title: Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro.
abstract: Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-depolymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes. Different isoforms display non-redundant functions and partially non-overlapping localization patterns, for example within the stress fiber network [6, 7]. Based on cell biological studies, it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [2]. To test this hypothesis, we analyzed the properties of actin filaments decorated by stress-fiber-associated tropomyosins (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2). These proteins bound F-actin with high affinity and competed with α-actinin for actin filament binding. Importantly, total internal reflection fluorescence (TIRF) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms cannot co-polymerize with each other on actin filaments. These isoforms also bind actin with different dynamics, which correlate with their effects on actin-binding proteins. The long isoforms Tpm1.6 and Tpm1.7 displayed stable interactions with actin filaments and protected filaments from ADF/cofilin-mediated disassembly, but did not activate non-muscle myosin IIa (NMIIa). In contrast, the short isoforms Tpm3.1, Tpm3.2, and Tpm4.2 displayed rapid dynamics on actin filaments and stimulated the ATPase activity of NMIIa, but did not efficiently protect filaments from ADF/cofilin. Together, these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations.
authors.affiliation: Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland.
authors.forename: Gergana
authors.initials: G
authors.lastname: Gateva
authors.affiliation: Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland.
authors.forename: Elena
authors.initials: E
authors.lastname: Kremneva
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Theresia
authors.initials: T
authors.lastname: Reindl
authors.affiliation: Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland.
authors.forename: Tommi
authors.initials: T
authors.lastname: Kotila
authors.affiliation: Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kogan
authors.affiliation: Biosciences and Biotechnology Institute of Grenoble, LPCV/CNRS/CEA/UGA/INRA, 38054 Grenoble, France.
authors.forename: Laurène
authors.initials: L
authors.lastname: Gressin
authors.affiliation: Oncology Research Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW 2052, Australia.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Gunning
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Dietmar J
authors.initials: DJ
authors.lastname: Manstein
authors.affiliation: Aix-Marseille University, CNRS, IBDM, 13284 Marseille, France.
authors.forename: Alphée
authors.initials: A
authors.lastname: Michelot
authors.affiliation: Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland. Electronic address: pekka.lappalainen@helsinki.fi.
authors.forename: Pekka
authors.initials: P
authors.lastname: Lappalainen
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000595:Amino Acid Sequence; D006801:Humans; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D014335:Tropomyosin
keywords: TIRF microscopy; actin; adhesion; cell migration; cofilin; cytoskeleton; myosin; stress fiber; tropomyosin; α-actinin