Brush border myosin-I microinjected into cultured cells is targeted to actin-containing surface structures

1994 ◽  
Vol 107 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
M. Footer ◽  
A. Bretscher
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Surface Structures ◽  
Specific Expression ◽  
Cultured Fibroblasts ◽  
Myosin I ◽  
Section Electron

The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When introduced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the consequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Fodrin: axonally transported polypeptides associated with the internal periphery of many cells.

1981 ◽  
Vol 90 (3) ◽  
pp. 631-642 ◽  
Author(s):  
J Levine ◽  
M Willard
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cortical Actin ◽  
Cultured Fibroblasts ◽  
Tissue Sections ◽  
Parallel Arrays ◽  
Guinea Pig Brain

Fodrin (formerly designated 26 and 27) comprises two polypeptides (250,000 and 240,000 mol wt) that are axonally transported at a maximum time-averaged velocity of 40 mm/d--slower than the most rapidly moving axonally transported proteins, but faster than at least three additional groups of proteins. In this communication, we report the intracellular distribution of fodrin. Fodrin was purified from guinea pig brain, and a specific antifodrin antibody was produced in rabbit and used to localize fodrin in tissue sections and cultured cells by means of indirect immunofluorescence. Fodrin antigens were highly concentrated in the cortical cytoplasm of neurons and also nonneuronal tissues (e.g., skeletal muscle, uterus, intestinal epithelium). Their disposition resembles a lining of the cell: hence, the designation fodrin (from Greek fodros, lining). In cultured fibroblasts, immunofluorescently labeled fodrin antigens were arranged in parallel arrays of bands in the plane of the plasma membrane, possibly reflecting an exclusion of labeled fodrin from some areas occupied by stress fibers. The distribution of fodrin antigens in mouse 3T3 cells transformed with simian virus 40 was more diffuse, indicating that the disposition of fodrin is responsive to altered physiological states of the cell. When mixtures of fodrin and F-actin were centrifuged, fodrin cosedimented with the actin, indicating that these proteins interact in vitro. We conclude that fodrin is a specific component of the cortical cytoplasm of many cells and consider the possibilities: (a) that fodrin may be indirectly attached to the plasma membrane via cortical actin filaments; (b) that fodrin may be mobile within the cortical cytoplasm and that, in axons, a cortical lining may be in constant motion relative to the internal cytoplasm; and (c) that fodrin could serve to link other proteins and organelles to a submembrane force-generating system.

scholarly journals The 110-kD protein-calmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme.

1989 ◽  
Vol 108 (6) ◽  
pp. 2395-2400 ◽  
Author(s):  
M S Mooseker ◽  
T R Coleman
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Average Rate ◽  
Skeletal Muscle Myosin ◽  
Intestinal Brush Border ◽  
Myosin I ◽  
Actin Cables ◽  
Muscle Myosin

The 110-kD protein-calmodulin complex (110K-CM) of the intestinal brush border serves to laterally tether microvillar actin filaments to the plasma membrane. Results from several laboratories have demonstrated that this complex shares many enzymatic and structural properties with myosin. The mechanochemical potential of purified avian 110K-CM was assessed using the Nitella bead motility assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature (Lond.). 303:31-35). Under low Ca2+ conditions, 110K-CM-coated beads bound to actin cables, but no movement was observed. Using EGTA/calcium buffers (approximately 5-10 microM free Ca2+) movement of 110K-CM-coated beads along actin cables (average rate of approximately 8 nm/s) was observed. The movement was in the same direction as that for beads coated with skeletal muscle myosin. The motile preparations of 110K-CM were shown to be free of detectable contamination by conventional brush border myosin. Based on these and other observations demonstrating the myosin-like properties of 110K-CM, we propose that this complex be named "brush border myosin I."

Proteins involved in the attachment of actin to the plasma membrane

1982 ◽  
Vol 299 (1095) ◽  
pp. 291-299 ◽  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Cell Types ◽  
Brain Protein ◽  
Cultured Fibroblasts ◽  
Microfilament Bundles ◽  
Different Cell Types ◽  
Adhesion Plaque ◽  
The Brain

Proteins that may be involved in two types of actin-membrane association are discussed. The first set includes α-actinin, vinculin, fimbrin and a new cytoskeletal protein that are all concentrated in adhesion plaques, those regions of cultured fibroblasts where bundles of actin microfilaments terminate and where the plasma membrane comes close to the underlying substrate. The properties of non-muscle α-actinin suggest that it functions to cross-link actin filaments and thereby stabilize microfilament bundles rather than functioning in their attachment to the membrane. Fimbrin also appears to be involved in bundling of filaments rather than in attachment. In contrast, vinculin binds to the ends of actin filaments in vitro and is probably the best candidate for a role in the attachment of actin to membranes at the adhesion plaque. The discovery of a new protein, 215k, of unknown function, in the adhesion plaque suggests that many more proteins remain to be identified in this region. Attachment of actin filaments to other regions of the plasma membrane is also considered and a protein is described that seems to be a spectrin homologue in brain and other tissues. The brain protein resembles erythrocyte spectrin in its physical properties, in binding actin, in being associated with cell membranes and in crossreacting immunologically. We suggest that the brain protein and erythrocyte spectrin both belong to a family of related proteins (the spectrins) which function in the attachment of actin to membranes in many different cell types.

scholarly journals Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks.

1981 ◽  
Vol 91 (3) ◽  
pp. 695-705 ◽  
Author(s):  
J V Small
Keyword(s):  
Electron Microscopy ◽  
Actin Filament ◽  
Actin Filaments ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Leading Edge ◽  
Cultured Fibroblasts ◽  
Cell Biol ◽  
Filament Bundles

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

scholarly journals Cofilin, But Not Profilin, Is Required for Myosin-I-Induced Actin Polymerization and the Endocytic Uptake in Yeast

2002 ◽  
Vol 13 (11) ◽  
pp. 4074-4087 ◽  
Author(s):  
Fatima-Zahra Idrissi ◽  
Bianka L. Wolf ◽  
M. Isabel Geli
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Cortical Actin ◽  
Myosin I ◽  
Robust Evidence

Mutations in the budding yeast myosins-I (MYO3 andMYO5) cause defects in the actin cytoskeleton and in the endocytic uptake. Robust evidence also indicates that these proteins induce Arp2/3-dependent actin polymerization. Consistently, we have recently demonstrated, using fluorescence microscopy, that Myo5p is able to induce cytosol-dependent actin polymerization on the surface of Sepharose beads. Strikingly, we now observed that, at short incubation times, Myo5p induced the formation of actin foci that resembled the yeast cortical actin patches, a plasma membrane-associated structure that might be involved in the endocytic uptake. Analysis of the machinery required for the formation of the Myo5p-induced actin patches in vitro demonstrated that the Arp2/3 complex was necessary but not sufficient in the assay. In addition, we found that cofilin was directly involved in the process. Strikingly though, the cofilin requirement seemed to be independent of its ability to disassemble actin filaments and profilin, a protein that closely cooperates with cofilin to maintain a rapid actin filament turnover, was not needed in the assay. In agreement with these observations, we found that like the Arp2/3 complex and the myosins-I, cofilin was essential for the endocytic uptake in vivo, whereas profilin was dispensable.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

scholarly journals Regulation of Actin Filament Cross-linking and Bundle Shape in Drosophila Bristles

2000 ◽  
Vol 148 (1) ◽  
pp. 87-99 ◽  
Author(s):  
Lewis G. Tilney ◽  
Patricia S. Connelly ◽  
Kelly A. Vranich ◽  
Michael K. Shaw ◽  
Gregory M. Guild
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Cross Linking ◽  
Limited Area ◽  
Actin Bundles ◽  
Electron Microscopy Analysis ◽  
Section Electron ◽  
Cross Links ◽  
Bundle Size ◽  
Cross Linker

Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker–specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.

scholarly journals Effect of Stretching Force on the Cells of Epithelial Rests of Malassez In Vitro

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Teruyoshi Koshihara ◽  
Kenichi Matsuzaka ◽  
Toru Sato ◽  
Takashi Inoue
Keyword(s):  
Actin Filaments ◽  
Cultured Cells ◽  
Control Group ◽  
Rt Pcr ◽  
Vegf Mrna ◽  
Hsp70 Mrna ◽  
Epithelial Rests Of Malassez

Background and Objective. The aim of this study was to investigate the behavior of cells from epithelial rest of Malassez (ERM) against stretching force.Material and Methods. ERM-cultured cells were stretched for 1 hour, at the cycle of 18% elongation for 1 second followed by 1-second relaxation. The cells without addition of stretching force were used as controls. The cells were observed by immunohistochmical staining using actin 0, 12, 24, 36, 48, and 72 hours. Furthermore, expressions of HSP70-, VEGF-, and OPN-mRNAs of cells were also evaluated using quantitative RT-PCR.Results. Actin filaments were randomly orientated in the cytoplasm in the control group, whereas in the stretching group, actin filaments were orientated comparatively parallel to the stretching direction. Expression of HSP70-mRNA in the stretching group was significantly higher than that of control group at 12, 24, 36 hours (P<.05). Expression of VEGF-mRNA in the stretching group was significantly higher than that of control group at 24, 36, 48, and 72 hours (P<.05). Expression of OPN-mRNA in the stretching group was significantly higher than that of control group at 12 and 24 hours (P<.05).Conclusion. ERM cells response against the stretching force by expressing HSP70, VEGF, and OPN.

scholarly journals Metabolism and intracellular localization of a fluorescently labeled intermediate in lipid biosynthesis within cultured fibroblasts.

1981 ◽  
Vol 91 (3) ◽  
pp. 872-877 ◽  
Author(s):  
R E Pagano ◽  
K J Longmuir ◽  
O C Martin ◽  
D K Struck
Keyword(s):  
Endoplasmic Reticulum ◽  
Phosphatidic Acid ◽  
Cultured Cells ◽  
Large Fraction ◽  
Intracellular Localization ◽  
Chinese Hamster ◽  
Cultured Fibroblasts ◽  
Acid Analogue ◽  
Unique Method

In this paper we report on the uptake and distribution of an exogenously supplied fluorescent phosphatidic acid analogue by Chinese hamster fibroblasts. Under appropriate in vitro incubation conditions, 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidic acid was rapidly and preferentially transferred from phospholipid vesicles to cells at 2 degrees C. However, unlike similar fluorescent derivatives of phosphatidylcholine and phosphatidylethanolamine that remain restricted to the plasma membrane under such incubation conditions (Struck, D. K., and R. E. Pagano. 1080. J. Biol. Chem. 255:5405--5410), most of the phosphatidic acid-derived fluorescence was localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. This was shown by labeling cells with rhodamine-containing probes specific for mitochondria or endoplasmic reticulum, and comparing the patterns of intracellular NBD and rhodamine fluorescence. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid was converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Our findings suggest that fluorescent phosphatidic acid may be useful in correlating biochemical studies of lipid metabolism in cultured cells and studies of the Intracellular localization of the metabolites by fluorescence microscopy. In addition, this compound provides a unique method for visualizing the endoplasmic reticulum in living cells.

To bead or not to bead? Lens-specific intermediate filaments revisited

1997 ◽  
Vol 110 (21) ◽  
pp. 2629-2634 ◽  
Author(s):  
S.D. Georgatos ◽  
F. Gounari ◽  
G. Goulielmos ◽  
U. Aebi
Keyword(s):  
Plasma Membrane ◽  
Intermediate Filaments ◽  
Cultured Cells ◽  
Fiber Cells ◽  
New Information ◽  
Specific Proteins ◽  
Beaded Filaments ◽  
Nodular Appearance

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to ‘mainstream’ IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

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