Snyder J A, Cohen L
Department of Biological Sciences, University of Denver, Colorado 80208, USA.
Cell Motil Cytoskeleton. 1995;32(4):245-57. doi: 10.1002/cm.970320402.
PtK1 cells were treated with 10 micrograms/ml cytochalasin J (CJ) for 15 min at various stages of mitosis. When applied at nuclear envelope breakdown (NEB) chromosome congression was blocked or substantially slowed, and chromosomes failed to show organization patterns typical of prometaphase. Spindle microtubule (MT) numbers appeared unaffected as judged by the pattern of birefringent retardation. However, ultrastructural analysis showed MTs to be reorganized within the spindle domain with some exhibiting fragmentation and others failing to interact with poorly defined kinetochore laminae. The spindle domain took on a curved, almost banana-like shape, as related to the position of the centrosomes and lack of orientation of chromosomes. Serial section analysis of kinetochore regions showed reduced contour length and maturation of the kinetochore plate with few MTs associated with this structure. Cells similarly treated with 10 micrograms/ml CJ at NEB for 15 min and then released into conditioned medium for 15 min showed the most chromosomes resumed congression to the metaphase plate. Ultrastructural analysis revealed a more normal organization of spindle MTs, but kinetochore structure remained affected. CJ treatment of cells in prometaphase slightly affected chromosomes congression with most chromosomes aligning at the metaphase plate after 10-15 min of treatment. Ultrastructural analysis showed that astral MTs were disrupted and spindle MTs were fragmented; few MTs coursed from kinetochore to pole. Kinetochore structure was also affected with only small numbers of short MTs seen associated with kinetochores. Application of CJ at anaphase onset had little effect on anaphase A and B, but cytokinesis failed to occur. Anti-tubulin staining of a monolayer of cells treated with 10 micrograms/ml CJ for 15 min showed that over 60% of mitotic figures exhibited changes in MT organization. Cells showing the greatest effect of treatment had several foci of bundles of MTs, as if the spindle were multipolar. Chromosomes were arranged near the periphery of the spindle which could be a result of abnormalities of kinetochore structure. Improper association of spindle MTs with kinetochores and, thus, changes in kinetochore position could account for these changes in spindle architecture.
将PtK1细胞在有丝分裂的不同阶段用10微克/毫升的细胞松弛素J（CJ）处理15分钟。在核膜破裂（NEB）时施加，染色体的汇聚被阻断或显著减慢，并且染色体未能呈现典型的前中期组织模式。通过双折射延迟模式判断，纺锤体微管（MT）数量似乎未受影响。然而，超微结构分析显示，纺锤体区域内的微管发生了重组，一些微管出现片段化，另一些微管未能与定义不清的动粒层相互作用。与中心体的位置和染色体缺乏定向有关，纺锤体区域呈现出弯曲的、几乎像香蕉一样的形状。动粒区域的连续切片分析显示，动粒板的轮廓长度减小且成熟度降低，与此结构相关的微管很少。在NEB时用10微克/毫升CJ同样处理15分钟，然后释放到条件培养基中15分钟的细胞，显示大多数染色体恢复向中期板汇聚。超微结构分析显示纺锤体微管的组织更正常，但动粒结构仍然受到影响。在前中期用CJ处理细胞对染色体汇聚略有影响，处理10 - 15分钟后，大多数染色体排列在中期板上。超微结构分析显示星体微管被破坏，纺锤体微管片段化；从动粒到极的微管很少。动粒结构也受到影响，仅见少量短微管与动粒相关。在后期开始时施加CJ对后期A和B影响不大，但胞质分裂未能发生。用10微克/毫升CJ处理15分钟的单层细胞的抗微管蛋白染色显示，超过60%的有丝分裂图像呈现微管组织变化。显示处理效果最显著的细胞有几个微管束焦点，就好像纺锤体是多极的。染色体排列在纺锤体周边附近，这可能是动粒结构异常的结果。纺锤体微管与动粒的不当结合，以及由此导致的动粒位置变化，可能解释了纺锤体结构的这些变化。
