Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein.

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F-actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.

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Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

The Journal of Cell Biology ◽

10.1083/jcb.139.5.1243 ◽

1997 ◽

Vol 139 (5) ◽

pp. 1243-1253 ◽

Cited By ~ 45

Author(s):

R.J. Eddy ◽

J. Han ◽

J.S. Condeelis

Keyword(s):

Actin Cytoskeleton ◽

Actin Polymerization ◽

De Novo ◽

Leading Edge ◽

Actin Assembly ◽

Directed Movement ◽

Capping Protein ◽

End Capping

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

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A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

Molecular Biology of the Cell ◽

10.1091/mbc.e02-05-0300 ◽

2003 ◽

Vol 14 (1) ◽

pp. 118-128 ◽

Cited By ~ 34

Author(s):

Roberta Hopmann ◽

Kathryn G. Miller

Keyword(s):

Actin Cytoskeleton ◽

Actin Filament ◽

Actin Polymerization ◽

Genetic Interaction ◽

Actin Binding ◽

Actin Assembly ◽

Loss Of Function ◽

Capping Protein ◽

Filament Dynamics

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of thecpb loss-of-function phenotype. The interaction betweencpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.

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Non Diaphanous Formin Delphilin Acts as a Barbed End Capping Protein

10.1101/093104 ◽

2016 ◽

Author(s):

Priyanka Dutta ◽

Sankar Maiti

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Actin Polymerization ◽

Pdz Domains ◽

Capping Protein ◽

C Terminus ◽

End Capping

ABSTRACTFormins are important for actin polymerization. Delphilin is a unique formin having PDZ domains and FH1, FH2 domains at its N and C terminus respectively. In this study we observed that Delphilin binds to actin filaments, and have negligible actin filament polymerizing activity. Delphilin inhibits actin filament elongation like barbed end capping protein CapZ. In vitro, Delphilin stabilized actin filaments by inhibiting actin filament depolymerisation. Therefore, our study demonstrates Delphilin as an actin-filament capping protein.

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Visualization and Molecular Analysis of Actin Assembly in Living Cells

The Journal of Cell Biology ◽

10.1083/jcb.143.7.1919 ◽

1998 ◽

Vol 143 (7) ◽

pp. 1919-1930 ◽

Cited By ~ 127

Author(s):

Dorothy A. Schafer ◽

Matthew D. Welch ◽

Laura M. Machesky ◽

Paul C. Bridgman ◽

Shelley M. Meyer ◽

...

Keyword(s):

Actin Filament ◽

Eukaryotic Cell ◽

Cell Periphery ◽

Actin Assembly ◽

Cortical Actin ◽

Lim Kinase ◽

Permeabilized Cells ◽

Capping Protein ◽

Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

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Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽

10.1182/blood.v97.9.2648 ◽

2001 ◽

Vol 97 (9) ◽

pp. 2648-2656 ◽

Cited By ~ 22

Author(s):

Juan A. Rosado ◽

Else M. Y. Meijer ◽

Karly Hamulyak ◽

Irena Novakova ◽

Johan W. M. Heemskerk ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Actin Polymerization ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Human Platelets ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Integrin Αiibβ3 ◽

Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

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A new form of actin assembly around the Shigella-containing vacuole regulates its intracellular niche

10.1101/2020.03.12.988097 ◽

2020 ◽

Author(s):

Sonja Kühn ◽

John Bergqvist ◽

Laura Barrio ◽

Stephanie Lebreton ◽

Chiara Zurzolo ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Actin Polymerization ◽

De Novo ◽

Shigella Flexneri ◽

Host Cells ◽

Host Recognition ◽

Actin Assembly ◽

Actin Structure ◽

New Form ◽

Structure And Functions

SUMMARYThe enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterial containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host Cdc42, Toca-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV. This represents a novel microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon protects Shigella’s niche from canonical maturation or host recognition.

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Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

Molecular Biology of the Cell ◽

10.1091/mbc.e09-07-0596 ◽

2010 ◽

Vol 21 (1) ◽

pp. 165-176 ◽

Cited By ~ 235

Author(s):

Farida Korobova ◽

Tatyana Svitkina

Keyword(s):

Electron Microscopy ◽

Actin Cytoskeleton ◽

Actin Filament ◽

Dendritic Spines ◽

Dendritic Spine ◽

Myosin Ii ◽

Capping Protein ◽

Dominant Feature ◽

Presynaptic Boutons ◽

Spine Morphogenesis

Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.

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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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A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin.

The Journal of Cell Biology ◽

10.1083/jcb.99.3.994 ◽

1984 ◽

Vol 99 (3) ◽

pp. 994-1001 ◽

Cited By ~ 33

Author(s):

H Hosoya ◽

I Mabuchi

Keyword(s):

Sea Urchin ◽

Actin Filament ◽

Actin Polymerization ◽

Initial Rate ◽

Gel Filtration ◽

Molar Ratio ◽

Capping Protein ◽

Sea Urchin Egg ◽

Rate Of Polymerization ◽

Hemicentrotus Pulcherrimus

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.

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