Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

Kostyukova, Alla S.; Hitchcock‐DeGregori, Sarah E.

doi:10.1074/jbc.m311186200

Cited by 62 publications

(133 citation statements)

References 60 publications

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“…Tmod1 1-159 fragments were overexpressed in E.coli BL21(DE3)pLysS and additionally purified by reverse-phase high performance liquid chromatography according to (14). The yield of these peptides was low, their expression was poor and the purification was complicated due to the presence of a smaller peptide that was identified as a 1-146 residue fragment of Tmod1.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…To measure polymerization of actin at the pointed end, short filaments capped at the barbed ends with gelsolin were prepared by polymerization of 3 μM G-actin in the presence of 28 nM gelsolin according to (14). Polymerization was monitored by the increase in fluorescence when the filaments were diluted 5-fold with G-actin (10 % pyrenylactin) in Fbuffer (100 mM KCl, 2 mM MgCl 2 , 1 mM EGTA, 0.5 mM DTT, 0.2 mM ATP, 0.2 mM CaCl 2 , 1 mM NaN 3 , 10 mM imidazole, pH 7.0) containing TM and Tmod1.…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

“…9 % polyacrylamide gels that were polymerized in the presence of 10% glycerol without SDS were used (24). To prepare complexes for loading onto gels, equimolar amounts of Tmod in the absence or presence of GlyTM1b [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Zip were mixed in a 1:2 molar ratio. For titration, stock solutions of tropomodulin and GlyTM1b [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Zip were mixed in different ratios in buffer containing 100 mM NaCl.…”

Section: Binding Experimentsmentioning

confidence: 99%

“…AcTM1bzip and AcTM1azipboth form a coiled coil, melt as one cooperative unit, and have the major properties of full-length TM's N-terminus. They bind tropomodulin (9,14,24,26), bind C-terminal TM fragments, and form a ternary complex with troponin (41).Binding of Tmods to AcTM1bZip was directly analyzed using native polyacrylamide gel electrophoresis (Fig. 3).…”

mentioning

confidence: 99%

“…end (13,14). Tmod in combination with TM forms a cap at the pointed end, preventing addition or loss of G-actin (15)(16)(17).…”

mentioning

confidence: 99%

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Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

2006

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Tropomodulin, a tropomyosin-binding protein, caps the slow-growing (pointed) end of the actin filament regulating its dynamics. Tropomodulin, therefore, is important for determining cell morphology, cell movement, and muscle contraction. For the first time we show that one tropomodulin molecule simultaneously binds two tropomyosin molecules in a cooperative manner. On the basis of the tropomodulin solution structure and predicted secondary structure, we introduced a series of point mutations in regions important for tropomyosin-binding and actin-capping. Capping activity of these mutants was assayed by measuring actin polymerization using pyrene fluorescence. Using direct methods (circular dichroism and native gel-electrophoresis) for detecting tropomodulin/ tropomyosin binding, we localized the second tropomyosin-binding site to residues 109-144. Despite previous reports that the second binding site is for erythrocyte tropomyosin only, we found that both short non-muscle and long muscle α-tropomyosins bind there as well, though with different affinities. We propose a model for actin capping where one tropomodulin molecule can bind to two tropomyosin molecules at the pointed end. Keywordstropomodulin; tropomyosin; actin filament; pointed end Actin is a major component of the cell cytoskeleton and the sarcomeres of striated muscle, and plays a critical role in cell morphology, cell movement, and muscle contraction (1,2). It is known that actin filaments form vastly different structures in different cell types and in different locations within the cell. Regulation of the dynamics at actin's ends is of central importance in the assembly of these various structures. An actin filament has two distinct ends: a fastgrowing barbed end and a slow-growing pointed end (3). The exclusive properties of these ends are exploited by over 160 distinct actin binding proteins, including those that cap, sever, or cross-link filaments (4). Of particular interest is tropomodulin (Tmod), a tropomyosinbinding protein, which regulates actin filament dynamics by capping the pointed end.Tropomyosin (TM) is a two-chained coiled coil that binds head-to-tail (N to C terminus) along the length of both sides of actin filaments, spanning 6-7 actin monomers per TM molecule, depending on the isoform (5). There are a variety of TM isoforms encoded by different genes. The N-terminus of TM, which points towards the pointed end of the actin filament (6), is required for interaction with Tmod (7-9). Best known for its role in muscle contraction (10, 11), TM also regulates the stiffness of actin (12) and influences polymerization at the pointed To whom correspondence should be addressed: Alla S. Kostyukova, end (13,14). Tmod in combination with TM forms a cap at the pointed end, preventing addition or loss of G-actin (15-17).Tropomodulin, originally discovered as a TM-binding protein in erythrocyte membranes (18), is the only known pointed end specific capping protein (15). Presently, there are four known Tmod isoforms (16,19,20). These isofor...

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Fluorescence Measurementsmentioning

confidence: 99%

Section: Binding Experimentsmentioning

confidence: 99%

mentioning

confidence: 99%

“…end (13,14). Tmod in combination with TM forms a cap at the pointed end, preventing addition or loss of G-actin (15)(16)(17).…”

mentioning

confidence: 99%

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Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

2006

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Tropomodulins: Pointed‐end capping proteins that regulate actin filament architecture in diverse cell types

et al. 2012

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Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue‐specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed‐end capping activity of Tmods. Tmods are defined by a TM‐regulated/Pointed‐End Actin Capping (TM‐Cap) domain in their unstructured N‐terminal portion, followed by a compact, folded Leucine‐Rich Repeat/Pointed‐End Actin Capping (LRR‐Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods' functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod‐based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. © 2012 Wiley Periodicals, Inc

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Purification of tropomyosin Br‐3 and 5NM1 and characterization of their interactions with actin

et al. 2013

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Tropomyosins were first identified in neuronal systems in 1973. Although numerous isoforms were found and described since then, many aspects of their function and interactions remained unknown. Tropomyosin isoforms show different sorting pattern in neurogenesis. As one example, TM5NM1/2 is present in developing axons, but it is replaced by TMBr‐3 in mature neurons, suggesting that these tropomyosin isoforms contribute differently to the establishment of the functional features of the neuronal actin networks. We developed a method for the efficient purification of TMBr‐3 and TM5NM1 as recombinant proteins using bacterial expression system and investigated their interactions with actin. We found that both isoforms bind actin filaments, however, the binding of TM5NM1 was much stronger than that of TMBr‐3. TMBr‐3 and TM5NM1 modestly affected actin assembly kinetics, in an opposite manner. Consistently with the higher affinity of TM5NM1 it inhibited actin filament disassembly more efficiently than TMBr‐3. Similarly to other previously studied tropomyosins TM5NM1 inhibited the Arp2/3 complex‐mediated actin assembly. Notably, TMBr‐3 did not influence the Arp2/3 complex‐mediated polymerization. This is a unique feature of TMBr‐3, since so far it is the only known tropomyosin supporting the activity of the Arp2/3 complex, indicating that TMBr‐3 may colocalize and work simultaneously with Arp2/3 complex in neuronal cells. © 2013 Wiley Periodicals, Inc.

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Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

Cited by 62 publications

References 60 publications

Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

Tropomodulins: Pointed‐end capping proteins that regulate actin filament architecture in diverse cell types

Purification of tropomyosin Br‐3 and 5NM1 and characterization of their interactions with actin

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