BACKGROUND: In many types of tumors, the expression patterns of actin-binding proteins -fascin-1 and various isoforms of tropomyosin - are altered. Fascin-1 is an actin-bundling protein that promotes cancer cell motility, whereas tropomyosin functions as a tumor and metastasis suppressor. However, the mechanisms by which tropomyosin isoforms regulate fascin-1 remain poorly understood. This study aimed to investigate the reciprocal effects of fascin-1 and tropomyosin isoforms on their interactions with actin and on the formation of actin bundles. METHODS: Recombinant fascin-1 and the cytoskeletal tropomyosin isoforms encoded by TPM2 (Tpm2.1, Tpm2.3, and Tpm2.4) were expressed in BL21-DE3 cells and purified. High-speed centrifugation was employed to assess the actin affinities of fascin-1 and the Tpm2 isoforms. Actin filament bundling was analyzed using low-speed centrifugation and fluorescence microscopy. A pull-down assay was performed to examine direct interactions between fascin-1 and the Tpm2 isoforms. Confocal microscopy was used to analyze the localization of fascin-1 in the metastatic SAOS-2 LM5 cell line overexpressing Tpm2 isoforms. RESULTS: Among the three recombinant, acetylated Tpm2 isoforms, Tpm2.4 exhibited the highest affinity for F-actin. All Tpm2 isoforms strongly inhibited fascin-1-mediated actin bundling at low fascin-1 concentrations, with bundling restored only at substantially higher fascin-1 levels. The resulting actin bundles contained both Tpm2 and fascin-1; however, the number of filaments per bundle was reduced in the presence of any Tpm2 isoform. Fascin-1's affinity for actin was decreased in the presence of Tpm2 isoforms, and increased Tpm2 occupancy on actin filaments partially displaced fascin-1. In contrast, fascin-1 binding did not affect the affinity of Tpm2 isoforms for actin. Pull-down assays revealed that Tpm2 isoforms can directly interact with fascin-1, with Tpm2.4 showing the highest affinity. The inhibitory effect of Tpm2 on fascin-1-actin interactions was further supported by cellular data, which showed that overexpression of cytoplasmic Tpm2.1, Tpm2.3, or Tpm2.4 in SAOS-2 LM5 cells reduced fascin co-localization with actin. CONCLUSION: Cytoplasmic Tpm2 isoforms regulate actin bundling activity of fascin-1 by organizing protein composition in the bundles, a mechanism that may contribute to the suppression of metastatic phenotype in cancer cells.

Department of Biochemistry and Cell Biology Faculty of Biological Sciences Kazimierz Wielki University Ks Józefa Poniatowskiego 12 85 671 Bydgoszcz Poland

Department of Biophysics Medical School University of Pécs Pécs Hungary

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Pathological Physiology Faculty of Medicine Masaryk University Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

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