The Effect of Formins on the Polymerisation and Dynamic Properties of Actin Filaments

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Forminok hatása az aktin filamentum konformációjára = The effect of formin fragments on the conformation of actin filaments

A pályázat keretei között végzett vizsgálatainkban fluoreszcencia spektroszkópiai, gyors-kinetika és EPR módszereket alkalmaztunk motor fehérjék, és az aktin citoszkeleton felépítésében meghatározó szerepet betöltő aktin nukleációs faktorok tanulmányozására. Kinetikai módszerekkel jellemeztük a speciális és eddig ritkán vizsgált XIV-es miozint, valamint részletesen leírtuk és összevetettük különböző fajokból származó emlős izom miozin izoformák tulajdonságait. Megmutattuk, hogy az aktin nukleációs faktorok közé tartozó forminok kötődése fellazítja az aktin filamentumok szerkezetét. EPR vizsgálataink arra is rávilágítottak, hogy ezek a konformációs módosulások heterogén és összetett módon valósulnak meg. Azt is megmutattuk, hogy forminok által kiváltott konformációs változások visszafordíthatóak tropomiozin kötődésével. | The investigations involved in the project were based on the applications of fluorescence spectroscopic, rapid kinetic and EPR methods. We have studied the kinetic and conformational properties of motor proteins and actin nucleation factors. We described the kinetic behaviour of a special class of myosins, the myosin XIV, and also many isoforms isolated from mammalian muscles of various species. We sowed that the binding of formins - a family of actin nucleation factors - to the actin filaments made the structure of the filaments looser. The EPR data showed that these conformational modifications are complex and heterogeneous. Further studies provided evidence, that the forming-induced structural changes could be reversed by the binding of tropomyosin

The other side of the coin : Functional and structural versatility of ADF/cofilins

Several cellular processes rely on the fine tuning of actin cytoskeleton. A central component in the regulation of this cellular machinery is the ADF-H domain proteins. Despite sharing the same domain, ADF-H domain proteins produce a diverse functional landscape in the regulation of the actin cytoskeleton. Recent findings emphasize that the functional and structural features of these proteins can differ not only between ADF-H families but even within the same family. The structural and evolutional background of this functional diversity is poorly understood. This review focuses on the specific functional characteristics of ADF-H domain proteins and how these features can be linked to structural differences in the ADF-H domain and also to different conformational transitions in actin. In the light of recent discoveries we pay special attention to the ADF/cofilin proteins to find tendencies along which the functional and structural diversification is governed through the evolution

Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners

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Az aktin kölcsönhatása aktin-kötő fehérjékkel és peptidekkel: fluoreszcencia spektroszkópiai vizsgálatok = The Interaction of Actin with Actin-Binding Proteins and Peptides as Revealed by Spectroscopic Methods

Az OTKA K60968 pályázat keretei között az eredeti terveinknek megfelelően tanulmányoztuk az aktin monomereknek és filamentumoknak más fehérjékkel és peptidekkel való kölcsönhatásait. A kutatások során elsősorban fluoreszcencia spektroszkópiai módszereket alkalmaztunk, de az adott kérdéskörtől függően ezen módszerek eredményeit kiegészítettük elektron paramágneses rezonancia spektroszkópiai és kalorimetriai vizsgálatokkal is. Részletes vizsgálatokban jellemeztük az aktin filamentumoknak a forminokkal való kölcsönhatását, és megállapítottuk, hogy a forminok kötődésével a filamentumok szerkezete lazábbá válik. Azt is megfigyeltük, hogy a forminok által kiváltott konformációs módosulásokat a tropomiozin vagy a miozin kötődése megszünteti. Tanulmányoztunk és leírtunk továbbá egy eddig nem jellemzett formin családot, a DAAM forminokat. Ezen vizsgálataink mellett jellemeztük és értelmeztük az aktinnak a kölcsönhatását egyes mérgező toxinokkal, valamint új megfigyeléseket tettünk az aktin-miozin kölcsönhatás szerkezeti és kinetikai sajátságait illetően is. | According to the project plans we have studied the interactions between actin monomers / filaments and actin binding proteins and peptides. In these investigations we applied fluorescence spectroscopic methods, in conjunction with electron paramagnetic resonance and calorimetric assays. We described in details the interaction between actin filaments and formin, and found that the binding of formins made the actin filaments ore flexible. We also observed that the formin induced conformational changes were reversed by the binding of tropomyosin or myosin. We studied and described a novel formin family, the DAAM formins. Furthermore, we described the interactions between actin and toxic actin-binding peptides, and also characterised the interactions of myosin with actin in terms of the conformational and kinetic properties

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short.

Drosophila melanogaster Sarcomere Length Short (SALS) is a recently identified Wiskott - Aldrich syndrome protein homology 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcomeric actin filaments. Here we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer : filament equilibrium towards monomeric actin. In addition, SALS-WH2 can bind to but fails to depolymerize phalloidin-, or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning

DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin

DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin

Myosin XVI in the Nervous System

The myosin family is a large inventory of actin-associated motor proteins that participate in a diverse array of cellular functions. Several myosin classes are expressed in neural cells and play important roles in neural functioning. A recently discovered member of the myosin superfamily, the vertebrate-specific myosin XVI (Myo16) class is expressed predominantly in neural tissues and appears to be involved in the development and proper functioning of the nervous system. Accordingly, the alterations of MYO16 has been linked to neurological disorders. Although the role of Myo16 as a generic actin-associated motor is still enigmatic, the N-, and C-terminal extensions that flank the motor domain seem to confer unique structural features and versatile interactions to the protein. Recent biochemical and physiological examinations portray Myo16 as a signal transduction element that integrates cell signaling pathways to actin cytoskeleton reorganization. This review discusses the current knowledge of the structure-function relation of Myo16. In light of its prevalent localization, the emphasis is laid on the neural aspects