Snyder J A, Armstrong L, Stonington O G, Spurck T P, Pickett-Heaps J D
Department of Biological Sciences, University of Denver, CO 80208.
Eur J Cell Biol. 1991 Jun;55(1):122-32.
Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region. Changes in organization of MTs in the irradiated region were analyzed by EM serial section analysis coupled with 3-D computer reconstruction. Metaphase cells irradiated 2 to 4 microns below the spindle pole (imaged by polarization optics) lost birefringence in the irradiated region. Peripheral spindle fibers, previously curved to focus on the pole, immediately splayed outwards when severed. We demonstrate via serial section analysis that following irradiation the lesion was devoid of MTs. Within 30 s to 1 min, recovery in live cells commenced as the severed spindle pole moved toward the metaphase plate closing the lesion. This movement was concomitant with the recovery of spindle birefringence and some of the severed fibers becoming refocused at the pole. Ultrastructurally we confirmed that this movement coincided with bridging of the lesion by MTs presumably growing from the pole. The non-irradiated half spindle also lost some birefringence and shortened until it resembled the recovered half spindle. Anaphase cells similarly irradiated did not show recovery of birefringence, and the pole remained disconnected from the remaining mitotic apparatus. Reconstructions of spindle structure confirmed that there were no MTs in the lesion which bridged the severed spindle pole with the remaining mitotic apparatus. These results suggest the existence of chromosome-to-pole spindle forces are dependent upon the existence of a MT continuum, and to a lesser extent to the loss of MT initiation capacity of the centrosome at the metaphase/anaphase transition.
在中期和后期，当染色体与纺锤体极之间时，对有丝分裂的PtK1纺锤体进行紫外线（285纳米）照射。照射区域为一个与中期板平行的1.4×5微米的矩形，该区域内90%至100%的纺锤体微管（MTs）被切断。通过电子显微镜连续切片分析结合三维计算机重建，分析照射区域内微管组织的变化。在纺锤体极下方2至4微米处照射的中期细胞（通过偏振光学成像），照射区域失去双折射。先前弯曲以聚焦于极的外周纺锤体纤维，在被切断后立即向外展开。我们通过连续切片分析证明，照射后损伤部位没有微管。在30秒至1分钟内，活细胞开始恢复，因为被切断的纺锤体极向中期板移动，闭合损伤部位。这种移动与纺锤体双折射的恢复以及一些被切断的纤维重新聚焦于极同时发生。超微结构上我们证实，这种移动与可能从极生长的微管桥接损伤部位一致。未照射的半纺锤体也失去了一些双折射并缩短，直到它类似于恢复的半纺锤体。类似照射的后期细胞没有显示双折射的恢复，并且极与其余有丝分裂装置保持分离。纺锤体结构的重建证实，在连接被切断的纺锤体极与其余有丝分裂装置的损伤部位没有微管。这些结果表明，染色体到极的纺锤体力量的存在依赖于微管连续体的存在，并且在较小程度上依赖于中期/后期转换时中心体微管起始能力的丧失。
