Koury S T, Koury M J, Bondurant M C
Vanderbilt University, Nashville, Tennessee.
J Cell Biol. 1989 Dec;109(6 Pt 1):3005-13. doi: 10.1083/jcb.109.6.3005.
We have used murine splenic erythrolasts infected with the anemia-inducing strain of Friend virus (FVA cells), as an in vitro model to study cytoskeletal elements during erythroid maturation and enucleation. FVA cells are capable of enucleating in suspension culture in vitro, indicating that associations with an extracellular matrix or accessory cells are not required for enucleation to occur. The morphology of FVA cells undergoing enucleation is nearly identical to erythroblasts enucleating in vivo. The nucleus is segregated to one side of the cell and then appears to be pinched off resulting in an extruded nucleus and reticulocyte. The extruded nucleus is surrounded by an intact plasma membrane and has little cytoplasm associated with it. Newly formed reticulocytes have an irregular shape, are vacuolated and contain all cytoplasmic organelles. The spatial distribution of several cytoskeletal proteins was examined during the maturation process. Spectrin was found associated with the plasma membrane of FVA cells at all stages of maturation but was segregated entirely to the incipient reticulocyte during enucleation. Microtubules formed cages around nuclei in immature FVA cells and were found primarily in the incipient reticulocyte in cells undergoing enucleation. Reticulocytes occasionally contained microtubules, but a generalized diffuse distribution of tubulin was more common. Vimentin could not be detected at any time in FVA cell maturation. Filamentous actin (F-actin) had a patchy distribution at the cell surface in the most immature erythroblasts, but F-actin bundles could be detected as the cells matured. F-actin was found concentrated between the extruding nucleus and incipient reticulocyte in enucleating erythroblasts. Newly formed reticulocytes exhibited punctate actin fluorescence whereas extruded nuclei lacked F-actin. Addition of colchicine, vinblastine, or taxol to cultures of FVA cells did not affect enucleation. In contrast, cytochalasin D caused a complete inhibition of enucleation that could be reversed by washing out the cytochalasin D. These results demonstrate that F-actin plays a role in enucleation while the complete absence of microtubules or excessive numbers of polymerized microtubules do not affect enucleation.
我们使用感染了贫血诱导型弗氏病毒的小鼠脾红细胞（FVA细胞）作为体外模型，来研究红细胞成熟和去核过程中的细胞骨架成分。FVA细胞能够在体外悬浮培养中去核，这表明去核过程并不需要与细胞外基质或辅助细胞相关联。正在去核的FVA细胞的形态与体内正在去核的成红细胞几乎相同。细胞核被分隔到细胞的一侧，然后似乎被挤压掉，从而产生一个挤出的细胞核和网织红细胞。挤出的细胞核被完整的质膜包围，与之相关的细胞质很少。新形成的网织红细胞形状不规则，有空泡，并且含有所有细胞质细胞器。在成熟过程中检测了几种细胞骨架蛋白的空间分布。血影蛋白在FVA细胞成熟的所有阶段都与质膜相关，但在去核过程中完全分隔到初始网织红细胞中。微管在未成熟的FVA细胞中围绕细胞核形成笼状结构，并且在正在去核的细胞中的初始网织红细胞中主要被发现。网织红细胞偶尔含有微管，但微管蛋白的普遍弥漫分布更为常见。在FVA细胞成熟的任何时候都检测不到波形蛋白。丝状肌动蛋白（F-肌动蛋白）在最不成熟的成红细胞表面呈斑点状分布，但随着细胞成熟可以检测到F-肌动蛋白束。在正在去核的成红细胞中，F-肌动蛋白集中在挤出的细胞核和初始网织红细胞之间。新形成的网织红细胞表现出点状肌动蛋白荧光，而挤出的细胞核缺乏F-肌动蛋白。向FVA细胞培养物中添加秋水仙碱、长春碱或紫杉醇并不影响去核。相反，细胞松弛素D导致去核完全受到抑制，通过洗去细胞松弛素D可以逆转这种抑制。这些结果表明F-肌动蛋白在去核中起作用，而微管的完全缺失或过多的聚合微管并不影响去核。
