Department of Biochemistry, Ghent University, Faculty of Medicine and Health Sciences, Ghent, Belgium.
PLoS One. 2013 Nov 13;8(11):e78108. doi: 10.1371/journal.pone.0078108. eCollection 2013.
Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.
足突是作为单核细胞降解“热点”的细胞结构。它们出现在细胞腹面的点状结构中，富含 F-肌动蛋白和肌动蛋白调节剂，包括凝胶蛋白和 L-肌动蛋白结合蛋白。凝胶蛋白是一种普遍存在的切断和盖帽蛋白，而 L-肌动蛋白结合蛋白是白细胞特异性的肌动蛋白结合蛋白。足突中盖帽蛋白 CapG 的存在尚未被研究过。我们使用一种创新的方法，通过纳米抗体或骆驼科单域抗体来研究这些蛋白质在巨噬细胞足突中的作用。针对凝胶蛋白、L-肌动蛋白结合蛋白或 CapG 不同结构域的纳米抗体被稳定表达在巨噬样 THP-1 细胞中。CapG 没有在足突中富集。凝胶蛋白纳米抗体对足突的形成或功能没有影响，但在追踪不同的凝胶蛋白群体方面非常有效。一种特异性针对结合肌动蛋白的凝胶蛋白纳米抗体有效地富集在足突中。一种阻止凝胶蛋白-G-肌动蛋白相互作用的凝胶蛋白纳米抗体没有在足突中富集，这表明钙激活和结合肌动蛋白的凝胶蛋白构象是足突的组成部分。表达抑制性 L-肌动蛋白结合蛋白纳米抗体的 THP-1 细胞在形成稳定的足突方面受到阻碍。纳米抗体没有扰乱 L-肌动蛋白结合蛋白的 Ser5 磷酸化，尽管磷酸化的 L-肌动蛋白在足突中高度富集。此外，纳米抗体诱导的 L-肌动蛋白结合蛋白功能抑制导致足突中肌动蛋白周转率不规则和不稳定，从而减少了基底基质的降解。总之，这些结果表明 L-肌动蛋白结合蛋白对于巨噬细胞中足突的形成和功能是不可或缺的。
