Gadsby Jonathan R, Ioannou Pantelis Savvas, Butler Richard, Mason Julia, Smith Alison J, Dobramysl Ulrich, Chin Stacey E, Dobson Claire, Gallop Jennifer L
Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, U.K.
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K.
Biochem J. 2024 Dec 23;481(24):1977-1995. doi: 10.1042/BCJ20240367.
Filopodia, microspikes and cytonemes are implicated in sensing the environment and in dissemination of morphogens, organelles and pathogens across tissues. Their major structural component is parallel bundles of actin filaments that assemble from the cell membrane. Whilst the length of filopodia is central to their function, it is not known how their lengths are determined by actin bundle dynamics. Here, we identified a set of monoclonal antibodies that lengthen filopodia-like structures formed in a cell-free reconstitution system, and used them to uncover a key molecular switch governing length regulation. Using immunolabelling, enzyme-linked immunosorbent assays, immunoprecipitation and immunoblock experiments, we identified four antibodies that lengthen actin bundles by selectively binding the open DNase 1-binding loop (D-loop) of actin filaments. The antibodies inhibit actin disassembly and their effects can be alleviated by providing additional actin or cofilin. This work indicates that maintaining an open state of the actin filament D-loop is a mechanism of generating long filopodia-like actin bundles.
丝状伪足、微刺突和丝状伪足参与感知环境以及形态发生素、细胞器和病原体在组织中的传播。它们的主要结构成分是从细胞膜组装而成的平行肌动蛋白丝束。虽然丝状伪足的长度对其功能至关重要，但尚不清楚它们的长度是如何由肌动蛋白束动力学决定的。在这里，我们鉴定出一组单克隆抗体，这些抗体可延长在无细胞重构系统中形成的丝状伪足样结构，并利用它们揭示了一个控制长度调节的关键分子开关。通过免疫标记、酶联免疫吸附测定、免疫沉淀和免疫阻断实验，我们鉴定出四种通过选择性结合肌动蛋白丝的开放脱氧核糖核酸酶1结合环（D环）来延长肌动蛋白束的抗体。这些抗体抑制肌动蛋白解聚，并且通过提供额外的肌动蛋白或丝切蛋白可以减轻它们的作用。这项工作表明，维持肌动蛋白丝D环的开放状态是产生长丝状伪足样肌动蛋白束的一种机制。
