title: Purification of tropomyosin Br-3 and 5NM1 and characterization of their interactions with actin.
abstract: 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.
authors.affiliation: Department of Biophysics, University of Pécs, Medical School, Pécs, Hungary.
authors.forename: Nikolett
authors.initials: N
authors.lastname: Kis-Bicskei
authors.forename: Andrea
authors.initials: A
authors.lastname: Vig
authors.forename: Miklós
authors.initials: M
authors.lastname: Nyitrai
authors.forename: Beáta
authors.initials: B
authors.lastname: Bugyi
authors.forename: Gábor C
authors.initials: GC
authors.lastname: Talián
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D005091:Exons / Q000235:genetics; D005453:Fluorescence; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D058105:Polymerization; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D013997:Time Factors; D014335:Tropomyosin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D020033:Protein Isoforms; C411923:Tpm3 protein, mouse; D014335:Tropomyosin; C000592490:tropomyosin Br-3, mouse
keywords: actin; actin nucleation factors; fluorescence spectroscopy; purification; tropomyosin