Siatkowska Małgorzata, Robaszkiewicz Katarzyna, Rousová Andrea, Navrátil Jiří, Knopfová Lucia, Talián Gábor, Beneš Petr, Moraczewska Joanna
Department of Biochemistry and Cell Biology, Faculty of Biological Sciences, Kazimierz Wielki University, Ks. Józefa Poniatowskiego 12, 85-671, Bydgoszcz, Poland.
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
Biol Res. 2025 Aug 31;58(1):60. doi: 10.1186/s40659-025-00640-3.
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.
背景：在多种肿瘤类型中，肌动蛋白结合蛋白——肌动蛋白束蛋白-1（fascin-1）以及原肌球蛋白的各种同工型的表达模式会发生改变。Fascin-1是一种肌动蛋白成束蛋白，可促进癌细胞迁移，而原肌球蛋白发挥肿瘤和转移抑制因子的作用。然而，原肌球蛋白同工型调节fascin-1的机制仍知之甚少。本研究旨在探究fascin-1和原肌球蛋白同工型对它们与肌动蛋白相互作用以及肌动蛋白束形成的相互影响。 方法：重组fascin-1以及由TPM2编码的细胞骨架原肌球蛋白同工型（Tpm2.1、Tpm2.3和Tpm2.4）在BL21-DE3细胞中表达并纯化。采用高速离心法评估fascin-1和Tpm2同工型对肌动蛋白的亲和力。使用低速离心和荧光显微镜分析肌动蛋白丝的成束情况。进行下拉实验以检测fascin-1与Tpm2同工型之间的直接相互作用。利用共聚焦显微镜分析在过表达Tpm2同工型的转移性SAOS-2 LM5细胞系中fascin-1的定位。 结果：在三种重组的、乙酰化的Tpm2同工型中，Tpm2.4对F-肌动蛋白表现出最高的亲和力。在低fascin-1浓度下，所有Tpm2同工型均强烈抑制fascin-1介导的肌动蛋白成束，只有在fascin-1水平显著更高时成束才恢复。形成的肌动蛋白束同时包含Tpm2和fascin-1；然而，在存在任何Tpm2同工型的情况下，每束中的丝数量减少。在存在Tpm2同工型的情况下，fascin-1对肌动蛋白的亲和力降低，并且Tpm2在肌动蛋白丝上占有率的增加部分取代了fascin-1。相反，fascin-1的结合并不影响Tpm2同工型对肌动蛋白的亲和力。下拉实验表明，Tpm2同工型可直接与fascin-1相互作用，其中Tpm2.4表现出最高的亲和力。细胞实验数据进一步支持了Tpm2对fascin-1-肌动蛋白相互作用的抑制作用，该数据表明在SAOS-2 LM5细胞中过表达细胞质Tpm2.1、Tpm2.3或Tpm2.4会减少fascin与肌动蛋白的共定位。 结论：细胞质Tpm2同工型通过组织肌动蛋白束中的蛋白质组成来调节fascin-1的肌动蛋白成束活性，这一机制可能有助于抑制癌细胞的转移表型。
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