An actin barrier to resealing

2001 ◽  
Vol 114 (19) ◽  
pp. 3487-3494 ◽  
Author(s):  
Katsuya Miyake ◽  
Paul L. McNeil ◽  
Kazunori Suzuki ◽  
Rikiya Tsunoda ◽  
Naonori Sugai
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell Injury ◽  
Fusion Reaction ◽  
Physical Contact ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Cortical Array ◽  
Membrane Contacts ◽  
Local Cell

Plasma membrane disruption is a common form of cell injury in many normal biological environments, including many mammalian tissues. Survival depends on the initiation of a rapid resealing response that is mounted only in the presence of physiological levels of extracellular Ca2+. Vesicle-vesicle and vesicle-plasma membrane fusion events occurring in cortical cytoplasm surrounding the defect are thought to be a crucial element of the resealing mechanism. However, in mammalian cells, the vesicles used in this fusion reaction (endosomes/lysosomes) are not present in a ‘pre-docked’ configuration and so must be brought into physical contact with one another and with the plasma membrane. We propose that a requisite prelude to fusion is the disassembly in local cell cortex of the physical barrier constituted by filamentous actin. Consistent with this hypothesis, we found that rat gastric epithelial (RGM1) cell cortical staining with phalloidin was apparently reduced at presumptive disruption sites. Moreover, flow cytofluorometric analysis of wounded RGM1 populations revealed a small, but significant, Ca2+-dependent reduction in whole cell phalloidin staining. The functional significance of this disruption-induced depolymerization response was confirmed in several independent tests. Introduction into RGM1 cells of the filamentous actin-depolymerizing agent, DNase1, enhanced resealing, although cytochalasin treatment, by itself, had no effect. By contrast, when the filamentous actin cytoskeleton was stabilized experimentally, using phalloidin or jasplakinolide, resealing was strongly inhibited. Cells in wounded cultures displayed an enhanced cortical array of filamentous actin, and resealing by such cells was enhanced strongly by both cytochalasin and DNase 1, demonstrating the specific reversibility of a biologically mediated, polymerization-induced inhibition of resealing. We conclude that localized filamentous actin disassembly removes a cortical barrier standing in the way of membrane-membrane contacts leading to resealing-requisite homotypic and exocytotic fusion events.

Repairing a torn cell surface: make way, lysosomes to the rescue

2002 ◽  
Vol 115 (5) ◽  
pp. 873-879 ◽  
Author(s):  
Paul L. McNeil
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Actin Polymerization ◽  
Surface Membrane ◽  
Cell Cortex ◽  
Yolk Granule ◽  
Protein Markers ◽  
Filamentous Actin ◽  
Animal Cells ◽  
Fusion Plasma

Biological membranes are often described as `self-sealing' structures. If indeed membranes do have an inherent capacity for repair, does this explain how a cell can rapidly reseal a very large (1-1000 μm2)disruption in its plasma membrane? It is becoming increasingly clear that, in nucleated animal cells, the cytoplasm plays an active and essential role in resealing. A rapid and apparently chaotic membrane fusion response is initiated locally in the cytoplasm by the Ca2+ that floods in through a disruption: cytoplasmic vesicles are thereby joined with one another(homotypically) and with the surrounding plasma membrane (exocytotically). As a consequence, internal membrane is added to cell surface membrane at the disruption site. In the case of large disruptions, this addition is hypothesized to function as a `patch'. In sea urchin eggs, the internal compartment used is the yolk granule. Several recent studies have significantly advanced our understanding of how cells survive disruption-inducing injuries. In fibroblasts, the lysosome has been identified as a key organelle in resealing. Protein markers of the lysosome membrane appear on the surface of fibroblasts at sites of disruption. Antibodies against lysosome-specific proteins, introduced into the living fibroblast,inhibit its resealing response. In gastric eptithelial cells, local depolymerization of filamentous actin has been identified as a crucial step in resealing: it may function to remove a barrier to lysosome-plasma membrane contact leading to exocytotic fusion. Plasma membrane disruption in epithelial cells induces depolymerization of cortical filamentous actin and, if this depolymerization response is inhibited, resealing is blocked. In the Xenopus egg, the cortical cytoskeleton has been identified as an active participant in post-resealing repair of disruption-related damage to underlying cell cortex. A striking, highly localized actin polymerization response is observable around the margin of cortical defects. A myosin powered contraction occurring within this newly formed zone of F-actin then drives closure of the defect in a purse-string fashion.

Maintenance of the filamentous actin cytoskeleton is necessary for the activation of store-operated Ca2+ channels, but not other types of plasma-membrane Ca2+ channels, in rat hepatocytes

2002 ◽  
Vol 363 (1) ◽  
pp. 117-126 ◽  
Author(s):  
Ying-Jie WANG ◽  
Roland B. GREGORY ◽  
Greg J. BARRITT
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Critical Factor ◽  
Rat Hepatocytes ◽  
Cytochalasin D ◽  
Cation Channels ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Ca2 Channels ◽  
Intracellular Messenger

The roles of the filamentous actin (F-actin) cytoskeleton and the endoplasmic reticulum (ER) in the mechanism by which store-operated Ca2+ channels (SOCs) and other plasma-membrane Ca2+ channels are activated in rat hepatocytes in primary culture were investigated using cytochalasin D as a probe. Inhibition of thapsigargin-induced Ca2+ inflow by cytochalasin D depended on the concentration and time of treatment, with maximum inhibition observed with 0.1μM cytochalasin D for 3h. Cytochalasin D (0.1μM for 3h) did not inhibit the total amount of Ca2+ released from the ER in response to thapsigargin but did alter the kinetics of Ca2+ release. The effects of cytochalasin D (0.1μM) on vasopressin-induced Ca2+ inflow were similar to those on thapsigargin-induced Ca2+ inflow, except that cytochalasin D did inhibit vasopressin-induced release of Ca2+ from the ER. Cytochalasin D (0.1μM) inhibited vasopressin-induced Mn2+ inflow (predominantly through intracellular messenger-activated non-selective cation channels), but the degree of inhibition was less than that of vasopressin-induced Ca2+ inflow (predominantly through Ca2+-selective SOCs). Maitotoxin- and hypotonic shock-induced Ca2+ inflow were enhanced rather than inhibited by 0.1μM cytochalasin D. Treatment with 0.1μM cytochalasin D substantially reduced the amount of F-actin at the cell cortex, whereas 5μM cytochalasin D increased the total amount of F-actin and caused an irregular distribution of F-actin at the cell cortex. Cytochalasin D (0.1μM) caused no significant change in the overall arrangement of the ER {monitored using 3′,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] in fixed cells} but disrupted the fine structure of the smooth ER and reduced the diffusion of DiOC6(3) in the ER in live hepatocytes after photobleaching. It is concluded that (i) the concentration of cytochalasin D is a critical factor in the use of this agent as a probe to disrupt the cortical F-actin cytoskeleton in rat hepatocytes, (ii) a reduction in the amount of cortical F-actin inhibits SOCs but not intracellular messenger-activated non-selective cation channels, and (iii) inhibition of the activation of SOCs and reduction in the amount of cortical F-actin is associated with disruption of the organization of the ER.

scholarly journals Function and interactions of the Ysc84/SH3yl1 family of actin- and lipid-binding proteins

2015 ◽  
Vol 43 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Agnieszka N. Urbanek ◽  
Rebekah Chan ◽  
Kathryn R. Ayscough
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Morphological Changes ◽  
Lipid Binding ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Membrane Morphology

Understanding how actin filaments are nucleated, polymerized and disassembled in close proximity to cell membranes is an area of growing interest. Protrusion of the plasma membrane is required for cell motility, whereas inward curvature or invagination is required for endocytic events. These morphological changes in membrane are often associated with rearrangements of actin, but how the many actin-binding proteins of eukaryotes function in a co-ordinated way to generate the required responses is still not well understood. Identification and analysis of proteins that function at the interface between the plasma membrane and actin-regulatory networks is central to increasing our knowledge of the mechanisms required to transduce the force of actin polymerization to changes in membrane morphology. The Ysc84/SH3yl1 proteins have not been extensively studied, but work in both yeast and mammalian cells indicate that these proteins function at the hub of networks integrating regulation of filamentous actin (F-actin) with changes in membrane morphology.

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

scholarly journals A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane.

1986 ◽  
Vol 103 (5) ◽  
pp. 1829-1835 ◽  
Author(s):  
P G Woodman ◽  
J M Edwardson
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Trichloroacetic Acid ◽  
Plasma Membranes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Viral Glycoprotein ◽  
Acetylneuraminic Acid ◽  
Donor And Acceptor ◽  
A Cell

A cell-free assay has been developed for the delivery of influenza virus neuraminidase to the plasma membrane. Two types of postnuclear supernatant, which acted as donor and acceptor of the enzyme, were prepared from baby hamster kidney cells. Donor preparations were obtained from cells infected with influenza virus and containing neuraminidase en route to the plasma membrane. Acceptor preparations were obtained from cells containing, bound to their plasma membranes, Semliki Forest virus with envelope glycoproteins bearing [3H]N-acetylneuraminic acid. Fusion between vesicles from these two preparations permits access of the enzyme to its substrate, which results in the release of free [3H]N-acetylneuraminic acid. This release was detected through the transfer of radioactivity from a trichloroacetic acid-insoluble to a trichloroacetic acid-soluble fraction. An ATP-dependent component of release was found, which appears to be a consequence of vesicle fusion. This component was enhanced when the donor was prepared from cells in which the enzyme had been concentrated in a compartment between the Golgi complex and the plasma membrane, which indicates that a specific exocytic fusion event has been reconstituted. The extent of fusion is greatly reduced by pre-treatment of donor and acceptor preparations with trypsin, which points to the involvement of proteins in the fusion reaction.

An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. II. Cortical wound healing

1979 ◽  
Vol 37 (1) ◽  
pp. 59-67
Author(s):  
M. Geuskens ◽  
R. Tencer
Keyword(s):  
Wound Healing ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Xenopus Laevis ◽  
Ultrastructural Study ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Cell Cortex ◽  
Animal Pole ◽  
Annular Zone

Uncleaved fertilized eggs of Xenopus laevis treated with wheat germ agglutinin (WGA) have been pricked at the animal pole both inside and outside the regressed furrow region. The wounded cortex of both regions has been studied with the electron microscope and compared with the same region of wounded, untreated eggs. In all 3 cases, filaments are organized in an annular zone in the damaged cortex. When the surface is pricked outside the regressed furrow of WGA-treated embryos, bundles of microfilaments radiate from the ring and extend in deep folds which form a ‘star’ around the wound at the surface of the embryo. However, when the surface is pricked in the new membrane of the regressed furrow, filaments are intermingled with internalized portions of the plasma membrane. It is suggested that, when the surface is pricked outside the furrow region, more filaments are mobilized to counteract the tangential retraction of the membrane which has acquired more rigidity after WGA binding.

scholarly journals Epiplasts: Membrane Skeletons and Epiplastin Proteins in Euglenids, Glaucophytes, Cryptophytes, Ciliates, Dinoflagellates, and Apicomplexans

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Ursula Goodenough ◽  
Robyn Roth ◽  
Thamali Kariyawasam ◽  
Amelia He ◽  
Jae-Hyeok Lee
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Amino Acid ◽  
Secondary Structure ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Random Coil ◽  
Cell Cortex ◽  
Tail Domain ◽  
Membrane Skeletons

ABSTRACTAnimals and amoebae assemble actin/spectrin-based plasma membrane skeletons, forming what is often called the cell cortex, whereas euglenids and alveolates (ciliates, dinoflagellates, and apicomplexans) have been shown to assemble a thin, viscoelastic, actin/spectrin-free membrane skeleton, here called the epiplast. Epiplasts include a class of proteins, here called the epiplastins, with a head/medial/tail domain organization, whose medial domains have been characterized in previous studies by their low-complexity amino acid composition. We have identified two additional features of the medial domains: a strong enrichment of acid/base amino acid dyads and a predicted β-strand/random coil secondary structure. These features have served to identify members in two additional unicellular eukaryotic radiations—the glaucophytes and cryptophytes—as well as additional members in the alveolates and euglenids. We have analyzed the amino acid composition and domain structure of 219 epiplastin sequences and have used quick-freeze deep-etch electron microscopy to visualize the epiplasts of glaucophytes and cryptophytes. We define epiplastins as proteins encoded in organisms that assemble epiplasts, but epiplastin-like proteins, of unknown function, are also encoded in Insecta, Basidiomycetes, andCaulobactergenomes. We discuss the diverse cellular traits that are supported by epiplasts and propose evolutionary scenarios that are consonant with their distribution in extant eukaryotes.IMPORTANCEMembrane skeletons associate with the inner surface of the plasma membrane to provide support for the fragile lipid bilayer and an elastic framework for the cell itself. Several radiations, including animals, organize such skeletons using actin/spectrin proteins, but four major radiations of eukaryotic unicellular organisms, including disease-causing parasites such asPlasmodium, have been known to construct an alternative and essential skeleton (the epiplast) using a class of proteins that we term epiplastins. We have identified epiplastins in two additional radiations and present images of their epiplasts using electron microscopy. We analyze the sequences and secondary structure of 219 epiplastins and present an in-depth overview and analysis of their known and posited roles in cellular organization and parasite infection. An understanding of epiplast assembly may suggest therapeutic approaches to combat infectious agents such asPlasmodiumas well as approaches to the engineering of useful viscoelastic biofilms.

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