在裂殖酵母中Cytokinetic活动的时空分析
Summary
裂殖酵母， 粟酒裂殖酵母是研究胞质极好的模型系统，在细胞分裂的最后阶段。在这里，我们描述了一个显微镜的方法来分析活裂殖酵母细胞不同cytokinetic事件。
Abstract
胞质，在细胞分裂的最后一步是维持基因组完整性的关键。正确的胞质分裂是细胞分化和发育非常重要。胞质分裂涉及一系列在时间上和空间上很好地协调活动。胞质涉及在分割部位的肌动球蛋白环的形成，随后通过环收缩，膜沟的形成和细胞外基质重塑。裂殖酵母， 裂殖酵母 （ 裂殖酵母）是揭示有大量的清晰度在胞质分裂的初始事件充分研究的模型系统。但是，我们不明白cytokinetic事件显然有什么不同spatiotemporally协调。为了确定这一点，需要分析很详细的不同cytokinetic事件在时间和空间。在这里，我们描述了一个显微镜的方法来研究不同cytokinetic EV需求测试的活细胞。使用这种方法，可以不同的时间cytokinetic事件和确定胞质中不同蛋白质的募集时间。此外，我们描述的协议在细胞分裂的部位比较蛋白质的定位和分布。这是一个基本的协议来研究胞质裂殖酵母，也可以用于其它酵母和真菌的系统。
Introduction
胞质，在细胞分裂的最后一步，是适当的分化，发育，和生物体的存活一个复杂的过程是必不可少的。胞质分裂涉及到的组织，以确保成功细胞分离，同时保持基因组的完整性1多个事件。胞质涉及事件，其中一次组装的肌动球蛋白环经历收缩，这是并发膜膨胀和开沟，和细胞外细胞基质重塑，最后接着细胞脱落1，2，3。 cytokinetic事件的不当组织可导致细胞的分离和倍性的缺陷，并可能导致的疾病，如癌症4，5，6，7，8。的基本原则，使Òcytokinetic事件rganization都不能很好地理解，因此导致路障中的这些疾病的病因学的理解。
裂殖酵母粟酒裂殖酵母 （ 裂殖酵母）是研究胞质优异的模型系统，由于涉及1蛋白质的保守性质。裂殖酵母，肌动球蛋白环组件后，该环进入成熟/停留期，此时它不收缩9。成熟与肌动球蛋白环收缩，并发膜开沟和隔侵入开始结束。多年来开创性的工作给了我们肌动球蛋白环组件的一个相当不错的理解裂殖酵母1,9，10。在一些真核生物，包括裂殖酵母中，肌动球蛋白环的成功组装不足以膜开沟。 f中ission酵母，环缩单独不提供足够的力来克服对沟形成11内部膨压。最近的模型表明，这股力量代替由隔侵入11提供。在另一个模型中，质膜的扩展的作用已建议向沟形成12，13。环收缩和膜开沟不允许BGS1 / CPS1温度敏感突变cps1-191，对于主隔膜形成14，15中的主要酶发生。缺乏BGS1细胞显示缺陷主要隔，延长环收缩15，16。 BGS1是肌动球蛋白环组件17,18后成熟过程中招募到细胞分裂站点隔侵入。 SimilarlY，在果蝇胚胎细胞化过程中，环形缩窄是双相有显著缓慢的初始速度收缩19，类似在裂殖酵母中观察到的成熟阶段。双相环收缩可能会减慢膜开沟以确保足够的膜扩张20和细胞外基质的修改。这表明，肌动球蛋白环组件后，发生环收缩有效地仅当电池已经满足了沟形成的要求。它不能很好地理解，需要什么样的条件对于环组件后肌动球蛋白环收缩，也不是调节该过程的分子事件。我们最近表明，以下肌动球蛋白环组件中的小GTP酶的Cdc42经历一种独特的时空激活图案21。此模式是由Cdc42的胍核苷酸交换因子的独特的定位图案（建立全环基金）的激活Cdc42的。全球环境基金GEF1定位于组装肌动球蛋白环和促进环收缩和隔侵入发病，而SCD1定位于开沟膜和促进正常的隔膜形成。我们发现，通过其环基金建立的Cdc42的激活模式导致不同cytokinetic事件的调节。
理解的事件，最终导致细胞分离以下肌动球蛋白环组件的分子机制，需要按照时间和空间上的不同cytokinetic事件。裂殖酵母，胞质分裂首先涉及细胞核周围前驱节点，最终招II型肌球蛋白，所述formin CDC12，以及肌动球蛋白环组件所需的其他蛋白质的组装。到时的不同cytokinetic事件和提供一个参考帧，的纺锤体极体标志物（纺锤体形成）分离被认为是时间0 22。个的组件È肌动球蛋白环可通过监测在一段时间，如II型肌球蛋白调节轻链RLC1一个荧光标记的肌动球蛋白环蛋白的强度。在这里，我们描述了微观的方法随时间推移分析胞质的不同阶段。
Protocol
Representative Results
Discussion
在这里，我们已经描述了协议来研究在裂殖酵母cytokinetic事件以时间的方式。这里所描述的协议提供了参照彼此不同cytokinetic事件的时间分辨率;蛋白质招聘或参照cytokinetic缺相的时间;整个胞质的不同阶段的环的结构;和胞质的参照的有丝分裂的进展。与此协议是可能的精确定义可以在不同的突变体而改变，从而提供涉及不同cytokinetic相蛋白质的更详细的了解cytokinetic相。另外，为了在时间上区分不同…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by startup funds from The University of Tennessee and TN-SCORE, a multi-disciplinary research program sponsored by NSF-EPSCoR (EPS-1004083).
Materials
| Yeast extract media | Sunrise Science Products | YES 225 | 0.5% w/v yeast extract, 3% w/v glucose, 225mg/L adenine, histidine, leucine, uracil, and lysine |
| Agarose | SeaKem LE agarose, Lonza | 50001 | |
| Ascorbic acid | Sigma-Aldrich | A4544 | |
| Glass Bottomed culture dish | MatTek Corporation | P35G-1.5-14-C | Coverslip No. 1.5 was used. This will vary as per the microscope specifications used. |
| VT-Hawk 2D array laser scanning confocal microscopy system | Visitech International, UK | with an Olympus IX-83 inverted microscope with a 100X / numerical aperture 1.49 UAPO lens (Olympus) and EM-CCD digital camera (Hamamatsu). | |
| ImageJ | NIH | Image analysis software |
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