Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA.
Eur J Cell Biol. 2023 Dec;102(4):151367. doi: 10.1016/j.ejcb.2023.151367. Epub 2023 Oct 20.
How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.
细胞如何利用复杂的肌动蛋白结合蛋白混合物来组装和维持具有不同结构和动力学的功能多样化的肌动蛋白丝网络，这是细胞生物学中的一个长期存在的问题。细胞中复杂而专门的肌动蛋白结构的一个引人注目的例子是丝状伪足，它可以感知细胞外的化学和机械信号，帮助引导运动细胞。丝状伪足具有独特的肌动蛋白结构，由长而平行的肌动蛋白丝组成，这些肌动蛋白丝由 fascin 束成束，形成指状的膜突起。丝状伪足中平行肌动蛋白丝的伸长可以由两种连续的肌动蛋白丝伸长因子——formin 和 Ena/VASP 介导，它们定位于丝状伪足的尖端。目前仍存在争议，即丝状伪足的结构是如何产生的，一种假说认为丝状伪足是从由 Arp2/3 复合物核化的紧密堆积、分支的肌动蛋白丝组成的片足中产生的，而由盖帽蛋白保持短。目前尚不清楚是否需要不同的肌动蛋白丝伸长因子来促进从高度密集的短分支盖帽纤维网络中出现具有不同特征的丝状伪足。为了解决这个问题，我们结合了珠子运动和微图案仿生测定与多色全内反射荧光显微镜成像，成功地从密集分支的片足样网络（LLN）中重建了丝状伪足样网络（FLN）的形成，使用了八种纯化蛋白（肌动蛋白、原肌球蛋白、Arp2/3 复合物、Wasp pWA、 fascin、盖帽蛋白、VASP 和 formin mDia2）。饱和的盖帽蛋白浓度抑制 FLN 组装，但添加 formin 或 Ena/VASP 会使 LLN 形成的 FLN 不同程度地恢复。具体来说，我们发现 formin/mDia2 生成的 FLN 相对较长且缺乏盖帽蛋白，而 VASP 生成的 FLN 相对较短且含有盖帽蛋白，这表明肌动蛋白伸长因子可以影响从 LLN 中出现的 FLN 的结构和组成。我们的仿生重建系统表明，formin 或 VASP 是诱导从 LLN 到 FLN 转变所必需和充分的，并且建立了强大的体外平台来研究 FLN 组装机制。
