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

在25℃、75 MPa高压处理30 min后，再进行90 min的后培养，主要观察到约8倍于正常长度的长丝状细胞。FtsZ环的形成在丝状细胞中大大减少。我们已经报告了这些观察结果。FtsZ蛋白与微管蛋白α（或β）具有低水平的序列相似性。因此，可以说FtsZ是细菌的细胞骨架。我们的目的是测试是否cytokerone也是最敏感的HPT在真核微生物，如裂殖酵母。肌动蛋白用罗丹明-鬼笔环定荧光染色。用YFP（黄色荧光蛋白）结合的微管蛋白α观察微管。HPT（100 MPa，30 min）后，微管、肌动蛋白索和收缩环（由肌动蛋白和肌球蛋白组成）解聚消失。在液体培养基中的后生长中，微管在6小时后恢复，肌动蛋白索和收缩环在18小时后重新出现。结论：肌动蛋白索和收缩环对HPT最为敏感。在对裂殖酵母进行HPT实验时，DAPI染色显示DNA时，我们发现HPT诱导的染色体凝集。后生长30 min后，冷凝得到缓解。众所周知，染色体凝聚发生在有丝分裂的前期。HPT诱导的染色体凝集不受细胞周期的影响，并具有较高的致密性。核膜在HPT后没有收缩，如通过YFP连接的输入蛋白的核膜的可视化所证实的，表明染色体DNA本身确实浓缩。HPT诱导的染色体凝集不需要细胞周期依赖的凝集素（Cut 3-Cut 14复合物）。HPT诱导型冷凝将是对高压胁迫的紧急保护反应。

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.