MRC Laboratory of Molecular Biology, Cambridge, UK.
Department of Biology and Integrative Program for Biological and Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
EMBO Rep. 2023 Nov 6;24(11):e57264. doi: 10.15252/embr.202357264. Epub 2023 Sep 13.
Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.
细胞质微管是管状聚合物，其内腔可以容纳小分子蛋白质或纤维丝。这些物质的具体身份和它们的积累机制尚未得到明确证实。在这里，我们使用 Drosophila S2 细胞突起的低温电子断层扫描技术，发现微管腔内存在类似于最近在人类 HAP1 细胞中报道的纤维丝。用小分子药物 thapsigargin 抑制肌浆/内质网 Ca2+-ATP 酶后，这些纤维丝的频率增加。亚断层平均法显示，内腔纤维丝呈卷曲螺旋结构，类似于结合肌动蛋白的胞质动力蛋白（cofilactin）。与此一致的是，我们观察到在相同条件下，细胞中的胞质动力蛋白去磷酸化，这是一种激活修饰，增加了内腔纤维丝的出现。此外，在相同条件下，用 RNA 干扰敲低胞质动力蛋白会降低具有 cofilactin 形态的内腔纤维丝的频率。这些结果表明，胞质动力蛋白的激活刺激了其在微管腔内肌动蛋白纤维上的积累。
