A direct proof that sole actin dynamics drive membrane deformations

AbstractCell membrane deformations are crucial for proper cell function. Specialized protein assemblies initiate inward or outward membrane deformations that turn into, for example, filopodia or endocytic intermediates. Actin dynamics and actin-binding proteins are involved in this process, although their detailed role remains controversial. We show here that a dynamic, branched actin network is sufficient, in absence of any membrane-associated proteins, to initiate both inward and outward membrane deformation. With actin polymerization triggered at the membrane of liposomes, we produce inward filopodia-like structures at low tension, while outward endocytosis-like structures are robustly generated regardless of tension. Our results are reminiscent of endocytosis in mammalian cells, where actin polymerization forces are required when membrane tension is increased, and in yeast, where they are always required to overcome the opposing turgor pressure. By combining experimental observations with physical modeling, we propose a mechanism for actin-driven endocytosis under high tension or high pressure conditions.

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Platelet adhesion: Structural and functional diversity of short dystrophin and utrophins in the formation of dystrophinassociated-protein complexes related to actin dynamics

Thrombosis and Haemostasis ◽

10.1160/th04-11-0765 ◽

2005 ◽

Vol 94 (12) ◽

pp. 1203-1212 ◽

Cited By ~ 18

Author(s):

Doris Cerecedo ◽

Dalila Martínez-Rojas ◽

Oscar Chávez ◽

Francisco Martínez-Pérez ◽

Francisco García-Sierra ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Platelet Adhesion ◽

Protein Complexes ◽

Human Platelets ◽

Actin Dynamics ◽

Actin Binding ◽

Dynamic Cell ◽

Associated Proteins ◽

Coated Surfaces ◽

Dynamic Structures

SummaryPlatelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71d, as well as the Up71 isoform and the dystrophin-associated proteins, α and β-dystrobrevins. Distribution of Dp71d/Dp71Δ110 m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71Δ110 m ~DAPC and Up400/Up71~DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71Δ110 m ~DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71Δ110 m ~DAPC and Up400/Up71~DAPC in the biological roles of the platelets is discussed.

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Actin Cytoskeleton Regulates Calcium Dynamics and NFAT Nuclear Duration

Molecular and Cellular Biology ◽

10.1128/mcb.24.4.1628-1639.2004 ◽

2004 ◽

Vol 24 (4) ◽

pp. 1628-1639 ◽

Cited By ~ 56

Author(s):

Fabiola V. Rivas ◽

James P. O'Keefe ◽

Maria-Luisa Alegre ◽

Thomas F. Gajewski

Keyword(s):

T Cell ◽

Actin Cytoskeleton ◽

T Cell Activation ◽

Actin Polymerization ◽

Cell Activation ◽

Cell Function ◽

Immunological Synapse ◽

Interleukin 2 ◽

Actin Dynamics ◽

Activated T Cells

ABSTRACT T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca2+ ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca2+-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.

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Lymphocyte-Specific Protein 1 Expression in Eukaryotic Cells Reproduces the Morphologic and Motile Abnormality of NAD 47/89 Neutrophils

Blood ◽

10.1182/blood.v91.12.4786 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4786-4795 ◽

Cited By ~ 23

Author(s):

Thomas H. Howard ◽

John Hartwig ◽

Casey Cunningham

Keyword(s):

Actin Polymerization ◽

Cell Function ◽

Specific Protein ◽

Polymorphonuclear Neutrophils ◽

Actin Binding ◽

Actin Network ◽

Cell Surfaces ◽

Actin Bundles ◽

Mixed Polarity ◽

Low Levels

Abstract Despite its name, the actin-binding protein lymphocyte-specific protein1 (LSP1) is found in all hematopoetic cells, and yet its role in cell function remains unclear. Recently, LSP1 was identified as the 47-kD protein overexpressed in the polymorphonuclear neutrophils of patients with a rare neutrophil disorder, neutrophil actin dysfunction with abnormalities of 47-kD and 89-kD proteins (NAD 47/89). These neutrophils are immotile, defective in actin polymerization in response to agonists, and display distinctive, fine, “hairlike” F-actin-rich projections on their cell surfaces. We now show that overexpression of LSP1 produces F-actin bundles that are likely responsible for the morphologic and motile abnormalities characteristic of the NAD 47/89 phenotype. Coincident with LSP1 overexpression, cells from each of several different eukaryotic lines, including a highly motile human melanoma line, develop hairlike surface projections that branch distinctively and contain F-actin and LSP1. The hairlike projections are supported at their core by thick actin bundles, composed of actin filaments of mixed polarity, which periodically anastomose to generate a branching structure. The motility of the melanoma cells is inhibited even at low levels of LSP1 expression. Therefore, these studies show that overexpression of LSP1 alone can recreate the morphologic and motile defects seen in NAD 47/89 and suggest that LSP1 is distinct from other known actin binding proteins in its effect on F-actin network structure.

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Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009167117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19904-19913 ◽

Cited By ~ 2

Author(s):

Caner Akıl ◽

Linh T. Tran ◽

Magali Orhant-Prioux ◽

Yohendran Baskaran ◽

Edward Manser ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Actin Filament ◽

Calcium Binding ◽

Mammalian Cells ◽

Actin Polymerization ◽

Calcium Release ◽

Duplication Event ◽

Cellular Level ◽

Actin Dynamics

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.

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Changes in Vasodilator-Stimulated Phosphoprotein Phosphorylation, Profilin-1, and Cofilin-1 in Accreta and Protection by DHA

Reproductive Sciences ◽

10.1177/1933719118792095 ◽

2018 ◽

Vol 26 (6) ◽

pp. 757-765 ◽

Cited By ~ 1

Author(s):

Mehboob Ali ◽

Lynette K. Rogers ◽

Kathryn M. Heyob ◽

Catalin S. Buhimschi ◽

Irina A. Buhimschi

Keyword(s):

Actin Polymerization ◽

Cellular Proliferation ◽

Placenta Accreta ◽

Gestational Trophoblastic Disease ◽

Actin Dynamics ◽

Actin Binding ◽

Migration And Invasion ◽

Aberrant Expression ◽

Vasp Phosphorylation ◽

And Migration

Accreta and gestational trophoblastic disease (ie, choriocarcinoma) are placental pathologies characterized by hyperproliferative and invasive trophoblasts. Cellular proliferation, migration, and invasion are heavily controlled by actin-binding protein (ABP)-mediated actin dynamics. The ABP vasodilator-stimulated phosphoprotein (VASP) carries key regulatory role. Profilin-1, cofilin-1, and VASP phosphorylated at Ser157 (pVASP-S157) and Ser239 (pVASP-S239) are ABPs that regulate actin polymerization and stabilization and facilitate cell metastases. Docosahexaenoic acid (DHA) inhibits cancer cell migration and proliferation. We hypothesized that analogous to malignant cells, ABPs regulate these processes in extravillous trophoblasts (EVTs), which exhibit aberrant expression in placenta accreta. Placental–myometrial junction biopsies of histologically confirmed placenta accreta had significantly increased immunostaining levels of cofilin-1, VASP, pVASP-S239, and F-actin. Treatment of choriocarcinoma-derived trophoblast (BeWo) cells with DHA (30 µM) for 24 hours significantly suppressed proliferation, migration, and pVASP-S239 levels and altered protein profiles consistent with increased apoptosis. We concluded that in accreta changes in the ABP expression profile were a response to restore homeostasis by counteracting the hyperproliferative and invasive phenotype of the EVT. The observed association between VASP phosphorylation, apoptosis, and trophoblast proliferation and migration suggest that DHA may offer a therapeutic solution for conditions where EVT is hyperinvasive.

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EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

The Journal of Cell Biology ◽

10.1083/jcb.200212057 ◽

2003 ◽

Vol 160 (3) ◽

pp. 399-407 ◽

Cited By ~ 92

Author(s):

Raymond S. Maul ◽

Yuhong Song ◽

Kurt J. Amann ◽

Sachi C. Gerbin ◽

Thomas D. Pollard ◽

...

Keyword(s):

Actin Filament ◽

Actin Polymerization ◽

Binding Activity ◽

Stress Fibers ◽

Actin Dynamics ◽

Actin Binding ◽

Membrane Ruffling ◽

Cross Links ◽

As Stress ◽

Kinetics Of

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

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Lymphocyte-Specific Protein 1 Expression in Eukaryotic Cells Reproduces the Morphologic and Motile Abnormality of NAD 47/89 Neutrophils

Blood ◽

10.1182/blood.v91.12.4786.412k25_4786_4795 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4786-4795 ◽

Cited By ~ 1

Author(s):

Thomas H. Howard ◽

John Hartwig ◽

Casey Cunningham

Keyword(s):

Actin Polymerization ◽

Cell Function ◽

Specific Protein ◽

Polymorphonuclear Neutrophils ◽

Actin Binding ◽

Actin Network ◽

Cell Surfaces ◽

Actin Bundles ◽

Mixed Polarity ◽

Low Levels

Despite its name, the actin-binding protein lymphocyte-specific protein1 (LSP1) is found in all hematopoetic cells, and yet its role in cell function remains unclear. Recently, LSP1 was identified as the 47-kD protein overexpressed in the polymorphonuclear neutrophils of patients with a rare neutrophil disorder, neutrophil actin dysfunction with abnormalities of 47-kD and 89-kD proteins (NAD 47/89). These neutrophils are immotile, defective in actin polymerization in response to agonists, and display distinctive, fine, “hairlike” F-actin-rich projections on their cell surfaces. We now show that overexpression of LSP1 produces F-actin bundles that are likely responsible for the morphologic and motile abnormalities characteristic of the NAD 47/89 phenotype. Coincident with LSP1 overexpression, cells from each of several different eukaryotic lines, including a highly motile human melanoma line, develop hairlike surface projections that branch distinctively and contain F-actin and LSP1. The hairlike projections are supported at their core by thick actin bundles, composed of actin filaments of mixed polarity, which periodically anastomose to generate a branching structure. The motility of the melanoma cells is inhibited even at low levels of LSP1 expression. Therefore, these studies show that overexpression of LSP1 alone can recreate the morphologic and motile defects seen in NAD 47/89 and suggest that LSP1 is distinct from other known actin binding proteins in its effect on F-actin network structure.

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N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

AJP Cell Physiology ◽

10.1152/ajpcell.00426.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1562-C1566 ◽

Cited By ~ 32

Author(s):

Christopher J. Guerriero ◽

Ora A. Weisz

Keyword(s):

Membrane Trafficking ◽

Actin Polymerization ◽

Cell Function ◽

Actin Dynamics ◽

Cellular Functions ◽

Intact Cells ◽

Atp Levels ◽

Dependent Inhibition

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo.

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Burkholderia pseudomalleitype III secreted protein BipC: role in actin modulation and translocation activities required for the bacterial intracellular lifecycle

PeerJ ◽

10.7717/peerj.2532 ◽

2016 ◽

Vol 4 ◽

pp. e2532 ◽

Cited By ~ 5

Author(s):

Wen Tyng Kang ◽

Kumutha Malar Vellasamy ◽

Lakshminarayanan Rajamani ◽

Roger W. Beuerman ◽

Jamuna Vadivelu

Keyword(s):

Actin Polymerization ◽

Cell Interaction ◽

Binding Activity ◽

Host Cells ◽

Actin Dynamics ◽

Actin Binding ◽

Virulence Determinants ◽

Secretion Systems ◽

Number Of Patients

Melioidosis, an infection caused by the facultative intracellular pathogenBurkholderia pseudomallei, has been classified as an emerging disease with the number of patients steadily increasing at an alarming rate.B. pseudomalleipossess various virulence determinants that allow them to invade the host and evade the host immune response, such as the type III secretion systems (TTSS). The products of this specialized secretion system are particularly important for theB. pseudomalleiinfection. Lacking in one or more components of the TTSS demonstrated different degrees of defects in the intracellular lifecycle ofB. pseudomallei. Further understanding the functional roles of proteins involved inB. pseudomalleiTTSS will enable us to dissect the enigma ofB. pseudomallei-host cell interaction. In this study, BipC (a translocator), which was previously reported to be involved in the pathogenesis ofB. pseudomallei, was further characterized using the bioinformatics and molecular approaches. ThebipCgene, coding for a putative invasive protein, was first PCR amplified fromB. pseudomalleiK96243genomic DNA and cloned into an expression vector for overexpression inEscherichia coli. The soluble protein was subsequently purified and assayed for actin polymerization and depolymerization. BipC was verified to subvert the host actin dynamics as demonstrated by the capability to polymerize actinin vitro. Homology modeling was also attempted to predict the structure of BipC. Overall, our findings identified that the protein encoded by thebipCgene plays a role as an effector involved in the actin binding activity to facilitate internalization ofB. pseudomalleiinto the host cells.

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TetraThymosinβ Is Required for Actin Dynamics in Caenorhabditis elegans and Acts via Functionally Different Actin-binding Repeats

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0225 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4735-4748 ◽

Cited By ~ 28

Author(s):

Marleen Van Troys ◽

Kanako Ono ◽

Daisy Dewitte ◽

Veronique Jonckheere ◽

Natalie De Ruyck ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filament ◽

Actin Polymerization ◽

Actin Dynamics ◽

Actin Binding ◽

In Vitro Assays ◽

Filamentous Actin ◽

Lethal Mutant ◽

Interaction Sites

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinβ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinβ has four repeats, each related to the actin monomer-sequestering protein thymosinβ 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinβ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinβ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinβ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinβ is of crucial importance at specific developmental stages requiring actin polymerization.

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