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. 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. 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. 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.
Siatkowska et al. (Sun,) studied this question.