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V M Fowler

Publications and source records attributed to V M Fowler.

At least 19 recordsLinked to original sources
PubMed1997

Purification and characterization of an alpha 1 beta 2 isoform of CapZ from human erythrocytes: cytosolic location and inability to bind to Mg2+ ghosts suggest that erythrocyte actin filaments are capped by adducin.

CapZ ("capping protein") is a heterodimeric actin capping protein that blocks actin filament assembly and disassembly at the fast growing (barbed) filament ends and is proposed to function in regulating actin filament dynamics as well as in stabilizing actin filament lengths in muscle and nonmuscle cells. We show here that erythrocytes contain a nonmuscle isoform of capZ (EcapZ) that is present exclusively in the cytosol and is not associated with the short actin filaments in the erythrocyte membrane skeleton. This is unlike other cell types where capZ is associated with cytoskeletal actin filaments and suggests that cytosolic EcapZ may be inactive, or alternatively, that the barbed ends are capped by adducin, a membrane skeleton protein that was shown recently to cap actin filament barbed ends in vitro [Kuhlman, P. A., Hughes, C. A., Bennett, V., & Fowler, V. M. (1996) J. Biol. Chem. 271, 7986]. To distinguish between these possibilities, we purified EcapZ from erythrocyte cytosol and characterized its biochemical and functional properties. Two-dimensional gel electrophoresis and western blotting reveals the EcapZ subunit composition to be alpha1beta2, as described for capZ from many other nonmuscle cells, with no evidence for posttranslational modifications. Purified EcapZ is fully functional in blocking actin elongation from barbed filament ends (Kcap approximately 1-5 nM) as well as in nucleating actin polymerization. Furthermore, cytosolic EcapZ binds to actin filament barbed ends, indicating that sequestering of EcapZ by a cytosolic inhibitory factor or insufficient amounts of EcapZ in cytosol also cannot account for its absence from the membrane skeleton. To test directly whether the barbed ends of the erythrocyte actin filaments were already capped, we measured binding of purified EcapZ to isolated membranes. Purified EcapZ does not cosediment with membranes prepared by hypotonic lysis in the presence of magnesium, suggesting that the barbed ends of the erythrocyte actin filaments are capped under these conditions but not by EcapZ. In contrast, purified EcapZ stoichiometrically reassociates with all the actin filament barbed ends in membranes prepared by hypotonic lysis in 5 mM sodium phosphate, pH 8.0 (5P8), conditions in which the barbed filament ends were previously reported to be uncapped. Comparison of the amounts of adducin associated with membranes prepared in the presence and absence of magnesium reveals that 60-80% of the adducin dissociates from the membrane during hemolysis and washing in 5P8 buffer, suggesting that the barbed ends become artifactually uncapped due to loss of adducin. The erythrocyte actin filaments may thus represent a specialized class of membrane-associated actin filaments that are capped by adducin instead of capZ.

Actin Cytoskeleton↗
PubMed1997

Two populations of beta-spectrin in rat skeletal muscle.

We use immunoblotting, immunoprecipitation, and centrifugation in sucrose density gradients to show that the product of the erythrocyte beta-spectrin gene in rat skeletal muscle (muscle beta-spectrin) is present in two states, one associated with fodrin, and another that is not associated with any identifiable spectrin or fodrin subunit. Immunofluorescence studies indicate that a significant amount of beta-spectrin without alpha-fodrin is present in the myoplasm of some muscle fibers, and, more strikingly, at distinct regions of the sarcolemma. These results suggest that alpha-fodrin and muscle beta-spectrin associate in muscle in situ, but that some muscle beta-spectrin without a paired alpha-subunit forms distinct domains at the sarcolemma.

Animals↗
PubMed1997

Capping actin filament growth: tropomodulin in muscle and nonmuscle cells.

Actin filament lengths are precisely regulated and very stable in the sarcomeres of striated muscle, in the erythrocyte membrane skeleton, and in cell protrusions such as microvilli in intestinal epithelial cells and stereocilia in hair cells of the inner ear. In contrast, in motile cells, actin filament lengths are dynamically regulated when cells extend lamellipodia. Control of actin filament lengths and dynamics in cells is expected to be achieved in part by capping proteins that prevent filament growth or shrinkage by blocking subunit exchange at both the fast growing (barbed) and slow growing (pointed) filament ends. Much is known about how barbed end capping proteins control actin filament assembly and length in many cells, but little is known about the significance of regulating actin filament assembly at the pointed end. Tropomodulin is the only known capping protein for the pointed ends of actin filaments and is a approximately 40-kD protein that is expressed in erythrocytes, striated muscle, lens fiber cells, some regions of the adult brain, as well as sensory neurons and epithelial cells of the inner ear. A related isoform (59% identical at the protein level) is expressed principally in neurons of both embryonic and adult brain. In striated muscle and in erythrocytes, tropomodulin is tightly associated with the actin filament pointed ends where it functions to maintain actin filament length in vivo (for recent reviews, see Fowler, 1996; Gregorio and Fowler, 1996). Unlike proteins that cap actin filament barbed ends, tropomodulin also binds tropomyosin and requires tropomyosin for tight capping of actin filament pointed ends. Mapping of functional domains on tropomodulin shows that the COOH-terminal end of tropomodulin is important for actin filament pointed end capping activity while the NH2-terminal portion of tropomodulin contains the tropomyosin binding domain. Searches of protein and EST databases for tropomodulin-like sequences reveal a number of proteins with homologies to both the tropomyosin binding and the actin filament capping portions of tropomodulin. In particular, we have identified a tropomodulin-like 64-kD protein that is principally expressed in smooth muscle cells. We anticipate that tropomodulin and this 64-kD protein are members of a larger family of tropomyosin and actin binding proteins that are responsible for capping actin filament pointed ends and regulating actin filament lengths in muscle and nonmuscle cells.

Actins↗
PubMed1996

A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments.

Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.

Actin Cytoskeleton↗
PubMed1996

Cytoskeleton.

Explore the source record for details and available documents.

Cytoskeleton↗
PubMed1996

Regulation of actin filament length in erythrocytes and striated muscle.

Actin filaments polymerize in vitro to lengths which display an exponential distribution, yet in many highly differentiated cells they can be precisely maintained at uniform lengths in elaborate supramolecular structures. Recent results obtained using two classic model systems, the erythrocyte membrane cytoskeleton and the striated muscle sarcomere, reveal surprising similarities and instructive differences in the molecules and mechanisms responsible for determining and maintaining actin filament lengths in these two systems. Tropomodulin caps the slow-growing, pointed filament ends in muscle and in erythrocytes. CapZ caps the fast-growing, barbed filament ends in striated muscle, whereas a newly discovered barbed end capping protein, adducin, may cap the barbed filament ends in erythrocytes. The mechanisms responsible for specifying the characteristic filament lengths in these systems are more elusive and may include strict control of the relative amounts of actin filament capping proteins and side-binding proteins, molecular templates (e.g. tropomyosin and nebulin) and/or verniers (e.g. tropomyosin).

Actins↗
PubMed1995

Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes.

Control of actin filament length and dynamics is important for cell motility and architecture and is regulated in part by capping proteins that block elongation and depolymerization at both the fast-growing (barbed) and slow-growing (pointed) ends. Tropomodulin is a capping protein for the pointed end of the actin filament; it is associated with the free, pointed ends of the thin filaments in striated muscle, where it is thought to bind to both tropomyosin and actin. In embryonic chick cardiac myocytes, tropomodulin assembles after the thin, as well as the thick, filaments have become organized into periodic I and A bands, suggesting that tropomodulin might be involved in maintaining actin filament length. Here we show that microinjection of an antibody that inhibits tropomodulin's pointed-end-capping activity in vitro results in a marked elongation of actin filaments from their pointed ends and a > 80% reduction in the percentage of beating cells. This demonstrates that pointed-end capping by tropomodulin is required to maintain actin filament length in vivo and that this is essential for contractile function in embryonic chick cardiac myocytes.

Actins↗
PubMed1995

Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly.

Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.

Actin Cytoskeleton↗
PubMed1994

Isoform-specific interaction of tropomodulin with skeletal muscle and erythrocyte tropomyosins.

Tropomodulin is a tropomyosin-binding protein that localizes to the pointed end of striated muscle thin filaments and caps the pointed end of tropomyosin-actin filaments in vitro. Results from previous studies have suggested that tropomyosin-tropomodulin interactions are isoform-specific. To investigate the molecular basis for the isoform specific interactions of tropomyosin and tropomodulin, we isolated a cDNA for chicken skeletal muscle tropomodulin. The derived amino acid sequence of muscle tropomodulin is 86% identical with that of human erythrocyte tropomodulin, indicating that tropomodulins are highly conserved proteins. Multiple mRNAs seen on Northern blots of chicken skeletal muscle mRNA can be accounted for by multiple polyadenylation signals in the 3'-untranslated region of the cDNA. 125I-Labeled skeletal muscle and erythrocyte tropomyosins were assayed for binding bacterially expressed muscle tropomodulin using a solid phase binding assay. Unexpectedly, skeletal muscle and erythrocyte tropomyosins bound with similar affinities to muscle tropomodulin (Kd values approximately 0.2 microM). However, cross-competition studies using erythrocyte and skeletal muscle tropomyosins indicated that they bound different sites on tropomodulin. Competition studies using recombinant fragments of tropomodulin to map the binding domains for the two tropomyosins demonstrated that residues 6-94 contained the skeletal muscle tropomyosin binding site and residues 90-184 contained the erythrocyte tropomyosin binding site. Binding of different regions of tropomodulin to muscle and non-muscle tropomyosins may permit interaction of tropomodulin with tissue-specific components or may influence the pointed end capping activity of tropomodulin.

Amino Acid Sequence↗
PubMed1994

Dynamic properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase C beta.

Ankyrin is a well characterized membrane skeletal protein which has been implicated in the anchorage of specific integral membrane proteins to the spectrin-based membrane skeleton in a number of systems. In this study, the organization of ankyrin was examined in lymphocytes in relation to T cell function. Light and electron microscope immunolocalization studies revealed extensive heterogeneity in the subcellular distribution of ankyrin in murine tissue-derived lymphocytes. While ankyrin can be localized at the lymphocyte plasma membrane, it can also be accumulated at some distance from the cell periphery, in small patches or in a single discrete, nonmembrane-bound structure. Double immunofluorescence studies demonstrated that ankyrin colocalizes with spectrin and with the signal transducing molecule protein kinase C beta (PKC beta) in tissue-derived lymphocytes, suggesting a functional association between these molecules in the lymphocyte cytoplasm. In addition, T lymphocyte activation-related signals and phorbol ester treatment, both of which lead to PKC activation, cause a rapid translocation of ankyrin, together with spectrin and PKC beta, to a single Triton X-100-insoluble aggregate in the cytoplasm. This finding suggests a mechanism for the reported appearance of PKC in the particulate fraction of cells after activation: activated lymphocyte PKC beta may interact with insoluble cytoskeletal elements like ankyrin and spectrin. Further evidence for a link between the subcellular organization of these proteins and PKC activity is provided by the observation that inhibitors of PKC activity cause their concomitant redistribution to the cell periphery. The dynamic nature of lymphocyte ankyrin and its ability to accumulate at sites distant from the plasma membrane are properties which may be unique to the lymphocyte form of the molecule. Its colocalization with PKC beta in the lymphocyte cytoplasm, together with its redistribution in response to physiological signals, suggests that structural protein(s) may play a role in signal transduction pathways in this cell type. Our data support the conclusion that ankyrin is not solely involved in anchorage of proteins at the plasma membrane in lymphoid cells.

Animals↗
PubMed1994

Tropomodulin caps the pointed ends of actin filaments.

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.

Actin Cytoskeleton↗
PubMed1994

Identification and characterization of tropomodulin and tropomyosin in the adult rat lens.

The lens fiber cells express all the major components of the erythrocyte membrane skeleton including spectrin, protein 4.1 and ankyrin. We have used immunoblot and immunoprecipitation analyses, as well as immunofluorescence localization to identify and characterize two additional components of the membrane skeleton in the rat lens: tropomyosin and the tropomyosin-binding protein tropomodulin. In the erythrocyte, tropomyosin and tropomodulin are proposed to stabilize and limit the lengths of the short actin filaments of the spectrin-actin network, thus influencing the organization and mechanical properties of the erythrocyte membrane skeleton. Antibodies directed against erythrocyte tropomodulin specifically recognize a M(r) 43,000 polypeptide from rat lens that comigrates with erythrocyte tropomodulin on SDS-gels. A non-muscle isoform of tropomyosin is also present in the lens. This tropomyosin isoform migrates on SDS-gels with a M(r) of approximately 28,000 and is distinct from the two erythrocyte isoforms of tropomyosin (M(r) 27,000 and 29,000). Indirect immunofluorescence staining of 5 microns cryosections of adult rat lens reveals that both tropomodulin and tropomyosin colocalize with rhodamine phalloidin staining for actin filaments on fiber cell plasma membranes. Lens tropomodulin exhibits many characteristics that are similar to its erythrocyte counterpart. For example, lens tropomodulin binds tropomyosin in a solid-phase blot binding assay, and extraction experiments with Triton X-100, urea and NaOH show that the membrane-bound tropomodulin in the lens is a tightly associated peripheral membrane protein that is a component of the Triton-insoluble cytoskeleton. However, unlike the erythrocyte, there are approximately 2000 actin monomers per tropomodulin in the lens.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Immunolocalization of tropomodulin, tropomyosin and actin in spread human erythrocyte skeletons.

The human erythrocyte membrane skeleton consists of a network of short actin filaments cross-linked into a hexagonal network by long, flexible spectrin molecules. The lengths of the short actin filaments (33 +/- 5 nm) at the central junctions are proposed to be stabilized and limited by association with tropomyosin and the tropomyosin-binding protein, tropomodulin. Here, we use immunogold labelling followed by negative staining to specifically localize tropomodulin, tropomyosin and actin to the sites of the central junctions in spread membrane skeletons. In addition to negative staining, immunogold labelling for tropomodulin at the sites of the central junctions was also visualized by a quick-freeze, deep-etch, rotary-replication technique. These experiments confirm previous indirect evidence that the short filaments at the central junctions are indeed actin filaments and provide the first direct evidence that tropomodulin and tropomyosin are associated with the erythrocyte actin filaments in situ.

Actins↗
PubMed1993

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle.

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.

Actin Cytoskeleton↗
PubMed1992

Tropomodulin binding to tropomyosins. Isoform-specific differences in affinity and stoichiometry.

Tropomodulin is a human erythrocyte membrane cytoskeletal protein that binds to one end of tropomyosin molecules and inhibits tropomyosin binding to actin filaments [Fowler, V. M. (1990) J. Cell Biol. 111, 471-482]. We have characterized the interaction of erythroid and non-erythroid tropomyosins with tropomodulin by non-denaturing gel electrophoresis and by solid-phase binding assays using 125I-tropomyosin. Non-denaturing gel analysis demonstrates that all tropomodulin molecules are able to bind tropomyosin and that tropomodulin forms complexes with tropomyosin isoforms from erythrocyte, brain, platelet and skeletal muscle tissue. Scatchard analysis of binding data using tropomyosin isoforms from these tissues indicate that tropomodulin binds preferentially to erythrocyte tropomyosin. Specificity is manifested by decreases in the apparent affinity or the saturation binding capacity of tropomodulin for non-erythrocyte tropomyosins. Erythrocyte tropomyosin saturates tropomodulin at approximate stoichiometric ratios of 1:2 and 1:4 tropomyosin/tropomodulin (apparent Kd = 14 nM-1 and 5 nM-1, respectively). Brain tropomyosin saturates tropomodulin at a 1:2 ratio of tropomyosin/tropomodulin, but with a threefold lower affinity than erythrocyte tropomyosin. Platelet tropomyosin saturates tropomodulin at a tropomyosin/tropomodulin ratio of 1:4, but with a sevenfold lower affinity than erythrocyte tropomyosin at the 1:4 ratio. These results correlate with oxidative cross-linking data which indicate that tropomodulin can self-associate to form dimers and tetramers in solution. Since tropomodulin interacts with one of the ends of tropomyosin, varying interactions of tropomyosin isoforms with tropomodulin probably reflect the heterogeneity in N-terminal or C-terminal sequences characteristic of the different tropomyosin isoforms. Isoform-specific interactions of tropomodulin with tropomyosins may represent a novel mechanism for selective regulation of tropomyosin/actin interactions.

Carrier Proteins↗
PubMed1992

Molecular cloning and characterization of human fetal liver tropomodulin. A tropomyosin-binding protein.

Human erythrocyte tropomodulin is a novel tropomyosin regulatory protein that binds to the end of erythrocyte tropomyosin and blocks heat-to-tail association of tropomyosin along actin filaments. It has been proposed to play a role in modulating the association of tropomyosin with the spectrin-actin complex in the erythrocyte membrane skeleton. Immunoscreening of a human fetal liver cDNA expression library in lambda gt11, followed by 5'-end extension by polymerase chain reaction from the same library, yielded a composite cDNA sequence of 2665 base pairs (bp). It contains a 34-bp 5'-untranslated region, a 1.6-kilobase (kb) 3'-untranslated region, and a complete open reading frame of 1077 bp that encodes a protein of 359 amino acids with a calculated molecular mass of 40.6 kDa and a pI of 4.8. Authenticity of the tropomodulin cDNA was confirmed by a complete sequence match of 49 predicted amino acids with the sequences of three tryptic peptides of the erythrocyte tropomodulin. The sequence has no internal repeats and no significant homology with any known proteins. Secondary structure predictions indicate that tropomodulin may consist of a series of seven or eight short alpha-helical segments and fold into a somewhat compact shape. The tropomyosin binding activity has been mapped to an N-terminal region containing residues 39-138. Nine independent PCR clones, five from a human reticulocyte cDNA library and four from the fetal liver cDNA library, revealed identical N-terminal 103 amino acids, suggesting that the sequence reported here may also be of erythrocyte tropomodulin. Northern analysis of human reticulocyte RNA showed two hybridizing bands of 2.7 and 1.6 kb, indicating that the 2665-bp cDNA sequence reported here was that of the longer transcript.

Amino Acid Sequence↗