Effect of Microtubule Disruption on Cell Adhesion and Spreading

Kadi, Amir; Pichard, Véronique; Lehmann, Maxime; Briand, Claudette; Braguer, Diane; Marvaldi, Jacques; Rognoni, J. B.; Luis, José

doi:10.1006/bbrc.1998.8536

Cited by 51 publications

(37 citation statements)

References 34 publications

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“…This is clearly the case as we observed that non conserved amino-acid residues are critical for the efficiency of the peptides. For example, efficiency of obtustatin to inhibit cell adhesion (IC50 ¼ 1.8 nM) was improved by about 10-fold by replacement of Leu 24 (present in obtustatin) by Arg 24 (present in lebestatin), leading to an IC50 of 0.22 nM, a value very similar to that of lebestatin (IC50 ¼ 0.20 nM). This structure/function relationship study will be described in detail elsewhere (O Ziri, manuscript in preparation).…”

Section: Discussionmentioning

confidence: 99%

“…23,24 Briefly, cells in single cell suspension were added to wells coated with purified extracellular matrix (ECM) proteins and allowed to adhere to the substrata for 1 h (HMEC-1, HT1080, IGROV1, CHO-a1 and PC12 cells) or 2 h (HT29-D4 and K562 cells) at 371C. After washing, attached cells were fixed, stained by 0.1% crystal violet and lysed with 1% SDS.…”

Section: Cell Adhesion and Migration Assaysmentioning

confidence: 99%

“…In vitro cell migration assays were performed in modified Boyden chambers (NeuroProbe Inc., Bethesda, MD, USA) with porous membranes precoated with 10 mg/ml of type IV collagen as previously described, 23,24 except that cells were stained with 0.1% crystal violet. Cell migration was then quantified by measurement the absorbance at 600 nm.…”

Section: Cell Adhesion and Migration Assaysmentioning

confidence: 99%

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Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis

Kallech-Ziri

Luis

Salma

et al. 2005

Laboratory Investigation

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Lebestatin, a new member of the lysine-threonine-serine (KTS)-disintegrin family, was purified to homogeneity from Tunisian snake (Macrovipera lebetina) venom. It is a single-chain polypeptide composed of 41 amino acids. The amino-acid sequence of lebestatin shows that it displays a pattern of cysteines similar to other short disintegrins, but contains the sequence KTS rather than RGD in its integrin-binding loop. Lebestatin presents a high homology with obtustatin and viperistatin. Lebestatin interacts specifically with the a1b1 integrin. It was thus able to inhibit both adhesion and migration of PC12 and a1b1 integrin-expressing CHO cells (CHO-a1) to type I and IV collagens. This disintegrin also affected adhesion and migration of endothelial cells and exhibited an anti-angiogenic effect in vivo when using the 8-day-old embryo chick chorioallantoic membrane model. Laboratory Investigation (2005) 85, 1507-1516.

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Section: Discussionmentioning

confidence: 99%

Section: Cell Adhesion and Migration Assaysmentioning

confidence: 99%

Section: Cell Adhesion and Migration Assaysmentioning

confidence: 99%

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Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis

Kallech-Ziri

Luis

Salma

et al. 2005

Laboratory Investigation

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“…Cell Adhesion Assay-Adhesion assays were performed as described previously (28,29). Briefly, cells were harvested in single cell suspension by treatment with 0.53 mM EDTA in PBS, added to wells coated with purified ECM proteins, and allowed to adhere to the substrata for 2 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Role of Endoproteolytic Processing in the Adhesive and Signaling Functions of αvβ5 Integrin

Berthet

Rigot

Champion

et al. 2000

Journal of Biological Chemistry

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Integrins are transmembrane glycoproteins, composed of noncovalently associated ␣ and ␤ subunits, that are involved in cell-extracellular matrix (ECM) 1 and cell-cell interactions (1). Many integrin ␣ chains undergo a post-translational endoproteolytic cleavage. The ␣ 3 , ␣ 5 , ␣ 6 , ␣ 7 , ␣ 8 , ␣ 9 , ␣ v , and ␣ IIb subunits are cleaved in the membrane-proximal extracellular region, resulting in a heavy chain that is disulfide-linked to a membrane spanning light chain (2). The ␣ 4 and ␣ E subunits can also be cleaved, but at unusual positions, near the middle and in the N-terminal region of the molecule, respectively (3, 4). Endoproteolytic cleavage of integrin ␣ subunits occurs at specific sites comprising pairs of basic amino acids.Post-translational proteolysis is a common mechanism required for the synthesis of biologically active proteins in bacteria, fungi, yeast, invertebrates, and mammals (5). However, the role of endoproteolytic cleavage of integrin ␣ subunits is not clear. The cleavage is conserved, not only in different ␣ chains but also across species, suggesting that it might be of functional importance. It has been established, by site-directed mutagenesis of cleavage sites, that uncleaved ␣ IIb ␤ 3 and ␣ 4

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“…When phorbol ester was added to disrupt the interaction of these two proteins (28), FRET disappeared. These data suggest that MacMARCKS and dynamitin indeed interact in the cells.…”

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confidence: 99%

In Vivo Interaction between Dynamitin and MacMARCKS Detected by the Fluorescent Resonance Energy Transfer Method

Jin

Yue

2001

Journal of Biological Chemistry

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Dynamitin is a subunit of the dynactin complex regulating microtubule-dependent motor functions, and MacMARCKS (Macrophage-enriched myristoylated alanine-rich protein kinase C substrate) is a major protein kinase C substrate regulating integrin activation. The interaction between dynamitin and MacMARCKS has been implicated in integrin-dependent cell spreading. However, the in vivo interaction of these two proteins in living cells has not been demonstrated. Spatial and temporal information about the interaction is also lacking. In this study, we used the fluorescent resonance energy transfer method to demonstrate in vivo interaction between MacMARCKS and dynamitin with cyan fluorescent protein (CFP)-conjugated dynamitin as the donor fluorophore and yellow fluorescent protein (YFP)-conjugated MacMARCKS as the acceptor fluorophore. The interaction of these two fusion proteins was studied both in vitro and in vivo, and typical fluorescent resonance energy transfer was observed; the CFP emission peak increased while the YFP emission peak decreased when protein interaction was abolished. Spatial and temporal information was obtained in RAW macrophage cells. In resting macrophage cells, dynamitin-Mac-MARCKS interaction is concentrated at the cell periphery, although the majority of dynamitin is distributed at the perinuclear region of the cells. When cells were treated with phorbol 12-myristate 13-acetate, both proteins concentrated to perinuclear regions of the cells, and yet the interaction disappeared as the cell spread. Similar events were also observed in 293 cells. Thus, we conclude that dynamitin and MacMARCKS indeed interact in living cells.The cytoskeleton plays a pivotal role in activating ␤ 2 integrin molecules by restraining the integrin molecules on the cell membrane in an inactivated state (1-3). Upon stimulation by PKC 1 activation, the cytoskeleton constraint is released and integrin is activated. Both actin and microtubule filaments are involved in constraining integrin molecules. The PKC signal is most likely transduced to cytoskeleton by MacMARCKS protein, a member of the MARCKS (myristoylated alanine-rich protein kinase C substrate) family of PKC substrates (4 -6), because mutation or lack of this protein prevents PKC-stimulated molecular mobility of integrin (2), as well as ligand binding by integrin (7-9). In addition, MARCKS protein, the homologue of MacMARCKS, has been shown to be involved in cell spreading (10, 11). MacMARCKS protein contains an N-terminal membrane-targeting domain that is myristoylated at its N-terminal glycine residue. The effector domain, in the center part of the protein, contains the PKC phosphorylation sites, two serine residues at positions 93 and 104.Interaction between MacMARCKS and dynamitin, a subunit of dynactin, has been observed (12). Dynactin (reviewed in Refs. 13-16), a complex of proteins, is an important regulator of dynein, a microtubule-dependent minus end-directed motor protein (17). The dynactin complex has been suggested as a "molecular bridge" connecting...

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Effect of Microtubule Disruption on Cell Adhesion and Spreading

Cited by 51 publications

References 34 publications

Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis

Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis

Role of Endoproteolytic Processing in the Adhesive and Signaling Functions of αvβ5 Integrin

In Vivo Interaction between Dynamitin and MacMARCKS Detected by the Fluorescent Resonance Energy Transfer Method

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