Cytoplasmic gels from macrophages

Pacaud, Michèle; Molla, Annie

doi:10.1111/j.1432-1033.1987.tb11204.x

Cited by 8 publications

(12 citation statements)

References 50 publications

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“…The discovery of a potential calcium-binding site in Lplastin is consistent with the identity of this isoform with a protein previously identified in human macrophages by Pacaud (13) and Pacaud and Molla (14) that exhibited the same molecular weight (approximately 70,000) and isoelectric point (pl 5.3) as L-plastin in two-dimensional gels. We have previously shown that L-plastin is one of the most…”

supporting

confidence: 62%

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Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Lin¹,

Aebersold²,

Leavitt³

1990

Mol. Cell. Biol.

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Plastins are a family of at least three cytoplasmic protein isoforms that are expressed differentially between cells of the hematopoietic lineages and cells of solid tissues. Expression of the L-plastin isoform appears to be restricted to replicating blood cells, and the two T-plastin isoforms appear to be restricted to replicating cells of solid tissues. However, L-plastin is induced in many human solid tumor-derived cells. We used the anchored polymerase chain reaction technique to amplify and clone the missing 5' ends of plastin mRNAs. We found that both plastin isoforms contain a potential calcium binding site near the N terminus.Plastin was first noted as a polypeptide that appeared to be induced in human fibrosarcomas and other human tumorderived cell lines (2,(6)(7)(8). Initially, expression of a single plastin species was thought to be restricted in normal cells to hematopoietic cell lineages and activated in other cell types as a consequence of neoplastic transformation (2, 8). When plastin cDNAs were isolated from the human fibrosarcoma cell line HuT-14, we discovered that at least two distinct isoforms were coexpressed in this neoplastic cell line: Lplastin, which was normally expressed in blood leukocytes, and T-plastin, which was found in normal cells derived from solid tissues such as fibroblasts, endothelial and epithelial cells, keratinocytes, and melanocytes (8; J. Leavitt and C.-S. Lin, unpublished results). The high cytoplasmic abundance of L-plastin and its normal restriction to expression in circulatory cells led to speculation that its induction in tumor cells may govern some phenotypic characteristics of neoplastic cells, such as reduced cytoplasmic spreading on substrates and reduced anchorage dependence, both of which are normal properties of blood cells (2, 4). Initially, no clues as to the cellular function of plastin isoforms were apparent when we examined the amino acid sequences of the two isoforms. Other than the appearance of L-plastin in human cancer cells and its cytoplasmic location, our knowledge of plastins was limited to the observation that L-plastin was a substrate for phosphorylation at serine residues (1,2,5,12).A number of subsequent observations led us to question whether we had correctly identified the N-terminal amino acid sequence and the 5' ends of these cDNAs for both plastin isoforms. Initially, we identified the N-terminal sequence (8) on the basis of the finding that the open reading frames for both isoform cDNAs ended at a methionine residue (amino acid 58 in Fig. 1). We were unable to identify the N terminus of L-plastin by microsequencing because of N blocking (8). Second, the L-plastin clone contained an insert cDNA of 3.7 kilobases, the size of the L-plastin mRNA. Because the L-plastin cDNA was the size of the mRNA, we assumed that at least part of the upstream noncoding sequence of this cDNA represented the 5' untranslated region of the L-plastin mRNA. We then attempted * Corresponding author. to use this putative 5' sequence to map the transcription star...

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confidence: 62%

“…The macrophage protein was shown to bind to polymerized actin in a calcium-dependent manner (13,14). Also, Matsushima and co-workers (12) have purified L-plastin from human macrophages (identified as L-plastin by sequence [8]) and have demonstrated that it is phosphorylated at serine residues.…”

mentioning

confidence: 99%

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Lin¹,

Aebersold²,

Leavitt³

1990

Mol. Cell. Biol.

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“…In addition to actin and filamin (Hartwig & Stossel, 1975, 1981, other major components of these gels include two actin-bundling proteins, a-actinin and a new 70-kDa1 protein, and two unidentified molecules of 55 kDa and 17 kDa (Pacaud, 1986). We were subsequently able to demonstrate that a rise in the free calcium concentration of cytosolic extracts, within the 0006-2960/93/0432-3448504.00/0 physiological range, could trigger sequential changes in the protein composition of actin gels (Pacaud & Molla, 1987). Further studies on isolated cytoplasmic gels and purified 70-kDA protein led us to suggest that Ca2+ control of the interactions between this protein and F-actin may exert a dominant influence in regulating the state of microfilament organization (Pacaud & Harricane, 1987).…”

mentioning

confidence: 82%

“…Further studies on isolated cytoplasmic gels and purified 70-kDA protein led us to suggest that Ca2+ control of the interactions between this protein and F-actin may exert a dominant influence in regulating the state of microfilament organization (Pacaud & Harricane, 1987). Despite the established view that non-muscle a-actinin is a calciummodulated protein (for a review see Pollard and Cooper (1986), calcium had no effect on the association of a-actinin with F-actin in cytoplasmic gels (Pacaud & Molla, 1987).…”

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confidence: 99%

Purification and further characterization of macrophage 70-kDa protein, a calcium-regulated, actin-binding protein identical to L-plastin

Pacaud¹,

Derancourt²

1993

Biochemistry

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We have previously identified a macrophage 70-kDa, actin-bundling protein as a constituent of actin-based cytoplasmic gel and showed that its association with or dissociation from cytoplasmic gels was remarkably affected by submicromolar calcium. In this study, we purified the 70-kDa protein from soluble cytosolic extracts and carried out a more detailed characterization. The amino acid sequences of four peptidic fragments, obtained from the purified protein by enzymatic or chemical cleavage, were completely or nearly identical to those of L-plastin, a protein initially identified in transformed cells from solid tumors (Goldstein & Leavitt, 1985). By Western blot analysis of normal cells and tissues using specific anti-70-kDa protein antibodies, the 70-kDa molecule was detected only in hematopoietic cells. The 70-kDa protein bound to actin with apparent Kd values of 1.8 and 5.5 microM in the absence and presence of 20 microM free calcium, respectively. Cross-linking activity measured by falling-ball viscosimetry was optimal at free calcium lower than 0.15 microM but was progressively inhibited at higher calcium concentrations, within the physiological range. Half-maximal inhibition occurred at 1.6 microM free calcium. No severing of actin filaments by the 70-kDa protein was observed in any of these assays or previously (Pacaud & Harricane, 1987). Major conformational changes of the protein, as measured by the fluorescence emission intensity of tyrosine residues, occurred at free calcium concentration ranging between 0.15 and 1.5 microM. Magnesium did not mimic the calcium effect. The results suggest that the 70-kDa protein possesses both high-affinity sites and selectivity for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Examples of known Ca 2 § dent interactions of CaM with actin-binding proteins include several proteins that cross-link actin filaments like erythrocyte adducin (Mische et al 1987), those that link actin filaments to other proteins (Anderson and Morrow 1987;Okabe and Sobue 1987), and those regulating smooth-muscle contraction including fodrin (Wang et al 1987), caldesmon (Sobue et al 1988), and myosin light-chain kinase (Yagi et al 1978). In addition, CaM is known to increase the rate of polymerization of F-actin in human platelets (Piazza and Wallace 1985) and to affect actin gelation (Kotani et al 1985;Pacaud and Molla 1987). In Ernodesmis CaM may bind to and activate a yet-unidentified protein, which in turn regulates actin-based motility in these cells.…”

Section: Discussionmentioning

confidence: 97%

Calmodulin and wound healing in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen: Ultrastructure of the cortical cytoskeleton and immunogold labeling

Goddard

Claire

1991

Planta

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Ultrastructural changes in the cortical cytoskeleton during wound-induced cytoplasmic contraction were examined in the coenocytic green alga Ernodesmis verticillata. Both calmodulin (CaM) and actin were localized in intact and contracting cells by immunogold labeling. Within 5 min after wounding, compact microfilament (MF) bundles were observed which increase in diameter as cytoplasmic contraction proceeds. Calmodulin labeling is associated with amorphous material studding the MF bundles, whereas actin labeling occurs along the individual MFs. No MF bundles were ever observed during contraction that were not also labeled with anti-CaM antibodies. In cells treated with the CaM antagonist W-7 (N-[6-aminohexyl]-5-chloro-1-naphtha-lenesulfonamide), MF bundles do not form, and the formation of loosely arranged MFs (similar to nascent bundles in untreated cells) is greatly retarded. We propose that CaM binds indirectly to actin by activating an actin-binding regulatory protein which functions in early stages of the transduction sequence leading to functional MF bundles. Additionally, ultrastructural evidence is presented for a plasma-membrane skeleton or undercoating in this alga.

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Cytoplasmic gels from macrophages

Cited by 8 publications

References 50 publications

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Purification and further characterization of macrophage 70-kDa protein, a calcium-regulated, actin-binding protein identical to L-plastin

Calmodulin and wound healing in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen: Ultrastructure of the cortical cytoskeleton and immunogold labeling

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