Arp2/3 Controls the Motile Behavior of N-WASP-Functionalized GUVs and Modulates N-WASP Surface Distribution by Mediating Transient Links with Actin Filaments

Delatour, Vincent; Helfer, Emmanuèle; Didry, Dominique; Le, Kim; Gaucher, Jonathan; Carlier, Marie-France; Romet-Lemonne, Guillaume

doi:10.1529/biophysj.107.118653

Cited by 49 publications

(49 citation statements)

References 47 publications

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“…To our knowledge, this is the first time that such a reconstituted active composite system has been described. Our results suggest that the actin cortex may influence plasma membrane organization not only through the interaction of lipids and proteins with a stable actin meshwork (9,42) or polymerizing actin (40,44), but also through the flows of actin filaments generated by myosininduced stresses. Importantly, the flows of myosin-driven short actin filaments influence only those membrane components (proteins and lipids) that can bind to actin such as HYE, whereas the mutant HYE(R579A) and the inert lipid RhoPE do not show a change in their dynamics.…”

Section: Discussionmentioning

confidence: 86%

“…A series of in vitro studies have explored the organization of confined, dynamic filaments (both actin and microtubules) (34)(35)(36)(37)(38)(39) or the role of actin architecture on membrane organization (40)(41)(42)(43)(44)(45)(46). Indeed, these studies have yielded insights into the nontrivial emergent configurations that mixtures of polar filaments and motors can adopt when fueled by ATP (34)(35)(36)(37), in particular constitutively remodeling steady states that display characteristics of active mechanics (38,39,47).…”

mentioning

confidence: 99%

“…In those experiments, the apparent advection of membrane bound ActA or WASP toward the site of actin polymerization is mainly due to the change in binding affinity of WASP to actin through Arp2/3 (44) and the spherical geometry resulting in the drag of actin to one pole of the vesicle after symmetry break of the actin shell. That this dynamic process changes the bulk properties of the bilayer, namely the critical temperature of a phase-separating lipid bilayer, was shown by Liu and Fletcher (40) when the actin nucleator N-WASP was connected to a lipid species (PIP 2 ) that was capable of partitioning into one of the two phases.…”

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confidence: 99%

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Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

Köster

Husain

Iljazi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

142

152

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The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actinbinding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phasesegregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.T he cell surface mediates interactions between the cell and the outside world by serving as the site for signal transduction. It also facilitates the uptake and release of cargo and supports adhesion to substrates. These diverse roles require that the cell surface components involved in each function are spatially and temporally organized into domains spanning a few nanometers (nanoclusters) to several micrometers (microdomains). The cell surface itself may be considered as a fluid-lipid bilayer wherein proteins are embedded (1). In the living cell, this multicomponent system is supported by an actin cortex, composed of a branched network of actin and a collection of filaments (2-4).Current models of membrane organization fall into three categories: those invoking lipid-lipid and lipid-protein interactions in the plasma membrane [e.g., the fluid mosaic model (1, 5) and the lipid raft hypothesis (6)], or those that appeal to the membrane-associated actin cortex (e.g., the picket fence model) (7), or a combination of these (8, 9). Although these models based on thermodynamic equilibrium principles have successfully explained the organization and dynamics of a range of membrane components and molecules, there is a growing class of phenomena that appears inconsistent with chemical and thermal equilibrium, which might warrant a different explanation. These include aspects of the organization and dynamics of outer leaflet...

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Section: Discussionmentioning

confidence: 86%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

Köster

Husain

Iljazi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

142

152

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“…The isolated C domain of SCAR VCA binds G-actin like a WH2 domain using its N-terminal ␣-helix (33). The VC domain of N-WASP binds the Arp2/3 complex via the C domain and branches filaments with a lower efficiency than VCA (13). The CA domain binds the Arp2/3 complex at two sites of different affinities (27), and two Alexa 488-labeled VCAs co-sediment with Arp2/3 in analytical centrifugation studies (26).…”

mentioning

confidence: 99%

Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Gaucher

Mauge

Didry

et al. 2012

Journal of Biological Chemistry

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“…[13] In contrast, there have been relatively few reports on the reconstitution of dynamically self-assembled cytoskeletal-like structures within protocell models. [14][15][16][17][18] Whilst these studies represent important steps towards the confirmation and refinement of biological mechanisms of cytoskeletal assembly and organization, the possibility of mimicking the cytoskeleton synthetically, that is, using the reversible noncovalent supramolecular assembly of nonbiological components, has not, to the best of our knowledge been explicitly explored. In contrast to previous studies on the encapsulation of polymer gels in vesicles, [20][21][22][23] herein we report the noncovalent assembly of a supramolecular network within the interior of phospholipid vesicles using the in situ enzymatic dephosphorylation of small-molecule, amino-acid-based components.…”

mentioning

confidence: 99%

Cytoskeletal‐like Supramolecular Assembly and Nanoparticle‐Based Motors in a Model Protocell

Kumar

Patil

et al. 2011

Angew Chem Int Ed

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Reconstitution of cellular functions within synthetic constructs such as lipid or surfactant vesicles represents a central objective of protocell research. [1][2][3][4] Realizing this goal has important implications for the design and construction of soft, water-based compartmentalized systems with lifelike properties, and should provide unique opportunities in synthetic biology and bionanotechnology, [5] as well as research on the origins of life.[6] Recent studies have developed synthetic protocell models based on vesicles capable of polymerase chain reaction (PCR) induced DNA amplification, [7] gene expression of single components [8] or cascading networks, [9,10] biochemical transformations, [11] poly(adenylic acid) synthesis, [12] or RNA replication. [13] In contrast, there have been relatively few reports on the reconstitution of dynamically self-assembled cytoskeletal-like structures within protocell models. [14][15][16][17][18] Whilst these studies represent important steps towards the confirmation and refinement of biological mechanisms of cytoskeletal assembly and organization, the possibility of mimicking the cytoskeleton synthetically, that is, using the reversible noncovalent supramolecular assembly of nonbiological components, has not, to the best of our knowledge been explicitly explored. In contrast to previous studies on the encapsulation of polymer gels in vesicles, [20][21][22][23] herein we report the noncovalent assembly of a supramolecular network within the interior of phospholipid vesicles using the in situ enzymatic dephosphorylation of small-molecule, amino-acid-based components. Moreover, we exploit supramolecular gelation within the vesicles to generate robust, soft microcompartments capable of chemically derived self-propulsion arising from the platinum-nanoparticle-catalyzed decomposition of hydrogen peroxide (H 2 O 2 ). [24][25][26][27][28][29][30] Aqueous suspensions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles comprising supramolecular hydrogel interiors were prepared using an inverted emulsion method [31,32] combined with in situ alkaline phosphatase-mediated dephosphorylation of N-fluorenylmethylcarbonyl-tyrosine-(O)-phosphate (Fmoc-TyrP; see Scheme 1 in the Supporting Information).[33] As described previously, [33,34] the latter process results in the noncovalent selfassembly of Fmoc-Tyr molecules into bundles of supramolecular nanofilaments that exhibit solid-like viscoelastic properties (see Figure S1 in the Supporting Information), and can be reversibly disassembled by heating the hydrogels above the gel-sol transition temperature (typically around 45 8C). Optical microscopy studies indicated that the amino acid/enzymecontaining hydrogelled vesicles were not aggregated, spherical in shape, 1-25 mm in diameter, and structurally stable when dispersed in water for several months at room temperature, or freeze-dried and investigated by SEM (Figure 1 a,b). The hydrogelled vesicles could be sedimented and rehydrated by slow centrifugation at 120 g for 30 min...

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Arp2/3 Controls the Motile Behavior of N-WASP-Functionalized GUVs and Modulates N-WASP Surface Distribution by Mediating Transient Links with Actin Filaments

Cited by 49 publications

References 47 publications

Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

Interactions of Isolated C-terminal Fragments of Neural Wiskott-Aldrich Syndrome Protein (N-WASP) with Actin and Arp2/3 Complex

Cytoskeletal‐like Supramolecular Assembly and Nanoparticle‐Based Motors in a Model Protocell

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