Walker Caleb, Mansour Daniel, Effiong Unyime, Jordan Dominique, Wang Liping, Lafer Eileen M, Alvarado Jose, Belardi Brian, Rangamani Padmini, Stachowiak Jeanne C
Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA.
Department of Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla, CA, USA.
bioRxiv. 2025 Jun 21:2025.06.18.660423. doi: 10.1101/2025.06.18.660423.
Protein condensates use multivalent binding and surface tension to assemble actin filaments into diverse architectures, reminiscent of filopodia and stress fibers. During this process, nucleation of new filaments and elongation of existing filaments inherently compete for a shared pool of actin monomers. Here we show that a balance between these competing processes is required to deform condensates of VASP, an actin binding protein, into structures with high aspect ratios. Addition of magnesium, which promotes filament nucleation, helped actin to deform condensates into high aspect ratio structures. In contrast, addition of profilin, which inhibits filament nucleation, allowing existing filaments to elongate, caused actin to assemble into ring-like bundles that failed to substantially increase condensate aspect ratio. Computational modeling helped to explain these results by predicting that a group of short linear filaments, which can apply asymmetric pressure to the condensate boundary, is needed to increase condensate aspect ratio. In contrast, a small number of long filaments with the same total actin content should fail to overcome the droplet surface tension, forming a ring-like bundle. To test these predictions, we introduced gelsolin, which severed log filaments within rings, creating new barbed ends. The resulting set of shorter filaments regained the ability to deform condensates into high aspect ratio structures. Collectively, these results suggest that a balance of actin filament nucleation and elongation is required to deform protein condensates. More broadly, these findings illustrate how protein condensates can balance multiple kinetic processes to direct the assembly of diverse cytoskeletal architectures.
蛋白质凝聚物利用多价结合和表面张力将肌动蛋白丝组装成各种结构，让人联想到丝状伪足和应力纤维。在此过程中，新丝的成核和现有丝的延长在本质上竞争肌动蛋白单体的共享池。我们在此表明，需要在这些相互竞争的过程之间取得平衡，才能将肌动蛋白结合蛋白VASP的凝聚物变形为高纵横比的结构。添加促进丝成核的镁，有助于肌动蛋白将凝聚物变形为高纵横比的结构。相反，添加抑制丝成核、使现有丝延长的肌动蛋白单体结合蛋白，会导致肌动蛋白组装成环状束，而无法显著增加凝聚物的纵横比。计算模型通过预测一组短的线性丝（其可对凝聚物边界施加不对称压力）来解释这些结果，这组短丝是增加凝聚物纵横比所必需的。相比之下，具有相同总肌动蛋白含量的少量长丝应无法克服液滴表面张力，从而形成环状束。为了验证这些预测，我们引入了凝溶胶蛋白，其切断环内的长丝，产生新的带刺末端。由此产生的一组较短的丝恢复了将凝聚物变形为高纵横比结构的能力。总体而言，这些结果表明，需要肌动蛋白丝成核和延长之间的平衡来使蛋白质凝聚物变形。更广泛地说，这些发现说明了蛋白质凝聚物如何平衡多个动力学过程以指导各种细胞骨架结构的组装。
