Tropomodulin Increases the Critical Concentration of Barbed End-capped Actin Filaments by Converting ADP·Pi-actin to ADP-actin at All Pointed Filament Ends

Weber, Annemarie; Pennise, Cynthia R.; Fowler, Velia M.

doi:10.1074/jbc.274.49.34637

Cited by 81 publications

(139 citation statements)

References 28 publications

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“…Another LIM domain containing protein present in the PSD fraction, the cysteine rich protein, has been shown to interact with the actin cytoskeleton and may be involved in muscle differentiation (57). Tropomodulin lowers the apparent affinity of pointed ends of the actin filament for actin monomers (58). Syndapin regulates endocytosis and actin dynamics (59).…”

Section: Discussionmentioning

confidence: 99%

Proteomics Analysis of Rat Brain Postsynaptic Density

Hornshaw

Schors

et al. 2004

Journal of Biological Chemistry

233

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The postsynaptic density contains multiple protein complexes that together relay the presynaptic neurotransmitter input to the activation of the postsynaptic neuron. In the present study we took two independent proteome approaches for the characterization of the protein complement of the postsynaptic density, namely 1) two-dimensional gel electrophoresis separation of proteins in conjunction with mass spectrometry to identify the tryptic peptides of the protein spots and 2) isolation of the trypsin-digested sample that was labeled with isotope-coded affinity tag, followed by liquid chromatography-tandem mass spectrometry for the partial separation and identification of the peptides, respectively. Functional grouping of the identified proteins indicates that the postsynaptic density is a structurally and functionally complex organelle that may be involved in a broad range of synaptic activities. These proteins include the receptors and ion channels for glutamate neurotransmission, proteins for maintenance and modulation of synaptic architecture, sorting and trafficking of membrane proteins, generation of anaerobic energy, scaffolding and signaling, local protein synthesis, and correct protein folding and breakdown of synaptic proteins. Together, these results imply that the postsynaptic density may have the ability to function (semi-) autonomously and may direct various cellular functions in order to integrate synaptic physiology.The majority of excitatory neurotransmission in the brain occurs via glutamatergic synapses. In the presynaptic element of the synapse, specialized secretion machinery determines the activity-dependent membrane fusion of glutamate-containing vesicles and the release of transmitter into the synaptic cleft. In the postsynaptic element, glutamate receptors and downstream signal transduction are organized by the protein assembly of the postsynaptic density (PSD). 1 Both the presynaptic release machinery and the PSD are electron-dense assemblies (1, 2), in which proteins are thought to be organized into distinct functional complexes (3-5) that may be dynamically regulated by neuronal activity (6 -8). The modulation of this molecular architecture of the synapse is at the basis of synaptic plasticity (6 -8), and aberrations thereof may underlie neuronal disorders.In view of the importance of the PSD in glutamatergic neurotransmission and its involvement in neuroplasticity, considerable efforts have been made to identify its protein constituents as a prelude to understand the molecular basis of PSD functioning. In the past several years, yeast two-hybrid technology has been extensively used to characterize proteins that interact with the glutamate receptors and may constitute core elements of the PSD involved in the regulation of receptor trafficking and signaling (reviewed in Refs. 9 -11). Based largely on these studies a protein-protein interaction map of the PSD has emerged. In brief, the model posits that the postsynaptic receptor complexes are localized by scaffolding proteins such as the s...

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Section: Discussionmentioning

confidence: 99%

Proteomics Analysis of Rat Brain Postsynaptic Density

Hornshaw

Schors

et al. 2004

Journal of Biological Chemistry

233

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“…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%

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

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confidence: 99%

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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“…A physical interpretation of this result is that profilin lowers A c to that of a barbed end with terminal subunits containing ATP, and there is evidence that the nucleotide content of terminal subunits influences A c (35). With a balanced energy square and insignificant contribution from pointed ends, A c ϭ A cb .…”

Section: Contribution Of An Energy Imbalance To the Effect Of Profilimentioning

confidence: 99%

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

Yarmola

Bubb

2004

Journal of Biological Chemistry

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The free actin concentration at steady state, A c , is a variable that determines how actin regulatory proteins influence the extent of actin polymerization. We describe a novel method employing fluorescence anisotropy to directly measure A c in any sample after the addition of a trace amount of labeled thymosin ␤ 4 or thymosin ␤ 4 peptide. Using this assay, we confirm earlier theoretical work on the helical polymerization of actin and confirm the effects of actin filament-stabilizing drugs and capping proteins on A c , thereby validating the assay. We also confirm a controversial prior observation that profilin lowers the critical concentration of Mg 2؉ -actin. A general mechanism is proposed to explain this effect, and the first quantitative dose-response curve for the effect of profilin on A c facilitates its evaluation. This mechanism also predicts the effect of profilin on critical concentration in the presence of the limited amount of capping protein, which is the condition often found in cells, and the effect of profilin on critical concentration in cell extracts is demonstrated for the first time. Additionally, nonlinear effects of thymosin ␤ 4 on the steady state amount of F-actin are explained by the observed changes in A c . This assay has potential in vivo applications that complement those demonstrated in vitro.Total cellular actin is equal to the sum of the concentration of free monomer (the critical concentration, A c ), the concentration of unpolymerized actin sequestered by various actin-binding proteins, and the concentration of actin polymer or F-actin. Changes in A c induced by actin regulatory proteins influence actin polymer dynamics by an amplification mechanism that results in large changes in the amount of sequestered, unpolymerized actin. Quantitative evaluation of models of cytoskeletal function requires accurate knowledge of the value of A c . Unfortunately, A c has not yet been measured in cells and has only been estimated by very indirect methods in cell extracts (1). In vitro methods have been developed that allow for the measurement of F-actin using birefringence (2), viscosity (3), light scattering (4), centrifugation (5), binding of labeled phalloidin (6), or the fluorescence of pyrenyl-labeled actin (7). Other methods such as the DNase I binding assay yield the sum of A c and an indeterminate fraction of sequestered actin monomer (8). Clever uses of combinations of data have in some cases allowed investigators to subtract F-actin content from total actin and then fractionate the contributions of A c and monomer sequestration (9, 10), but the results have proven controversial (11,12).Based on theoretical considerations, the critical concentration is expected to be equal to the ratio of the rate constants of association and dissociation of actin subunits from actin filaments. Measurement of these rate constants using a method such as electron microscopy can be used to indirectly evaluate A c , assuming the applicability of the theory (13,14). However, the participation of actin b...

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Tropomodulin Increases the Critical Concentration of Barbed End-capped Actin Filaments by Converting ADP·Pi-actin to ADP-actin at All Pointed Filament Ends

Cited by 81 publications

References 28 publications

Proteomics Analysis of Rat Brain Postsynaptic Density

Proteomics Analysis of Rat Brain Postsynaptic Density

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

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

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