Intracompartmental sorting of essential myosin light chains: Molecular dissection and in vivo monitoring by epitope tagging

Soldati, Thierry; Perriard, Jean‐Claude

doi:10.1016/0092-8674(91)90618-9

Cited by 71 publications

(63 citation statements)

References 39 publications

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“…The involvement of the COOH terminus of the ELC in heavy chain binding is consistent with previous observations (7,8), but participation of residues at NH 2 terminus of the ELC has not been previously demonstrated. We tested the possibility that the NH 2 -terminal residues are required for maintaining an overall conformation of the ELC rather than being in a direct association with the heavy chain.…”

Section: Resultssupporting

confidence: 80%

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Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Chen

Chisholm

1995

Journal of Biological Chemistry

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Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (Pollenz, R. S., Chen, T. L., Trivinos-Lagos, L., and Chisholm, R. L. (1992) Cell 69, 951-962). Since normal myosin function requires association of ELC, we investigated the domains of ELC that are necessary for binding to the myosin heavy chain (MHC). Deleting the NH 2 -terminal 11 or 28 amino acid residues (⌬N11 or ⌬N28) or the COOH-terminal 15 amino acid residues (⌬C15) abolished binding of the ELC to the MHC when the mutants were expressed in wild-type (WT) cells. In contrast, the ELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in WT cells, although less efficient competition with WT ELC suggested that the affinity for the MHC is reduced. When these mutants were expressed in ELC-minus (mlcE ؊ ) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous ELC, ⌬N28 and ⌬N11 bound to the MHC at 15% of WT levels and ⌬C15 did not bind to a significant degree. I4 and D4, however, bound with normal stoichiometry. These data indicate that residues at both termini of the ELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants were analyzed for their ability to complement the cytokinesis defect displayed by mlcE ؊ cells, a correlation to the level of ELC carried by the MHC was observed, indicating that a stoichiometric ELC-MHC association is necessary for normal myosin function in vivo.Myosin is a mechanochemical enzyme that generates force during muscle contraction and has a fundamental role in a variety of cell movements (for review, see Ref. 2). Conventional myosin is a hexameric protein composed of a pair of heavy chains (MHC) 1 and two pairs of light chains, called essential light chains (ELC) and regulatory light chains (RLC). Both light chains bind to the neck of myosin (3, 4). We have shown that ELC-deficient Dictyostelium cell lines are defective in cytokinesis, and myosin purified from these cells does not show significant actin-activated ATPase activity (1). Since normal myosin function requires association of the ELC, we set out to map the domains of the ELC that are critical for ELC-MHC interaction.Sequence comparisons of ELCs from various sources show that the COOH-terminal half of the molecule is more conserved than the NH 2 -terminal half (5, 6) and that it may represent the site required for association with MHC. Skeletal muscle alkali light chain A1 in which the COOH-terminal 14 residues were chemically removed did not bind the S1 heavy chain, although the conformation remained largely unchanged (7). Transfection studies also showed that a COOH-terminal truncation of the alkali light chain A1 failed to colocalize with acto-myosin structures in cultured myocytes (8).The ELC belongs to the calmodulin-troponin gene family, which contains four helix-loop-helix structures, also known as EF-hand...

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Section: Resultssupporting

confidence: 80%

“…Skeletal muscle alkali light chain A1 in which the COOH-terminal 14 residues were chemically removed did not bind the S1 heavy chain, although the conformation remained largely unchanged (7). Transfection studies also showed that a COOH-terminal truncation of the alkali light chain A1 failed to colocalize with acto-myosin structures in cultured myocytes (8).…”

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confidence: 89%

Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Chen

Chisholm

1995

Journal of Biological Chemistry

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“…Chaussepied and Kasprzak (1989) have shown that the presence of different MLC isoforms in a myosin molecule confers different properties of association with actin. A study of intracompartmental sorting of MLC isoforms in rat cardiomyocytes has suggested that differences in affinity between MHC and MLC isoforms affect the sorting process (Soldati and Perriard, 1991). Work on the body wall musculature of C .…”

Section: Significance Of the Differentiation Programmentioning

confidence: 99%

Identification of a program of contractile protein gene expression initiated upon skeletal muscle differentiation

Sutherland

Esser

Elsom

et al. 1993

Developmental Dynamics

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The functional diversity of skeletal muscle is largely determined by the combinations of contractile protein isoforms that are expressed in different fibers. Just how the developmental expression of this large array of genes is regulated to give functional phenotypes is thus of great interest. In the present study, we perform a comprehensive analysis of contractile protein isoform mRNA profiles in skeletal muscle systems representing each generation of fiber formed: primary, secondary, and regenerating fibers. We find that in each system examined there is a common pattern of isoform gene expression during early differentiation for 5 of the 6 gene families we have investigated: myosin light chain (MLCI1, MLC2, tropomyosin, troponin (Tn)C, and TnI. We suggest that the common isoform patterns observed together represent a genetic program of skeletal muscle differentiation that is independent of the mature fiber phenotype and is found in all newly formed myotubes. Within each of these contractile protein gene families the program is independent of the isoforms of myosin heavy chain (MHC) expressed. The maintenance of such a program may reflect a specific requirement of the initial differentiation process. 0 1993 Wiley-Liss, Inc.

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“…For transient and stable transfections, we used a pCMV␤ vector (Clontech Laboratories AG, Basel, CH), in which the neomycin gene was cloned in EcoRI and in which the ␤-galactosidase gene was replaced by full-length cDNA encoding rat ␣-SMA, human ␥-and ␤-cytoplasmic actins, and chicken ␣-cardiac muscle actin. The 3Ј untranslated region (3ЈUTR) of actin isoforms was replaced by cDNA coding for the vesicular stomatitis virus G-protein (VSV-G; Soldati and Perriard, 1991), thus tagging the actins on C terminus. 3T3 fibroblasts were transfected with the use of FuGene 6 (Boehringer Mannheim AG, Mannheim, Germany) according to the manufacturer's protocol.…”

Section: Actin Constructs and Transfectionmentioning

confidence: 99%

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

Hinz

Celetta

Tomasek³

et al. 2001

MBoC

1,135

947

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To evaluate whether ␣-smooth muscle actin (␣-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of ␣-SMA, with that of lung fibroblasts (LFs), expressing high levels of ␣-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of ␣-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for ␣-SMA. In a second approach, we measured the isotonic contraction of SCF-and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGF␤1 increased ␣-SMA expression and lattice contraction by SCFs to the levels of LFs; TGF␤-antagonizing agents reduced ␣-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with ␣-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with ␣-cardiac and ␤-or ␥-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased ␣-SMA expression is sufficient to enhance fibroblast contractile activity. INTRODUCTIONEarly during healing of an open wound, resident dermal fibroblasts proliferate from the wound margin and migrate into the provisional matrix composed of a fibrin clot. About 1 week after wounding, the provisional matrix is replaced by neo-formed connective tissue, known as granulation tissue, essentially composed of small vessels, extracellular matrix, and fibroblastic cells that become activated and modulate into myofibroblasts. The main feature of myofibroblasts is represented by an important contractile apparatus similar to that of smooth muscle (Gabbiani et al., 1971), and in particular by the neo-expression of ␣-smooth muscle actin (␣-SMA), the actin isoform typical of vascular smooth muscle cells (Skalli et al., 1986). Myofibroblasts are recognized to play a central role in closing the wound tissue, through their capacity to produce a strong contractile force (for review see Grinnell, 1994;Rønnov-Jessen et al., 1996;Powell et al., 1999;Serini and Gabbiani, 1999), possibly generated within stress fibers, similar to those present in cultured fibroblasts (Skalli et al., 1986;Serini and Gabbiani, 1999). Because wound contraction takes place when de novo expressed ␣-SMA is incorporated in stress fibers (Darby et al., 1990), it has been suggested that this actin isoform plays an important role in granulation tissue contraction (for review see Serini and Gabbiani, 1999). Although stress fibers have been proposed to function as contractile organelles (Burridge, 1981;Katoh et al., 1998) and their presence has been correlated with the production of isometric tension (Harris et al., 1981), at p...

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Intracompartmental sorting of essential myosin light chains: Molecular dissection and in vivo monitoring by epitope tagging

Cited by 71 publications

References 39 publications

Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Identification of a program of contractile protein gene expression initiated upon skeletal muscle differentiation

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

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