Regulation of actin filament turnover by cofilin-1 and cytoplasmic tropomyosin isoforms.
Tropomyosin and cofilin are actin-binding proteins which control dynamics of actin assembly and disassembly. Tropomyosin isoforms can either inhibit or enhance cofilin activity, but the mechanism of this diverse regulation is not well understood. In this work mechanisms of actin dynamics regulation by four cytoskeletal tropomyosin isoforms and cofilin-1 were studied with the use of biochemical and fluorescent microscopy assays. The recombinant tropomyosin isoforms were products of two genes: TPM1 (Tpm1.6 and Tpm1.8) and TPM3 (Tpm3.2 and Tpm3.4). Tpm1.6/1.8 bound to F-actin with higher apparent binding constants and lower cooperativities than Tpm3.2/3.4. In consequence, subsaturating concentrations of cofilin-1 removed 50% of Tpm3.2/3.4 from F-actin. By contrast, 2 and 5.5 molar excess of cofilin-1 over actin was required to dissociate 50% of Tpm1.6/1.8. All tropomyosins inhibited the rate of spontaneous polymerization of actin, which was reversed by cofilin-1. Products of TPM1 favored longer filaments and protected them from cofilin-induced depolymerization. This was in contrast to the isoforms derived from TPM3, which facilitated depolymerization. Tpm3.4 was the only isoform, which increased frequency of the filament severing by cofilin-1. Tpm1.6/1.8 inhibited, but Tpm3.2/3.4 enhanced cofilin-induced conformational changes leading to accelerated release of rhodamine-phalloidin from the filament. We concluded that the effects were executed through different actin affinities of tropomyosin isoforms and cooperativities of tropomyosin and cofilin-1 binding. The results obtained in vitro were in good agreement with localization of tropomyosin isoforms in stable or highly dynamic filaments demonstrated before in various cells.
Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Ks. J. Poniatowskiego 12 Str., 85-671 Bydgoszcz, Poland.
Zofia
Z
Ostrowska
Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Ks. J. Poniatowskiego 12 Str., 85-671 Bydgoszcz, Poland.
Katarzyna
K
Robaszkiewicz
Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Ks. J. Poniatowskiego 12 Str., 85-671 Bydgoszcz, Poland. Electronic address: joanna.moraczewska@ukw.edu.pl.
Joanna
J
Moraczewska
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D051338:Cofilin 1 / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D051379:Mice; D010590:Phalloidine / Q000031:analogs & derivatives / Q000737:chemistry; D058105:Polymerization; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012235:Rhodamines / Q000737:chemistry; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*
D051338:Cofilin 1; D020033:Protein Isoforms; D012235:Rhodamines; D014335:Tropomyosin; C504731:rhodamine-phalloidin; D010590:Phalloidine
Cofilin; Isoforms; Microfilament; Tropomyosin