Calponin Repeats Regulate Actin Filament Stability and Formation of Podosomes in Smooth Muscle Cells

Gimona, Mario; Kaverina, Irina; Resch, Guenter P.; Vignal, Emmanuel; Burgstaller, Gerald

doi:10.1091/mbc.e02-11-0743

Cited by 149 publications

(184 citation statements)

References 40 publications

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“…Tropomyosins bound to filamentous actin prevent access of ADF/cofilins to actin filaments, thereby stabilizing actin filaments and reducing actin dynamics (Ono and Ono, 2002). In addition to tropomyosins, calponin2 and ␣-actinin1, two other TGF-␤ targets identified in this study, have been also implicated in stabilization of actin filaments (Panasenko and Gusev, 2001;Gimona et al, 2003). It remains to be determined whether, in addition to tropomyosins, calponins and ␣-actinin play a role in formation of stress fibers in response to TGF-␤ by blocking ADF/cofilins and gelsolin from binding to actin filaments.…”

Section: Discussionmentioning

confidence: 80%

A Critical Role of Tropomyosins in TGF-β Regulation of the Actin Cytoskeleton and Cell Motility in Epithelial Cells

Bakin

Safina

Rinehart

et al. 2004

MBoC

135

208

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We have investigated transforming growth factor beta (TGF-␤)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-␤ via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, ␣-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-␤ induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-␤ induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-␤ induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-␤ induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-␤ control of cell motility, and the loss of this TGF-␤ response is a critical step in the acquisition of metastatic phenotype by tumor cells. INTRODUCTIONThere is solid evidence that the transforming growth factor beta (TGF-␤) signaling pathway is a major cellular growth inhibitory and proapoptotic pathway in epithelial, endothelial, hematopoeitic, and other cell types (Roberts and Wakefield, 2003). However, clinical and experimental studies indicate that metastatic cancers of the breast and other tissues express elevated levels of TGF-␤ that appears to support the metastatic behavior of the tumor cells (Saito et al., 2000;Derynck et al., 2001). This apparent paradox has been associated with a progressive decline in the antitumorigenic function and a gain of protumorigenic activities of TGF-␤, including induction of epithelial to mesenchymal transition (EMT) and tumor cell migration and invasion (Derynck et al., 2001;Wakefield and Roberts, 2002). Oncogenic Ras, Src, and ErbB2 as well as alterations in TGF-␤ signaling mediated by Smads, mitogen-activated protein kinases (Mapks), Rho kinases, and Akt/PKB are thought to contribute to the metastatic phenotype (Derynck and Zhang, 2003;Roberts and Wakefield, 2003).The actin cytoskeleton plays a central role in the regulation of cellular processes linked to metastasis including cell proliferation, apoptosis, anchorage-independent cell growth, and cell migration and invasion (Pawlak and Helfman, 2001;Jaffe and Hall, 2002). TGF-␤ induces a rapid reorganization of the actin cytoskeleton, leading to membrane ruffling at the cell edges in both nontumorigenic and tumorigenic epithelial cells, whereas a prolonged incubation with TGF-␤ results in the formation of stress fibers (Bakin et al., 2002;Edlund et a...

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Section: Discussionmentioning

confidence: 80%

A Critical Role of Tropomyosins in TGF-β Regulation of the Actin Cytoskeleton and Cell Motility in Epithelial Cells

Bakin

Safina

Rinehart

et al. 2004

MBoC

135

208

View full text Add to dashboard Cite

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“…Although the right tail of the (H/L) E /(H/L) U distribution contains relatively few members, they represent pharynx-specific proteins: of the 1,100 proteins in our dataset with known expression patterns, 7 of the 18 that are thought to be expressed exclusively in the pharynx have (H/L) E /(H/L) U values greater than two (P = 1.25 × 10 −8 ; Fisher's exact test). When we examined a test set of 34 proteins whose genes are highly expressed in body wall muscle, intestinal epithelia, and neuronal cells (27), we found only three in our dataset (SI Appendix, Table S5). Two of the three are also expressed in the pharynx (28,29).…”

Section: Resultsmentioning

confidence: 99%

Cell-specific proteomic analysis in Caenorhabditis elegans

Yuet¹,

Doma

Ngo³

et al. 2015

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

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Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.click chemistry | protein engineering | proteomics | cell-specific protein expression | nematode pharyngeal muscle I n a complex eukaryote like Caenorhabditis elegans, cell heterogeneity restricts the usefulness of large-scale, mass spectrometry-based proteomic analysis. Enriching for specific cells is challenging, and researchers cannot systematically identify low-abundance proteins expressed in specific cells from wholeorganism lysates. Cell-selective bioorthogonal noncanonical amino acid tagging (cell-selective BONCAT) offers a way to overcome these limitations (1, 2). We have previously engineered a family of mutant Escherichia coli methionyl-tRNA synthetases (MetRSs) capable of appending the azide-bearing L-methionine (Met) analog L-azidonorleucine (Anl) to its cognate tRNA in competition with Met (3, 4). Because Anl is a poor substrate for any of the natural aminoacyl-tRNA synthetases, it is excluded from proteins made in wild-type cells but is incorporated readily into proteins made in cells that express an appropriately engineered MetRS. Controlling expression of mutant MetRSs by expression only in specific cells restricts Anl labeling to proteins produced in those cells. Tagged proteins can be distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to alkynyl or cyclooctynyl probes that permit facile detection, isolation, and visualization of labeled proteins. This strategy has been used to selectively enrich microbial proteins from mixtures of bacterial and mammalian cells. For example, Ngo et al. (5) found that proteins made in an E. coli strain outf...

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“…However, they can also be found or induced in a variety of other cell types, including smooth muscle cells (Gimona et al, 2003) and endothelial cells (Moreau et al, 2003;Osiak et al, 2005).…”

Section: Podosomes and Invadopodiamentioning

confidence: 99%

“…One of the most upstream signals is probably PKC activity. This is underscored by the fact that podosome formation can be induced by PKC-activating agents, such as phorbol esters (Gimona et al, 2003). An important subsequent switch is activation of Src.…”

Section: Microtubulesmentioning

confidence: 99%