-Sonne

Moreno-Pescador

et al. 2020

Preprint

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“…To that end, using supported membrane models we have identified membrane binding and shaping features of other annexins that are independent of vesicle fusion events, pointing to a direct effect of annexins on membranes [34]. For example, ANXA4 was shown to generate a curvature force at free membrane edges because of its Ca 2+ -dependent homo-trimerization, while binding of ANXA6 led to a constriction force [35]. Therefore, it is likely that annexins possess different membrane-shaping properties, allowing for a tailored response that meets the needs of repair.…”

Section: Annexins In Repairmentioning

confidence: 98%

Interdisciplinary Synergy to Reveal Mechanisms of Annexin-Mediated Plasma Membrane Shaping and Repair

Bendix

Simonsen

Florentsen

et al. 2020

Cells

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The plasma membrane surrounds every single cell and essentially shapes cell life by separating the interior from the external environment. Thus, maintenance of cell membrane integrity is essential to prevent death caused by disruption of the plasma membrane. To counteract plasma membrane injuries, eukaryotic cells have developed efficient repair tools that depend on Ca 2+ -and phospholipid-binding annexin proteins. Upon membrane damage, annexin family members are activated by a Ca 2+ influx, enabling them to quickly bind at the damaged membrane and facilitate wound healing. Our recent studies, based on interdisciplinary research synergy across molecular cell biology, experimental membrane physics, and computational simulations show that annexins have additional biophysical functions in the repair response besides enabling membrane fusion. Annexins possess different membrane-shaping properties, allowing for a tailored response that involves rapid bending, constriction, and fusion of membrane edges for resealing. Moreover, some annexins have high affinity for highly curved membranes that appear at free edges near rupture sites, a property that might accelerate their recruitment for rapid repair. Here, we discuss the mechanisms of annexin-mediated membrane shaping and curvature sensing in the light of our interdisciplinary approach to study plasma membrane repair.

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“…These GPMVs fill a unique niche, maintaining the approximate lipid and protein content of living membranes while offering the experimental tractability of synthetic models (3). This combination has allowed investigation of PM composition (4), physical properties (5)(6)(7), dynamics (8,9), and potentially signaling and transport (8,(10)(11)(12)(13) in isolation from the complexity of living cells.…”

Section: Introductionmentioning

confidence: 99%

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

Skinkle

Levental

2019

Preprint

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Giant Plasma Membrane Vesicles (GPMVs) are a widely used model system for biochemical and biophysical analysis of the isolated mammalian plasma membrane (PM). A core advantage of these vesicles is that they maintain the native lipid and protein diversity of the plasma membrane while affording the experimental flexibility of synthetic giant vesicles. In addition to fundamental investigations of PM structure and composition, GPMVs have been used to evaluate the binding of proteins and small molecules to cell-derived membranes, and the permeation of drug-like molecules through them. An important assumption of such experiments is that GPMVs are sealed; i.e. that permeation occurs by diffusion through the hydrophobic core rather than through hydrophilic pores. Here we demonstrate that this assumption is often incorrect. We find that most GPMVs isolated using standard preparations are passively permeable to various hydrophilic solutes as large as 40 kDa, in contrast to synthetic giant unilamellar vesicles (GUVs). We attribute this leakiness to relatively large and heterogeneous pores formed by rupture of vesicles from cells. These pores are stable and persist throughout experimentally relevant time scales. Finally, we identify preparation conditions that minimize poration and allow evaluation of sealed GPMVs. These unexpected observations of GPMV poration are of critical importance for interpreting experiments utilizing GPMVs as plasma membrane models, particularly for drug permeation and membrane asymmetry. STATEMENT OF SIGNIFICANCE:A critical assumption in using Giant Plasma Membrane Vesicles to study membrane penetration and interactions is that these vesicles maintain the permeability barrier of the native membrane from which they form. Using large fluorescently-labeled hydrophilic probes, we demonstrate that this assumption is often incorrect and conclude that macromolecular solutes permeate GPMVs through stable pores formed during shear-induced rupture of vesicles from cells. Using these insights into the mechanisms of poration, we demonstrate an approach to isolate sealed GPMVs.

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Curvature- and Phase-Induced Protein Sorting Quantified in Transfected Cell-Derived Giant Vesicles

Cited by 41 publications

References 71 publications

Annexin A4 trimers are recruited by high membrane curvatures in Giant Plasma Membrane Vesicles

Annexin A4 trimers are recruited by high membrane curvatures in Giant Plasma Membrane Vesicles

Interdisciplinary Synergy to Reveal Mechanisms of Annexin-Mediated Plasma Membrane Shaping and Repair

Cell-derived plasma membrane vesicles are permeable to hydrophilic macromolecules

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