Spreading of fibroblasts in medium containing cytochalasin B: formation of lamellar cytoplasm as a combination of several functional different processes.

Bliokh, Z. L.; Domnina, L. V.; Ivanova, O. Yu.; Pletjushkina, Olga Yu.; Svitkina, T.M.; Smolyaninov, V. A.; Vasiliev, J. M.; Gelfand, Israel M.

doi:10.1073/pnas.77.10.5919

Cited by 23 publications

(8 citation statements)

References 15 publications

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“…Various studies have demonstrated cytochalasin effects on cellular adhesion and/or cell spreading (52)(53)(54)(55)(56)(57)(58). In addition, several groups have demonstrated an intact focal adhesion signaling pathway in 293 cells and its sensitivity to cytochalasin D (59 -61).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of the Myristoylated Alanine-rich C-kinase Substrate Inhibits Cell Adhesion to Extracellular Matrix Components

Spizz

Blackshear

2001

Journal of Biological Chemistry

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Mice lacking the myristoylated alanine-rich C-kinase substrate, or MARCKS protein, exhibit abnormalities consistent with a defect in the ability of neurons to migrate appropriately during forebrain development. To investigate the possibility that this phenotype could be due to disruption of normal cellular adhesion to extracellular matrix, an assay was developed in which 293 cells co-expressing MARCKS and green fluorescent protein were tested for their adhesion competence on various substrates. The myristoylated alanine-rich C-kinase substrate or MARCKS 1 protein was originally identified because of its prominence as a cellular substrate for protein kinase C (PKC) (reviewed in Refs. 1 and 2). Although cloned more than a decade ago (3-5), a functional role for MARCKS remains elusive. Gene targeting experiments have demonstrated that MARCKS is essential for central nervous system development and postnatal life in the mouse (6). Defects seen in the brains of developing MARCKS-deficient embryos included frequent abnormal neurulation and failure of fusion of forebrain hemispheres. There was also universal neuronal and retinal ectopia, resulting from the inappropriate migration of neurons, as well as deficiencies in laminin and chondroitin sulfate proteoglycans (7), two components of the extracellular matrix (ECM) involved in migration of developing neurons (8, 9). These experiments suggested that MARCKS can regulate the ability of neurons to migrate normally; however, it was unclear whether this abnormality was at the level of cell:cell interactions, cell: ECM interactions, or even in the regulation of proteases involved in the remodeling of the ECM.MARCKS and its related protein, the MARCKS-like protein (MLP; also known as F52, MRP, or MacMARCKS) constitute a small family of heat-stable, acidic proteins, containing three regions of near-identity (2). An eight-residue domain in the amino-terminal portion of the protein surrounds the single intron splice site and is identical to a region of unknown function within the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor II receptor. The other two regions include an amino-terminal consensus sequence, which directs the co-translational addition of the 14-carbon myristoyl moiety, and a highly basic region of 25 amino acids containing the PKC phosphorylatable serines, known as the phosphorylation site domain (PSD). These two regions are responsible, respectively, for the hydrophobic and electrostatic interactions of MARCKS with negatively charged lipids in cellular membranes (10 -17). PKC-mediated phosphorylation of the PSD decreases MARCKS affinity for the plasma membrane, calmodulin, and actin, and prevents cathepsin B cleavage at the PSD (14, 18 -30). However, a direct interaction between MARCKS and these proteins in intact cells has not been demonstrated convincingly.Based on the morphological defects seen in the MARCKSdeficient mice, we speculated that MARCKS could be directly involved in cell:ECM and/or cell:cell interactions. To begin to analyze...

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Section: Discussionmentioning

confidence: 99%

Overexpression of the Myristoylated Alanine-rich C-kinase Substrate Inhibits Cell Adhesion to Extracellular Matrix Components

Spizz

Blackshear

2001

Journal of Biological Chemistry

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“…One of the main challenges in obtaining quantitative measure of MT guidance is the spatial and temporal overlap of all involved structures -MTs, F-actin and FAswithin the cell. Specific practical difficulty with testing the potential role of F-actin bundles in MT guidance has been the inability to selectively depolymerize F-actin (for example, by using Latrunculin B) without causing cell retraction (Bliokh et al, 1980), thus making any quantification of the influence of F-actin on MT growth essentially meaningless. Here, we overcome both of these limitations by using micropatterned, triangular live cells (Fig.…”

Section: Introductionmentioning

confidence: 99%

Microtubule guidance tested through controlled cell geometry

Huda

Soh

Pilans

et al. 2012

Journal of Cell Science

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SummaryIn moving cells dynamic microtubules (MTs) target and disassemble substrate adhesion sites (focal adhesions; FAs) in a process that enables the cell to detach from the substrate and propel itself forward. The short-range interactions between FAs and MT plus ends have been observed in several experimental systems, but the spatial overlap of these structures within the cell has precluded analysis of the putative long-range mechanisms by which MTs growing through the cell body reach FAs in the periphery of the cell. In the work described here cell geometry was controlled to remove the spatial overlap of cellular structures thus allowing for unambiguous observation of MT guidance. Specifically, micropatterning of living cells was combined with high-resolution in-cell imaging and gene product depletion by means of RNA interference to study the long-range MT guidance in quantitative detail. Cells were confined on adhesive triangular microislands that determined cell shape and ensured that FAs localized exclusively at the vertices of the triangular cells. It is shown that initial MT nucleation at the centrosome is random in direction, while the alignment of MT trajectories with the targets (i.e. FAs at vertices) increases with an increasing distance from the centrosome, indicating that MT growth is a non-random, guided process. The guided MT growth is dependent on the presence of FAs at the vertices. The depletion of either myosin IIA or myosin IIB results in depletion of F-actin bundles and spatially unguided MT growth. Taken together our findings provide quantitative evidence of a role for long-range MT guidance in MT targeting of FAs.

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“…The zones of active edges are usually located at the distal parts of the lamellar cytoplasm; these are the zones where pseudopods, such as lamellopodia and ruffles, are formed. A study of spreading in medium containing cytochalasin B (CB) at 10 ,ug/ml suggested that formation of the lamellar cytoplasm can be regarded as a combination of two functionally different processes-CB-resistant rudimentary pseudopodial reactions leading to the extension and attachment of arborized outgrowths and CB-sensitive lamellization of these outgrowths leading to their transformation into flattened wide lamellas (2). Experiments presented in this paper show that lamellization, in its turn, can be subdivided into components that have different sensitivities to CB: a low concentration of CB (2 ,ug/ml) prevents the formation of ruffling active edges but does not inhibit the outgrowth of lamellas that lack this specialized distal part.…”

mentioning

confidence: 99%

Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.

Domnina¹,

Gelfand²,

Ivanova³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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The effects of low doses of cytochalasin B (2 ,ug/ ml) and cytochalasin D (0.2 lag/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors.Substrate-spread fibroblasts and epithelial cells have a special structure, the lamellar cytoplasm, at their periphery. Characteristic features of the lamellar cytoplasm. include a flattened shape, the absence of large organelles, the presence of numerous focal contacts with the substrate at the lower surface, and a distinctive ability of the surface to clear itself from patched membrane receptors (see review in ref. 1). The zones of active edges are usually located at the distal parts of the lamellar cytoplasm; these are the zones where pseudopods, such as lamellopodia and ruffles, are formed. A study of spreading in medium containing cytochalasin B (CB) at 10 ,ug/ml suggested that formation of the lamellar cytoplasm can be regarded as a combination of two functionally different processes-CB-resistant rudimentary pseudopodial reactions leading to the extension and attachment of arborized outgrowths and CB-sensitive lamellization of these outgrowths leading to their transformation into flattened wide lamellas (2). Experiments presented in this paper show that lamellization, in its turn, can be subdivided into components that have different sensitivities to CB: a low concentration of CB (2 ,ug/ml) prevents the formation of ruffling active edges but does not inhibit the outgrowth of lamellas that lack this specialized distal part. MATERIALS AND METHODSMouse embryo fibroblasts were cultured in a 1:1 mixture of Eagle's medium and 0.5% lactalbumin hydrolysate/10% bovine serum. CB (Serva, Heidelberg, Federal Republic of Germany) was added to this medium at 2 pig/ml (low-CB medium) or 10 jkg/ml (high-CB medium). Cytochalasin D (Sigma) was used at 0...

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Spreading of fibroblasts in medium containing cytochalasin B: formation of lamellar cytoplasm as a combination of several functional different processes.

Cited by 23 publications

References 15 publications

Overexpression of the Myristoylated Alanine-rich C-kinase Substrate Inhibits Cell Adhesion to Extracellular Matrix Components

Overexpression of the Myristoylated Alanine-rich C-kinase Substrate Inhibits Cell Adhesion to Extracellular Matrix Components

Microtubule guidance tested through controlled cell geometry

Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.

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