Tran Vivien D, Amofa Kwasi Y, Ling Lu, Duong Miles, Kumar Sanjay
UC Berkeley-UCSF Graduate Program in Bioengineering, San Francisco CA 94143.
Department of Bioengineering, University of California, Berkeley, CA 94720.
Mol Biol Cell. 2025 Sep 1;36(9):ar108. doi: 10.1091/mbc.E24-08-0384. Epub 2025 Jul 9.
Contributions of the actin turnover machinery to cell motility have been extensively studied in traditional two-dimensional (2D) culture paradigms. However, much remains unknown about how these proteins contribute to three-dimensional (3D) motility, particularly in matrices lacking strong contact guidance cues. Here, we explore this question in the context of glioblastoma (GBM) cell invasion through 3D hyaluronic acid (HA) hydrogels. We begin with a CRISPR screen to identify contributions of core actin turnover proteins to migration speed in 2D and 3D. Although suppression of most proteins reduced motility in both 2D and 3D, suppression of cofilin-1 (CFL) increased migration speed in 3D. CFL knockout cells uniquely formed longer and more protrusions in 3D compared with non-targeting control cells. Consistent with the screen, targeted CFL short hairpin RNA-mediated knockdown (KD) decreased motility on 2D HA but increased motility in 3D HA. This effect appears HA-specific, as CFL KD did not increase motility in 3D collagen or in a transwell assay. Myosin X, CD44, and hyaluronidase-2 all localized to a subset of protrusions irrespective of cellular CFL status, implying that CFL suppression promotes filopodia and microtentacle extension. We propose that loss of CFL promotes the actin filaments in these protrusions, enabling GBM cells to penetrate 3D HA matrices.
肌动蛋白周转机制对细胞运动性的贡献已在传统的二维（2D）培养模式中得到广泛研究。然而，关于这些蛋白质如何促进三维（3D）运动性，尤其是在缺乏强接触导向线索的基质中，仍有许多未知之处。在这里，我们在胶质母细胞瘤（GBM）细胞通过三维透明质酸（HA）水凝胶侵袭的背景下探讨这个问题。我们首先进行了一项CRISPR筛选，以确定核心肌动蛋白周转蛋白对二维和三维迁移速度的贡献。虽然大多数蛋白质的抑制降低了二维和三维中的运动性，但抑制丝切蛋白-1（CFL）却增加了三维中的迁移速度。与非靶向对照细胞相比，CFL基因敲除细胞在三维中独特地形成了更长且更多的突起。与筛选结果一致，靶向CFL短发夹RNA介导的敲低（KD）降低了二维HA上的运动性，但增加了三维HA中的运动性。这种效应似乎是HA特异性的，因为CFL KD在三维胶原蛋白或Transwell实验中并未增加运动性。肌球蛋白X、CD44和透明质酸酶-2均定位于一部分突起，而与细胞CFL状态无关，这意味着CFL抑制促进丝状伪足和微触手的延伸。我们提出，CFL的缺失促进了这些突起中的肌动蛋白丝，使GBM细胞能够穿透三维HA基质。
