Crystal structure and molecular interactions of tropomyosin

Phillips, George N.; Lattman, Eaton E.; Cummins, Peter L.; Lee, K. Y.; Cohen, Carolyn

doi:10.1038/278413a0

Cited by 134 publications

(80 citation statements)

References 31 publications

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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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Section: Introductionmentioning

confidence: 99%

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

Schevzov

Bryce

Almonte-Baldonado

et al. 2005

MBoC

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“…Of these, pI 10-1.3 and p1 10-1.5 bound both p85at and p85( ( Figure 8, (Bernstein et al, 1977) was also searched with the sequence of the inter-SH2 region. Here two of the top three hits were with tropomyosin, a two stranded oa-helical coiled-coil muscle protein whose structure has been solved to 15 A resolution (Philips et al, 1979(Philips et al, , 1986. Although the amino acid sequence identity with tropomyosin was quite low (20% over 175 amino acids), the heptad repeats in the inter-SH2 region and that of tropomyosin were in register for a considerable part of the alignment.…”

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PI 3-kinase: structural and functional analysis of intersubunit interactions.

et al. 1994

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Phosphatidylinositol (PI) 3-kinase has an 85 kDa subunit (p85a) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85a and a related protein p853 are shown to form stable complexes with recombinant pllO in vivo and in vitro. Using a panel of glutathione S-transferase (GST) fusion proteins of the inter-SH2 region of p85, 104 amino acids were found to bind directly the pllO protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85ct protein in mouse L cells showed it was unable to bind PI 3-kinase activity in vivo. Mapping of the complementary site of interaction on the pllO protein defined 88 amino acids in the N-terminal region of pllO which mediate the binding of this subunit to either the p85a or the p853 proteins. The inter-SH2 region of p85 is predicted to be an independently folded module of a coiled-coil of two long anti-parallel c-helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3-kinase complex is discussed.

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“…It is a dimeric protein forming an elongated ␣-helical coiled-coil (4,5). It is an actin-associated protein, the dimeric units interacting end-to-end along actin filaments to form a continuous strand that wraps around the surface of the actin filament (6,7).…”

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Ultra Short Yeast Tropomyosins Show Novel Myosin Regulation

Maytum

Hatch

Konrad

et al. 2008

Journal of Biological Chemistry

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Tropomyosin (Tm) is an ␣-helical coiled-coil actin-binding protein present in all eukaryotes from yeast to man. Its functional role has been best described in muscle regulation; however its much wider role in cytoskeletal actin regulation is still to be clarified. Isoforms vary in size from 284 or 248 amino acids in vertebrates, to 199 and 161 amino acids in yeast, spanning from 7 to 4 actin binding sites respectively. In Saccharomyces cerevisiae, the larger yTm1 protein is produced by an internal 38-amino acid duplication, corresponding to a single actin-binding site. We have produced an ultra-short Tm with only 125 amino acids by removing both of the 38 amino acid repeats from yTm1, with the addition of an Ala-Ser extension used to mimic the essential N-terminal acetylation. This short Tm, and an M1T mutant of it, bind to actin with a similar affinity to most Tms previously studied (K 50% ϳ 0.5 M). However, an equilibrium fluorescence binding assay shows a much greater inhibition of myosin binding to actin than any previously studied Tm. Actin cosedimentation assays show this is caused by direct competition for binding to actin. The M1T mutant shows a reduced inhibition, probably due to weaker end-to-end interactions making it easier for myosin to displace Tm. All previously characterized Tms, although able to sterically block the myosinbinding site, are able to bind to actin along with myosin. By showing that Tm can compete directly with myosin for the same binding site these new Tms provide direct evidence for the steric blocking model. Tropomyosin (Tm)2 appears to be as ubiquitous as actin in eukaryotes (1-3). It is a dimeric protein forming an elongated ␣-helical coiled-coil (4, 5). It is an actin-associated protein, the dimeric units interacting end-to-end along actin filaments to form a continuous strand that wraps around the surface of the actin filament (6, 7). In this way it acts to stabilize actin filaments, as shown by the effect of knockouts of TPM1 in yeast, which have a sickly phenotype due to loss of their actin stress fibers (8, 9). It has been shown to be an essential protein in yeast, with knockouts expressing no Tm being lethal (9). However Tm functions not just as a structural protein, but also a regulatory one. Its role is best understood in the regulation of muscle contraction, where it is directly involved in regulating the myosinactin interaction (reviewed in Ref. 10). Its wider role in regulation of non-muscle actin filaments is less well understood, although it is becoming clear that it plays an important role in functional regulation of the cytoskeleton (11).From its essential roles in cytoskeletal and muscle regulation it is clearly important to understand how Tm functions.Although structurally it appears like a simple rod-like molecule, higher vertebrates express a large diversity of Tm isoforms. Mammals express over 20 isoforms from four different genes (1). As highlighted in the skTm sequence shown in Fig. 1A, alternate splicing of the 9 exon mammalian tropomyosins is limited to ...

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Crystal structure and molecular interactions of tropomyosin

Cited by 134 publications

References 31 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

PI 3-kinase: structural and functional analysis of intersubunit interactions.

Ultra Short Yeast Tropomyosins Show Novel Myosin Regulation

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