Studies on cytokinetic injury caused by high pressure treatment on E.coli and fission yeast

在75 Mpa、25℃的高压处理30min后，对大肠杆菌细胞进行后培养90min，可观察到比正常长度长约8倍的长丝状细胞。丝状细胞中FtsZ环的形成明显减少。我们已经报道了这些观察结果。FtsZ蛋白与微管蛋白α(或β)的序列相似性较低。因此，可以说FtsZ是一种细菌细胞骨架。我们的目标是测试细胞骨架是否也对HPT最敏感，在真核微生物中，如分裂酵母。肌动蛋白用罗丹明-菲洛定进行荧光染色。用黄色荧光蛋白标记的微管蛋白α显示微管。HPT(100 Mpa，30min)后，微管、肌动蛋白缆索和收缩环(由肌动蛋白和肌球蛋白组成)因解聚而消失。在液体培养液中的后期生长中，微管在6h后恢复，18h后重新出现肌动蛋白绳索和收缩环。结论：肌动蛋白绳索和收缩环对HPT最为敏感。在裂解酵母的HPT实验中，DAPI染色显示DNA时，偶然发现HPT引起的染色体凝集。后生长30min后凝结减轻。众所周知，染色体凝集发生在有丝分裂的前期。HPT诱导的染色体凝聚不依赖于细胞周期，并出现较高的致密性。YFP标记的Importin对核膜的显示证实，HPT后核膜没有收缩，表明染色体DNA本身确实凝聚。HPT诱导的染色体凝聚不需要细胞周期依赖性凝集素(Cut3-Cut14复合体)。HPT诱导型冷凝将是对高压应激的一种紧急保护性反应。

If post-culture was made on E.coli cells for 90 min after they received high pressure treatment(HPT) at 75MPa for 30 min at 25℃, long filamentous cells of about 8 times longer than normal length were observed predominantly. FtsZ ring formation was greatly reduced in the filamentous cells. We already reported these observations. FtsZ protein has a low level of sequence similarity with tubulin α(or β). It can be said, therefore, that FtsZ is a bacterial cytoskeleton. We aimed to test whether cytoskeletones are also most sensitive to HPT in eukaryotic microorganisms such as fission yeast. Actin was fluorescently stained with rhodamine-phalloidine. Microtubule was visualized with YFP(yellow fluorescent protein) conjugated tubulin α. After HPT (100MPa for 30 min), microtubule, actin cable and contractile ring (constituted with actin and myosin) disappeared due to depolymerization. In the post-growth in a liquid culture medium, microtubule was restored after 6 hours and actin cable and contractile ring reappeared after 18 hours. It was concluded that actin cable and contractile ring were most sensitive to HPT. During the experiment of HPT on fission yeast, we happened to find chromosome condensation induced by HPT when DNA was visualized by DAPI staining. The condensation was relieved after 30 min of post-growth. It is well known that chromosome condensation occurs in the prophase of mitosis. HPT induced chromosome condensation was cell cycle-independent and occurred to higher compactness. Nuclear membrane did not shrink after HPT as confirmed by visualization of nuclear membrane by YFP-linked importin, indicating that chromosomal DNA itself did condense. HPT induced chromosome condensation did not require condensin (Cut3-Cut14 complex) that is necessary for cell cycle dependent one. HPT induced type condensation will be an urgent protective response against high pressure-stress.