Adaptive actin organization counteracts elevated membrane tension to ensure robust endocytosis

AbstractClathrin-mediated endocytosis (CME) remains robust despite variations in plasma membrane tension. Actin assembly-mediated force generation becomes essential for CME under high membrane tension, but the underlying mechanisms are not understood. We investigated actin network ultrastructure at each stage of CME by super-resolution imaging. Actin and N-WASP spatial organization indicate that polymerization initiates at the base of clathrin-coated pits and that the actin network then grows away from the plasma membrane. Actin network organization is not tightly coupled to endocytic clathrin coat growth and deformation. Membrane tension-dependent changes in actin organization explain this uncoupling. Under elevated membrane tension, CME dynamics slow down and the actin network grows higher, resulting in greater coverage of the clathrin coat. This adaptive mechanism is especially crucial during the initial membrane curvature-generating stages of CME. Our findings reveal that adaptive force generation by the actin network ensures robust CME progression despite changes in plasma membrane tension.Highlights-Clathrin coat surface area and actin ultra-structure adapt to elevated membrane tension.-The actin network is nucleated at the base of the clathrin-coated pit and grows upward.-Actin ultra-structural organization is not tightly coupled to CME progression.-Actin force generation is required earlier in CME progression under elevated membrane tension.SummaryKaplan et al. revealed that actin assembly compensates for changes in plasma membrane tension by an adaptive force generating mechanism to ensure robust endocytosis. Under elevated membrane tension the network grows deeper, even in early endocytic stages, from the base upward.

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Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

Essays in Biochemistry ◽

10.1042/bse0570189 ◽

2015 ◽

Vol 57 ◽

pp. 189-201 ◽

Cited By ~ 13

Author(s):

Jay Shankar ◽

Cecile Boscher ◽

Ivan R. Nabi

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Cancer Cell ◽

Cellular Response ◽

Spatial Organization ◽

Essential Feature ◽

Cell Signalling ◽

Coated Pits ◽

Signalling Molecules ◽

Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

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Type I myosins anchor actin assembly to the plasma membrane during clathrin-mediated endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.201810005 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1138-1147 ◽

Cited By ~ 13

Author(s):

Ross T.A. Pedersen ◽

David G. Drubin

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Force Generation ◽

Type I ◽

Actin Assembly ◽

Filamentous Actin ◽

Actin Networks ◽

Wide Range ◽

Assembly Factors ◽

Myosin Motors

The actin cytoskeleton generates forces on membranes for a wide range of cellular and subcellular morphogenic events, from cell migration to cytokinesis and membrane trafficking. For each of these processes, filamentous actin (F-actin) interacts with membranes and exerts force through its assembly, its associated myosin motors, or both. These two modes of force generation are well studied in isolation, but how they are coordinated in cells is mysterious. During clathrin-mediated endocytosis, F-actin assembly initiated by the Arp2/3 complex and several proteins that compose the WASP/myosin complex generates the force necessary to deform the plasma membrane into a pit. Here we present evidence that type I myosin is the key membrane anchor for endocytic actin assembly factors in budding yeast. By mooring actin assembly factors to the plasma membrane, this myosin organizes endocytic actin networks and couples actin-generated forces to the plasma membrane to drive invagination and scission. Through this unexpected mechanism, myosin facilitates force generation independent of its motor activity.

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Mechanics of Curved Plasma Membrane Vesicles: Resting Shapes, Membrane Curvature, and In-Plane Shear Elasticity

Journal of Biomechanical Engineering ◽

10.1115/1.1865197 ◽

2004 ◽

Vol 127 (2) ◽

pp. 229-236 ◽

Cited By ~ 14

Author(s):

Tadashi Kosawada ◽

Kohji Inoue ◽

Geert W. Schmid-Schönbein

Keyword(s):

Plasma Membrane ◽

Cell Membrane ◽

Membrane Curvature ◽

Membrane Tension ◽

Coated Pits ◽

Plane Shear ◽

Plasmalemmal Vesicles ◽

Cell Functions ◽

Outer Domain ◽

Shear Elasticity

Highly curved cell membrane structures, such as plasmalemmal vesicles (caveolae) and clathrin-coated pits, facilitate many cell functions, including the clustering of membrane receptors and transport of specific extracellular macromolecules by endothelial cells. These structures are subject to large mechanical deformations when the plasma membrane is stretched and subject to a change of its curvature. To enhance our understanding of plasmalemmal vesicles we need to improve the understanding of the mechanics in regions of high membrane curvatures. We examine here, theoretically, the shapes of plasmalemmal vesicles assuming that they consist of three membrane domains: an inner domain with high curvature, an outer domain with moderate curvature, and an outermost flat domain, all in the unstressed state. We assume the membrane properties are the same in these domains with membrane bending elasticity as well as in-plane shear elasticity. Special emphasis is placed on the effects of membrane curvature and in-plane shear elasticity on the mechanics of vesicle during unfolding by application of membrane tension. The vesicle shapes were computed by minimization of bending and in-plane shear strain energy. Mechanically stable vesicles were identified with characteristic membrane necks. Upon stretch of the membrane, the vesicle necks disappeared relatively abruptly leading to membrane shapes that consist of curved indentations. While the resting shape of vesicles is predominantly affected by the membrane spontaneous curvatures, the membrane shear elasticity (for a range of values recorded in the red cell membrane) makes a significant contribution as the vesicle is subject to stretch and unfolding. The membrane tension required to unfold the vesicle is sensitive with respect to its shape, especially as the vesicle becomes fully unfolded and approaches a relative flat shape.

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Wnt5A-Mediated Actin Organization Regulates Host Response to Bacterial Pathogens and Non-Pathogens

Frontiers in Immunology ◽

10.3389/fimmu.2020.628191 ◽

2021 ◽

Vol 11 ◽

Author(s):

Suborno Jati ◽

Soham Sengupta ◽

Malini Sen

Keyword(s):

Conditioned Medium ◽

Host Response ◽

Bacterial Pathogens ◽

The State ◽

Actin Assembly ◽

Actin Network ◽

Actin Organization ◽

E Coli ◽

Interspecies Variability

Wnt5A signaling facilitates the killing of several bacterial pathogens, but not the non-pathogen E. coli DH5α. The basis of such pathogen vs. non-pathogen distinction is unclear. Accordingly, we analyzed the influence of Wnt5A signaling on pathogenic E. coli K1 in relation to non-pathogenic E. coli K12-MG1655 and E. coli DH5α eliminating interspecies variability from our study. Whereas cell internalized E. coli K1 disrupted cytoskeletal actin organization and multiplied during Wnt5A depletion, rWnt5A mediated activation revived cytoskeletal actin assembly facilitating K1 eradication. Cell internalized E. coli K12-MG1655 and E. coli DH5α, which did not perturb actin assembly appreciably, remained unaffected by rWnt5A treatment. Phagosomes prepared separately from Wnt5A conditioned medium treated K1 and K12-MG1655 infected macrophages revealed differences in the relative levels of actin and actin network promoting proteins, upholding that the Wnt5A-Actin axis operates differently for internalized pathogen and non-pathogen. Interestingly, exposure of rWnt5A treated K1 and K12-MG1655/DH5α infected macrophages to actin assembly inhibitors reversed the scenario, blocking killing of K1, yet promoting killing of both K12-MG1655 and DH5α. Taken together, our study illustrates that the state of activation of the Wnt5A/Actin axis in the context of the incumbent bacteria is crucial for directing host response to infection.

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Mechanochemical feedback and control of endocytosis and membrane tension

10.1101/201509 ◽

2017 ◽

Author(s):

Joseph Jose Thottacherry ◽

Anita Joanna Kosmalska ◽

Alberto Elosegui-Artola ◽

Susav Pradhan ◽

Sumit Sharma ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Feedback Loop ◽

Negative Feedback Loop ◽

Dependent Manner ◽

Homeostatic Regulation ◽

Membrane Tension ◽

Cellular Processes ◽

Tightly Coupled ◽

And Control

AbstractPlasma membrane tension is an important factor that regulates many key cellular processes. Membrane trafficking is tightly coupled to membrane tension and can modulate the latter by addition or removal of the membrane. However, the cellular pathway(s) involved in these processes are poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin and clathrin-independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon reduction of tension. On the other hand, inhibition of the CG pathway results in lower membrane tension, while up regulation significantly enhances membrane tension. We find that vinculin, a well-studied mechanotransducer, mediates the tension-dependent regulation of the CG pathway. Vinculin negatively regulates a key CG pathway regulator, GBF1, at the plasma membrane in a tension dependent manner. Thus, the CG pathway operates in a negative feedback loop with membrane tension which leads to a homeostatic regulation of membrane tension.

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Nanoscale indentation of plasma membrane establishes a contractile actomyosin scaffold through selective activation of the Amphiphysin-Rho1-Dia/DAAM and Rok pathway

10.1101/2020.08.04.236125 ◽

2020 ◽

Author(s):

Tushna Kapoor ◽

Pankaj Dubey ◽

Seema Shirolikar ◽

Krishanu Ray

Keyword(s):

Plasma Membrane ◽

Cell Adhesion ◽

Somatic Cell ◽

Cultured Cells ◽

Cell Signalling ◽

Molecular Networks ◽

List Type ◽

Actin Assembly ◽

Mechanical Bending ◽

Somatic Cyst Cell

AbstractNanoscale bending of plasma membrane increases cell adhesion, induces cell-signalling, triggers F-actin assembly and endocytosis in tissue-cultured cells. The underlying mechanisms are not very well understood. Here, we show that stretching the plasma membrane of somatic cyst cell around rigid spermatid heads generates a stable, tubular endomembrane scaffold supported by contractile actomyosin. The structure resembles an actin-basket covering the bundle of spermatid heads. Genetic analysis suggests that the actomyosin organisation is nucleated exclusively by the Formins, Diaphanous and DAAM, downstream of Rho1, recruited by the Bin-Amphiphysin-Rvs (BAR)-domain protein, Amphiphysin, around the spermatid heads. Actomyosin activity at the actin-basket gathers the spermatid heads into a compact bundle and resists the invasion of the somatic cell by the intruding spermatids. These observations revealed a new response mechanism of nanoscale bending of the plasma membrane, which generates a novel cell adhesion strategy through active clamping.HighlightsStretching the plasma membrane around a spermatid head recruits Amphiphysin and Rho1.Rho1 activation triggers F-actin assembly in situ through Diaphanous and DAAM.Rho1-Rok activation assembles actomyosin scaffold around the folded plasma membrane.Contractile actomyosin enables plasma membrane to clamp onto the spermatid head.Author summarySperm released from the somatic enclosure is essential for male fertility. During differentiation, the somatic cell membrane, associated with dense F-actin scaffold, tightly hold each spermatid head before release. Kapoor et al., showed that the bending and stretching of the plasma membrane trigger the assembly of an actomyosin scaffold around the bent membrane, which clamps the spermatids together preventing the premature release and somatic cell penetration. This finding provides new insight into the molecular networks activated by mechanical bending of the plasma membrane.

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Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

Molecular Biology of the Cell ◽

10.1091/mbc.e15-03-0142 ◽

2015 ◽

Vol 26 (18) ◽

pp. 3229-3244 ◽

Cited By ~ 27

Author(s):

Yingpei He ◽

Yuan Ren ◽

Bingbing Wu ◽

Boris Decourt ◽

Aih Cheun Lee ◽

...

Keyword(s):

Tyrosine Kinases ◽

Growth Cones ◽

Network Density ◽

Retrograde Flow ◽

Actin Assembly ◽

Actin Network ◽

Cellular Mechanisms ◽

Down Regulation ◽

Actin Organization ◽

Neuronal Growth Cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.

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A viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion

10.1101/761502 ◽

2019 ◽

Author(s):

Ka Man Carmen Chan ◽

Sungmin Son ◽

Eva M. Schmid ◽

Daniel A. Fletcher

Keyword(s):

Membrane Proteins ◽

Actin Cytoskeleton ◽

Cell Fusion ◽

Conformational Changes ◽

Close Contact ◽

Cytoplasmic Domain ◽

Force Generation ◽

Actin Assembly ◽

Actin Network ◽

Cell Cell

AbstractCell-cell fusion, which is essential for tissue development and used by some viruses to form pathological syncytia, is typically driven by fusogenic membrane proteins with tall (>10 nm) ectodomains that undergo conformational changes to bring apposing membranes in close contact prior to fusion. Here we report that a viral fusogen with a short (<2 nm) ectodomain, the reptilian orthoreovirus p14, accomplishes the same task by hijacking the actin cytoskeleton. We show that the cytoplasmic domain of p14 triggers N-WASP-mediated assembly of a branched actin network, directly coupling local force generation with a short membrane-disruptive ectodomain. This work reveals that overcoming energetic barriers to cell-cell fusion does not require conformational changes of tall fusogens but can instead be driven by harnessing the host cytoskeleton.Impact StatementA viral fusogen drives cell-cell fusion by hijacking the actin machinery to directly couple actin assembly with a short fusogenic ectodomain.

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Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis

eLife ◽

10.7554/elife.49840 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 15

Author(s):

Matthew Akamatsu ◽

Ritvik Vasan ◽

Daniel Serwas ◽

Michael A Ferrin ◽

Padmini Rangamani ◽

...

Keyword(s):

Electron Tomography ◽

Force Production ◽

Multiscale Model ◽

Membrane Tension ◽

Actin Assembly ◽

Actin Network ◽

Physical Constraints ◽

Filament Assembly ◽

Attachment Sites ◽

Cryo Electron Tomography

Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.

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The phosphoinositide coincidence detector Phafin2 promotes macropinocytosis by coordinating actin organisation at forming macropinosomes

Nature Communications ◽

10.1038/s41467-021-26775-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kay Oliver Schink ◽

Kia Wee Tan ◽

Hélène Spangenberg ◽

Domenica Martorana ◽

Marte Sneeggen ◽

...

Keyword(s):

Plasma Membrane ◽

Extracellular Fluid ◽

Coincidence Detection ◽

Cancer Development ◽

Actin Assembly ◽

Actin Network ◽

Ph Domain ◽

Actin Reorganization ◽

Domain Protein ◽

Pass Through

AbstractUptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.

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