High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture

Schevzov, Galina; Lloyd, Catriona; Gunning, Peter W.

doi:10.1083/jcb.117.4.775

Cited by 126 publications

(124 citation statements)

References 44 publications

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“…This is entirely consistent with previous studies that have shown that Tm isoforms differ in their ability to restore cell shape to transformed cells (Gimona et al, 1996) and in their impact on neuroepithelial cells (Bryce et al, 2003; Figure 6). In addition, the original demonstration that ␤ and ␥-actin carry different structural information (Schevzov et al, 1992) may be accounted for by their differential impact on Tm isoform levels and organization (Schevzov et al, 1993). In all cases, the alterations in cell shape are consistent with changes in the organization of actin filaments.…”

mentioning

confidence: 69%

“…Previous studies that have manipulated actin and tropomyosin isoform expression in myogenic and neuroepithelial cells have led to global changes in cell dimension (Schevzov et al, 1992;von Arx et al, 1995;Mounier et al, 1997;Bryce et al, 2003). In each case, the manipulated gene products have altered the organization of actin filaments throughout the cell.…”

Section: Qualitative and Quantitative Impact Of Tropomyosin On Neuronmentioning

confidence: 99%

“…In each case, the manipulated gene products have altered the organization of actin filaments throughout the cell. The only exception has been the impact of ␥-actin on cell membrane morphology in two studies (Schevzov et al, 1992;von Arx et al, 1995). In Tm5NM1 expression in neurons, the protein is specifically sorted to growth cones, and it is this compartment alone that shows an increase in size.…”

Section: Qualitative and Quantitative Impact Of Tropomyosin On Neuronmentioning

confidence: 99%

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Specific Features of Neuronal Size and Shape Are Regulated by Tropomyosin Isoforms

Schevzov

Bryce

Almonte-Baldonado

et al. 2005

MBoC

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Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the ␤-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments. INTRODUCTIONThe actin cytoskeleton plays an essential role in the structural changes that initially establish neuronal shape and subsequently contribute to the morphological differentiation of neurons. Actin filaments have been implicated in the initial sprouting of neurites, whereas microtubules strengthen and support the new extensions (Smith, 1988(Smith, , 1994.Tropomyosin (Tm) isoforms, integral components of actin microfilaments, form coiled-coil head-to-tail dimers that bind along the major groove of actin polymers (Phillips et al., 1979). Tms are derived from four highly conserved genes known as the ␣Tm fast , ␤Tm, ␥Tm (Tm5NM), and ␦Tm genes that, via alternative splicing, give rise to Ͼ40 isoforms (LeesMiller and Helfman, 1991;Dufour et al., 1998;Cooley and Bergtrom, 2001). Despite the well understood function of Tms in muscle where, together with the troponin complex, they regulate contraction in a calcium-dependent manner, little is known about their role in nonmuscle cells. In vitro studies have implicated Tms in the stabilization of the actin cytoskeleton by protecting actin filaments from the severing action of gelsolin (Ishikawa et al., 1989) and the depolymerizing action of ADF/cofilin (Bernstein and Bamburg, 1982). Gene transfection studies have demonstrated that tropomyosin isoforms can regulate the organization of actin filaments in transformed cells (Prasad et al., 1993;Boyd et al., 1995;Gimona et al., 1996) and the neuroepithelial cell line B35 (Bryce et al., 2003).In neurons, a strict repertoire of Tm isoforms is known to be expressed. These include TmBr3, Tm5a and Tm5b from the ␣Tm fast gene; multiple products from the ␥Tm gene; and Tm4 from the ␦Tm gene. Most interestingly, Tm isoforms have been previously shown to be spatially and temporally regulated, identifying distinct subcellular compartments. The Tm5a and Tm5b isoforms are enric...

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confidence: 69%

Section: Qualitative and Quantitative Impact Of Tropomyosin On Neuronmentioning

confidence: 99%

Section: Qualitative and Quantitative Impact Of Tropomyosin On Neuronmentioning

confidence: 99%

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Specific Features of Neuronal Size and Shape Are Regulated by Tropomyosin Isoforms

Schevzov

Bryce

Almonte-Baldonado

et al. 2005

MBoC

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“…Examination of the wound healing process in endothelial cells showed that the ,B-actin isoform, together with its mRNA, was exclusively distributed in leading lamellae, membrane ruffles, and advancing pseudopods (Hoock et al, 1991). Functional diversity of ,B-actin and y-actin isoforms was demonstrated in C2 myoblasts where overexpression of the human f3-actin isoform gene induced an increase in cell surface area and loss of stress fibers, whereas overexpression of the y-actin isoform was associated with a decreased surface area (Schevzov et al, 1992). To verify the role of ,B-actin in generating an enlarged cell phenotype, these authors further demonstrated that transfection of C2 myoblasts with mutant ,3-actin caused the disruption of the actin microfilament network and a decrease in membrane surface area similar to that of C2 cells that were transfected with wild-type y-actin.…”

Section: Discussionmentioning

confidence: 98%

Polarized distribution of actin isoforms in gastric parietal cells.

Yao

Chaponnier

Gabbiani

et al. 1995

MBoC

109

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The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic ,B-actin and -y-actin. Densitometry revealed a ratio for f3-actin/ y-actin that equaled 0.73 + 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the ,B-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of ,B-actin were also identified near the basolateral membrane. The y-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of ,B-actin, but not y-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of 13-actin/ y-actin in the immunoprecipitate (3/,y = 2.14 + 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that ,B-and 'y-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between 1B-actin and ezrin in gastric parietal cells.Finally, our results suggest that the ,B-and y-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion. INTRODUCTION essential role in a variety of cellular processes such as maintaining cell shape, cell motility, and cytokinesis.

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“…Vertebrate cytoplasmic actins and muscle actins are divergent from each other by about 5% in amino acid sequence and 500 million years in time, and they have only a few charged residue interchanges located at their amino terminal ends (Meagher and Williamson, 1994). In addition, some functional differences have been demonstrated between muscle and cytoplasmic actin isovariants, and even between isovariants of cytoplasmic actins (Rubenstein, 1990;Schevzov et aL, 1992). Thus, based on the relatively extreme divergence of ACT2/ACT8 actins from other Arabidopsis actins and the preservation of many of the actin subclasses over long evolutionary periods (McDowell et al, 1996b), we propose that the ACT2/ACT8 protein subclass will show functional differences from the other Arabidopsis actin subclasses.…”

Section: Discussionmentioning

confidence: 99%

Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues

et al. 1996

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SummaryArabidopsis has a complex and ancient actin gene family encoding six divergent subclasses of proteins. One subclass is represented by ACT2 and ACT8, which encode nearly identical proteins. These two genes differ significantly in flanking and intron sequences and in silent nucleotide positions within codons. Gene-specific RNA gel blot hybridization and reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays showed that ACT2 and/or ACT8 mRNAs were coordinately and strongly expressed in leaves, roots, stems, flowers, pollen, and siliques. Together they account for greater than 80% of the actin mRNA in most Arabidopsis organs. The 5' flanking regions, including the promoter, the mRNA leader exon, an intron in the mRNA leader, and the first 19 codons, were coupled to a 13-glucuronidase (GUS) reporter gene and transformed into Arabidopsis. The ACT2/GUS construct was expressed strongly in nearly all the vegetative tissues in seedlings, juvenile plants, and mature plants. These activities persisted in older tissues. Little or no expression was observed in seed coats, hypocotyls, gynoecia, or pollen sacs. In contrast, the expression of the ACT8/GUS construct was weaker. It was observed only in a subset of the organs and tissues expressing ACT2/GUS and was not significantly expressed in the flower. ACT2, ACT8, and ACT8/GUS mRNAs were present at moderate to high levels in pollen, and yet neither ACT2/GUS nor ACT8/GUS enzyme expression could be detected in pollen.This suggested a mechanism of translational control affecting ACT2 and ACT8 expression in some tissues. The conservation of protein sequence and overlapping patterns of expression, in spite of significant DNA sequence divergence, suggests that the function and regulation of these two genes have been conserved during the evolution of the Brassicaceae.

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High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture

Cited by 126 publications

References 44 publications

Specific Features of Neuronal Size and Shape Are Regulated by Tropomyosin Isoforms

Specific Features of Neuronal Size and Shape Are Regulated by Tropomyosin Isoforms

Polarized distribution of actin isoforms in gastric parietal cells.

Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues

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