Nishizawa Naoko, Arai Riku, Hiranuma Koki, Toya Mika, Sato Masamitsu
Department of Life Science and Medical Bioscience, Laboratory of Cytoskeletal Logistics, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan.
Faculty of Science and Engineering, Global Center for Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan.
PLoS One. 2025 Jan 9;20(1):e0308150. doi: 10.1371/journal.pone.0308150. eCollection 2025.
In mammalian epithelial cells, cytoplasmic microtubules are mainly non-centrosomal, through the functions of the minus-end binding proteins CAMSAP2 and CAMSAP3. When cells enter mitosis, cytoplasmic microtubules are reorganized into the spindle composed of both centrosomal and non-centrosomal microtubules. The function of the CAMSAP proteins upon spindle assembly remains unknown, as these do not exhibit evident localization to spindle microtubules. Here, we demonstrate that CAMSAP2, but not CAMSAP3, is required for spindle assembly upon mitotic entry. CAMSAP2 knockout (KO) Caco-2 cells showed a delay in mitotic progression, whereas CAMSAP3 KO cells did not. The spindle in CAMSAP2 KO cells was short and displayed a reduced microtubule density, particularly around chromosomes. This indicated a loss of bridging fibers, which are known to assist alignment of sister kinetochores through interaction with kinetochore fibers. Consistent with this, live-cell imaging of CAMSAP2 KO cells captured slow elongation of the anaphase spindle and errors in chromosome segregation. Therefore, we propose that CAMSAP2 ensures efficient reorganization of cytoplasmic microtubules into the mitotic spindle through constructing bridging fibers that assist faithful segregation of sister chromatids.
在哺乳动物上皮细胞中，细胞质微管主要是非中心体的，这是通过负端结合蛋白CAMSAP2和CAMSAP3的功能实现的。当细胞进入有丝分裂时，细胞质微管会重新组织成由中心体和非中心体微管组成的纺锤体。CAMSAP蛋白在纺锤体组装过程中的功能尚不清楚，因为它们在纺锤体微管上没有明显的定位。在这里，我们证明了CAMSAP2而非CAMSAP3在有丝分裂进入时的纺锤体组装中是必需的。CAMSAP2基因敲除（KO）的Caco-2细胞在有丝分裂进程中出现延迟，而CAMSAP3基因敲除细胞则没有。CAMSAP2基因敲除细胞中的纺锤体较短，微管密度降低，尤其是在染色体周围。这表明桥连纤维缺失，已知桥连纤维通过与动粒纤维相互作用来协助姐妹动粒的排列。与此一致的是，对CAMSAP2基因敲除细胞的活细胞成像显示后期纺锤体伸长缓慢且染色体分离出现错误。因此，我们提出CAMSAP2通过构建有助于姐妹染色单体准确分离的桥连纤维来确保细胞质微管有效重组成有丝分裂纺锤体。
