scholarly journals α-Adducin dissociates from F-actin and spectrin during platelet activation

2003 ◽  
Vol 161 (3) ◽  
pp. 557-570 ◽  
Author(s):  
Kurt L. Barkalow ◽  
Joseph E. Italiano ◽  
Denise E. Chou ◽  
Yoichiro Matsuoka ◽  
Vann Bennett ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Actin Filament ◽  
Actin Filaments ◽  
Ternary Complex ◽  
Cell Activation ◽  
Active Form ◽  
Membrane Skeleton ◽  
Protease Activated Receptor 1

Aspectrin-based skeleton uniformly underlies and supports the plasma membrane of the resting platelet, but remodels and centralizes in the activated platelet. α-Adducin, a phosphoprotein that forms a ternary complex with F-actin and spectrin, is dephosphorylated and mostly bound to spectrin in the membrane skeleton of the resting platelet at sites where actin filaments attach to the ends of spectrin molecules. Platelets activated through protease-activated receptor 1, FcγRIIA, or by treatment with PMA phosphorylate adducin at Ser726. Phosphoadducin releases from the membrane skeleton concomitant with its dissociation from spectrin and actin. Inhibition of PKC blunts adducin phosphorylation and release from spectrin and actin, preventing the centralization of spectrin that normally follows cell activation. We conclude that adducin targets actin filament ends to spectrin to complete the assembly of the resting membrane skeleton. Dissociation of phosphoadducin releases spectrin from actin, facilitating centralization of spectrin, and leads to the exposure of barbed actin filament ends that may then participate in converting the resting platelet's disc shape into its active form.

scholarly journals Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton

Blood ◽  
2010 ◽  
Vol 116 (14) ◽  
pp. 2590-2599 ◽  
Author(s):  
Jeannette D. Moyer ◽  
Roberta B. Nowak ◽  
Nancy E. Kim ◽  
Sandra K. Larkin ◽  
Luanne L. Peters ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Membrane Skeleton ◽  
Actin Dynamics ◽  
Filament Length ◽  
Western Blots ◽  
Rbc Membrane ◽  
Cellular Dehydration ◽  
Rbc Membrane Skeleton

Abstract The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.

PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration

2004 ◽  
Vol 286 (1) ◽  
pp. C153-C163 ◽  
Author(s):  
Yong Qian ◽  
Linda Corum ◽  
Qiao Meng ◽  
John Blenis ◽  
Jenny Z. Zheng ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Filament ◽  
Actin Filaments ◽  
Essential Role ◽  
Molecular Mechanisms ◽  
Chicken Embryo Fibroblast ◽  
Active Form ◽  
Specific Inhibitor ◽  
Phosphatidylinositol 3 Kinase ◽  
Pi3k Activity

This study was designed to identify the molecular mechanisms of phosphatidylinositol 3-kinase (PI3K)-induced actin filament remodeling and cell migration. Expression of active forms of PI3K, v-P3k or Myr-P3k, was sufficient to induce actin filament remodeling to lead to an increase in cell migration, as well as the activation of Akt in chicken embryo fibroblast (CEF) cells. Either the inhibition of PI3K activity using a PI3K-specific inhibitor, LY-294002, or the disruption of Akt activity restored the integrity of actin filaments in CEF cells and inhibited PI3K-induced cell migration. We also found that expression of an activated form of Akt (Myr-Akt) was sufficient to remodel actin filaments to lead to an increase in cell migration, which was unable to be inhibited by the presence of LY-294002. Furthermore, we found that p70S6K1 kinase was a downstream molecule that can mediate the effects of both PI3K and Akt on actin filaments and cell migration. Overexpression of an active form of p70S6K1 was sufficient to induce actin filament remodeling and cell migration in CEF cells, which requires Rac activity. These results demonstrate that activation of PI3K activity alone is sufficient to remodel actin filaments to increase cell migration through the activation of Akt and p70S6K1 in CEF cells.

scholarly journals Role of Diacylglycerol Kinase α in the Attenuation of Receptor Signaling

2001 ◽  
Vol 153 (1) ◽  
pp. 207-220 ◽  
Author(s):  
Miguel Angel Sanjuán ◽  
David R. Jones ◽  
Manuel Izquierdo ◽  
Isabel Mérida
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Phosphatidic Acid ◽  
T Cell Activation ◽  
Cell Activation ◽  
Active Form ◽  
Diacylglycerol Kinase ◽  
Membrane Receptors ◽  
Type I ◽  
Cell Responses

Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGKα, an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGKα-mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH2-terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acid–mediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGKα exerts on diacylglycerol-derived cell responses. This study is the first to describe DGKα as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.

scholarly journals Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

2015 ◽  
Vol 26 (9) ◽  
pp. 1699-1710 ◽  
Author(s):  
David S. Gokhin ◽  
Roberta B. Nowak ◽  
Joseph A. Khoory ◽  
Alfonso de la Piedra ◽  
Ionita C. Ghiran ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Microfluidic Channel ◽  
Biomechanical Properties ◽  
Membrane Skeleton ◽  
Single Filament ◽  
Rbc Membrane ◽  
Filament Assembly ◽  
Membrane Oscillations ◽  
Resealed Ghosts

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.

scholarly journals Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

2006 ◽  
Vol 174 (6) ◽  
pp. 851-862 ◽  
Author(s):  
Nobuhiro Morone ◽  
Takahiro Fujiwara ◽  
Kotono Murase ◽  
Rinshi S. Kasai ◽  
Hiroshi Ike ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
High Speed ◽  
Plasma Membranes ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Single Particle Tracking ◽  
Coated Pits ◽  
Cytoplasmic Surface

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

The Platelet Cytoskeleton

1993 ◽  
Vol 70 (06) ◽  
pp. 0884-0893 ◽  
Author(s):  
Joan E B Fox
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Membrane Skeleton ◽  
The Other ◽  
Thymosin Β4 ◽  
Future Studies ◽  
Cytoplasmic Actin ◽  
Platelet Aggregate

SummaryThe platelet cytoskeleton contains two actin filament-based components. One is the cytoplasmic actin filaments which fill the cytoplasm and mediate contractile events. The other is the membrane skeleton, which coats the plasma membrane and regulates properties of the membrane such as its contours and stability. In the unstimulated platelet, only 30-40% of the actin is polymerized into filaments; the rest is thought to be prevented from polymerizing by the association of thymosin β4 with monomeric actin and by the association of gelsolin with the barbed ends of pre-existing actin filaments. When platelets are activated, there is a rapid increase in actin polymerization; new filaments fill the extending filopodia and form a network at the periphery of the platelet. As a result of activation, myosin binds to cytoplasmic actin filaments, causing them to move towards the center of the platelet. As platelets aggregate, additional cytoskeletal reorganizations occur: GP Ilb-IIIa associates with adhesive ligand in a platelet aggregate; this results in the association of GP Ilb-IIIa, membrane skeleton proteins, and signaling molecules with cytoplasmic actin. Future studies should help to elucidate the significance of the cytoskeleton in regulating signal transduction events in platelets.

scholarly journals Actin filaments elongate from their membrane-associated ends

1981 ◽  
Vol 90 (2) ◽  
pp. 485-494 ◽  
Author(s):  
LG Tilney ◽  
EM Bonder ◽  
DJ DeRosier
Keyword(s):  
Plasma Membrane ◽  
Actin Filament ◽  
Actin Filaments ◽  
The Other ◽  
Dense Material ◽  
Thin Sections ◽  
Vacuole Membrane ◽  
Filament Bundles ◽  
Filament Bundle

In limulus sperm an actin filament bundle 55 mum in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end. Thin sections of sperm fixed during stages in spermatid maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane. In micrographs fo these early stages in maturation, short bundles are seen extending posteriorly from the dense material. The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end "preferred" for addition of actin monomers during polymerization. The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up. Tapering is brought about by the capping of the peripheral filaments in the bundle.

scholarly journals Identification of a membrane skeleton in platelets.

1988 ◽  
Vol 106 (5) ◽  
pp. 1525-1538 ◽  
Author(s):  
J E Fox ◽  
J K Boyles ◽  
M C Berndt ◽  
P K Steffen ◽  
L K Anderson
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Binding Protein ◽  
Amorphous Material ◽  
Three Dimensional ◽  
Amorphous Layer ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Filamentous Form ◽  
Actin Binding Protein

Platelets have previously been shown to contain actin filaments that are linked, through actin-binding protein, to the glycoprotein (GP) Ib-IX complex, GP Ia, GP IIa, and an unidentified GP of Mr 250,000 on the plasma membrane. The objective of the present study was to use a morphological approach to examine the distribution of these membrane-bound filaments within platelets. Preliminary experiments showed that the Triton X-100 lysis buffers used previously to solubilize platelets completely disrupt the three-dimensional organization of the cytoskeletons. Conditions were established that minimized these postlysis changes. The cytoskeletons remained as platelet-shaped structures. These structures consisted of a network of long actin filaments and a more amorphous layer that outlined the periphery. When Ca2+ was present, the long actin filaments were lost but the amorphous layer at the periphery remained; conditions were established in which this amorphous layer retained the outline of the platelet from which it originated. Immunocytochemical experiments showed that the GP Ib-IX complex and actin-binding protein were associated with the amorphous layer. Analysis of the amorphous material on SDS-polyacrylamide gels showed that it contained actin, actin-binding protein, and all actin-bound GP Ib-IX. Although actin filaments could not be visualized in thin section, the actin presumably was in a filamentous form because it was solubilized by DNase I and bound phalloidin. These studies show that platelets contain a membrane skeleton and suggest that it is distinct from the network of cytoplasmic actin filaments. This membrane skeleton exists as a submembranous lining that, by analogy to the erythrocyte membrane skeleton, may stabilize the plasma membrane and contribute to determining its shape.

scholarly journals Movement of the actin filament bundle in Mytilus sperm: a new mechanism is proposed

1987 ◽  
Vol 104 (4) ◽  
pp. 981-993 ◽  
Author(s):  
LG Tilney ◽  
Y Fukui ◽  
DJ DeRosier
Keyword(s):  
Plasma Membrane ◽  
Actin Filament ◽  
Cell Elongation ◽  
Actin Filaments ◽  
Blue Mussel ◽  
Optical Diffraction ◽  
Acrosomal Reaction ◽  
Filament Bundle

An actin filament bundle approximately 2-5 microns in length is present in the sperm of the blue mussel, Mytilus. In unfired sperm this bundle extends from the midpiece through a canal in the center of the nucleus to terminate on the membrane limiting the inside of the cone-shaped acrosomal vacuole. The bundle is composed of 45-65 actin filaments which are hexagonally packed and regularly cross-bridged together to form an actin paracrystal so well ordered that it has six nearly equal faces. Upon induction of the acrosomal reaction, a needle-like process is formed in a few seconds. Within this process is the actin filament bundle which appears unchanged in filament number and packing as determined by optical diffraction methods. Using fluorescein-conjugated phalloidin we were able to establish that the bundle does not change length but instead is projected anteriorly out of the midpiece and nuclear canal like an arrow. Existing mechanisms to explain this extension cannot apply. Specifically, the bundle does not increase in length (no polymerization), does not change its organization (no change in actin twist), does not change filament number (no filament sliding), and cannot move by myosin (wrong polarity). Thus we are forced to look elsewhere for a mechanism and have postulated that at least a component of this movement, or cell elongation, is the interaction of the actin filament bundle with the plasma membrane.

Cytoskeletal Proteins and Platelet Signaling

2001 ◽  
Vol 86 (07) ◽  
pp. 198-213 ◽  
Author(s):  
Joan Fox
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Critical Role ◽  
Membrane Skeleton ◽  
Cytoskeletal Proteins ◽  
Rapid Changes ◽  
Platelet Signaling ◽  
Induced Changes ◽  
Cellular Components ◽  
Filament Network

SummaryThe actin filament network fills the cytoplasm of unstimulated platelets and connects with a submembranous latticework of short cross-linked actin filaments, known as the membrane skeleton. One function of the cytoskeleton is to direct the contours of the membrane in the unstimulated platelet and the rapid changes in shape in the activated platelet. Activation-induced changes result from events such as phosphorylation or calpain-induced cleavage of cytoskeletal proteins. The specific reorganizations depend upon the combination of signals to which platelets are exposed. A second function of the cytoskeleton is to bind other cellular components; it binds signaling molecules, localizing them to specific cellular locations; it binds the plasma membrane regulating properties of the membrane, maintaining microdomains in the membrane, or regulating activities of membrane proteins. In this way, the cytoskeleton plays a critical role in regulation of spatial organizations and, thus, in the integration of cellular activities.

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