Biomedical Analytical and High Resolution Microscopy Facility, University of the Basque Country, Leioa, Vizcaya, Spain.
Biol Cell. 2009 Oct 12;102(1):25-35. doi: 10.1042/BC20090077.
The execution phase of apoptosis is characterized by extensive blebbing of the plasma membrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes during this process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescently tagged proteins which were targeted to different subcellular locations.
The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blocking different phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were monitored by time-lapse confocal microscopy. Fluorescently labelled proteins were not mis-localized from their original locations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nucleolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyes confirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation of phosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period.
These results show that the functional integrity of the nuclear envelope and plasma membrane may be conserved until the end of the execution phase of apoptosis.
凋亡的执行阶段的特点是质膜广泛起泡，这通常导致体外的二次裂解。为了分析细胞在这个过程中的膜通透性，我们诱导了转染了荧光标记蛋白的人黑色素瘤 A375 细胞的凋亡，这些蛋白被靶向到不同的亚细胞位置。
白藜芦醇和丁酸盐的双重处理通过阻断细胞周期的不同阶段产生协同诱导凋亡的作用。通过延时共聚焦显微镜监测质膜、核膜和核仁的变化。在几个小时的起泡过程中，没有一个细胞中的荧光标记蛋白从其原始位置发生错误定位。因此，在起泡阶段，亲核蛋白和核仁蛋白在核内的保留，以及重要的选择性染色质染料的可及性，证实了核区室的功能保持，直到最终的坏死泡。在起泡期间未检测到磷脂酰丝氨酸向质膜外叶的易位。
这些结果表明，核膜和质膜的功能完整性可能在凋亡执行阶段结束之前得到保持。
