A Lipid Bound Actin Meshwork Organizes Liquid Phase Separation in Model Membranes

Honigmann, Alf; Sadeghi, Sina; Keller, Jan; Hell, Stefan W.; Eggeling, Christian; Vink, R. L. C.

doi:10.1016/j.bpj.2013.11.3506

Cited by 60 publications

(97 citation statements)

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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: 85%

“…After addition of polymerized actin, myosin II was added to the chamber in ATP-containing buffers and appropriate salt conditions to generate minifilaments (51). Phasesegregating lipid mixtures of multiple lipid species and cholesterol were prepared in chloroform, deposited on experimental chambers formed on freshly cleaved mica supports (∼100-μm thickness), and rehydrated for bilayer formation (42). Fluorescence imaging was carried out on a custom-designed total internal reflection fluorescence microscope, and electron microscopy on myosin II filaments was performed following established protocols (71).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

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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.

147

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: 85%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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.

147

152

View full text Add to dashboard Cite

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“…Indeed, syntaxin-1 molecules have been shown to concentrate under docked vesicles before fusion, suggesting that a pool of plasma membrane syntaxin-1 can be recruited by an unknown mechanism under SVs 43 . Another possible contributing factor could involve translocated actin exerting an effect on the domain organization of the plasma membrane 44 leading to efficient docking as previously speculated 45 . Consistent with earlier key reports using TIRF microscopy, we also found that both chromaffin cell and PC12 cell SVs become restricted in their lateral movement following stimulation, indicating that they are undergoing docking 34,35 .…”

Section: Discussionmentioning

confidence: 93%

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking

et al. 2015

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In neurosecretory cells, secretory vesicles (SVs) undergo Ca 2 þ -dependent fusion with the plasma membrane to release neurotransmitters. How SVs cross the dense mesh of the cortical actin network to reach the plasma membrane remains unclear. Here we reveal that, in bovine chromaffin cells, SVs embedded in the cortical actin network undergo a highly synchronized transition towards the plasma membrane and Munc18-1-dependent docking in response to secretagogues. This movement coincides with a translocation of the cortical actin network in the same direction. Both effects are abolished by the knockdown or the pharmacological inhibition of myosin II, suggesting changes in actomyosin-generated forces across the cell cortex. Indeed, we report a reduction in cortical actin network tension elicited on secretagogue stimulation that is sensitive to myosin II inhibition. We reveal that the cortical actin network acts as a 'casting net' that undergoes activity-dependent relaxation, thereby driving tethered SVs towards the plasma membrane where they undergo Munc18-1-dependent docking.

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“…We also measured fluxes between the cytoplasm and the nucleus and the delayed communication caused by the passage through the nuclear pore complex [7,16]. Other successful applications of the pCF approach have been reported by other labs [11,[17][18][19][20]. However, all previous work was aimed at detecting the existence of correlated fluctuations between two distant points without constructing an image of all correlated points.…”

Section: Introductionmentioning

confidence: 99%

Visualization of barriers and obstacles to molecular diffusion in live cells by spatial pair-cross-correlation in two dimensions

Malacrida

Hedde

Ranjit

et al. 2017

Biomed. Opt. Express

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Despite recent advances in optical super-resolution, we lack a method that can visualize the path followed by diffusing molecules in the cytoplasm or in the nucleus of cells. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level by averaging the behavior of many molecules over time at a single spot, thus achieving very good statistics but at only one point in the cell. Earlier image-based methods including raster-scan and spatiotemporal image correlation need spatial averaging over relatively large areas, thus compromising spatial resolution. Here, we use spatial pair-crosscorrelation in two dimensions (2D-pCF) to obtain relatively high resolution images of molecular diffusion dynamics and transport in live cells. The 2D-pCF method measures the time for a particle to go from one location to another by cross-correlating the intensity fluctuations at specific points in an image. Hence, a visual map of the average path followed by molecules is created.

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A Lipid Bound Actin Meshwork Organizes Liquid Phase Separation in Model Membranes

Cited by 60 publications

References 0 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

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking

Visualization of barriers and obstacles to molecular diffusion in live cells by spatial pair-cross-correlation in two dimensions

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