Name: spatiotemporal analysis of cytokinetic events in fission yeast
Uploaded: 2017-02-20T14:00:00
Description: Watch this Scientific Journal Video about Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast at JoVE.com

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).

1.样品的制备<ol><li>生长表达纺锤体极体标记Sad1在每个-mCherry 23和II型肌球蛋白调节轻链RLC1-番茄24中，YE液体培养基在32℃下8代裂殖酵母细胞。 注：对于温度敏感的突变体，在25℃生长细胞。生长细胞对数中期，以叶为0.5的OD 600。欲了解更多有关裂殖酵母的生长条件是指裂殖酵母实验手册25。 </li><li>豫备（或最少）的媒体用0.6％?...

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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Yeast extract media</td>
			<td>Sunrise Science Products</td>
			<td>YES 225</td>
			<td>0.5% w/v yeast extract, 3% w/v glucose, 225mg/L adenine, histidine, leucine, uracil, and lysine</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SeaKem LE agarose, Lonza</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottomed culture dish</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Coverslip No. 1.5 was used. This will vary as per the microscope specifications used.&#160;</td>
		</tr>
		<tr>
			<td>VT-Hawk 2D array laser scanning confocal microscopy system</td>
			<td>Visitech International, UK</td>
			<td></td>
			<td>with an Olympus IX-83 inverted microscope with a 100X / numerical aperture 1.49 UAPO lens (Olympus) and EM-CCD digital camera (Hamamatsu).&#160;</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
	</tbody>
</table>

spatiotemporal analysis of cytokinetic events in fission yeast

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。

表达该环标记裂殖酵母细胞，RLC1-GFP（绿色， 图2）和纺锤体极体标记Sad1在每个-mCherry（红， 图2）胞质中被成像。纺锤体极体标记的发病（白色箭头， 图2A，B），被认为是时间0 RLC1-GFP信号出现时间-4分钟参照纺锤极体的分离（红色箭头，图2A，2B）。 RLC1-GFP信号形成连续的环10分钟后纺锤极体的分离（空箭头， 图2A，B）的...

Watch this Scientific Journal Video about Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast at JoVE.com

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

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。

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

The overall goal of this live cell microscopy technique is to study to the spatiotemporal events that occur during cytokinesis in the fission yeast model system. This method can help answer key questions in the cytokinetic field such as the molecular details of the different stages of cytokinesis over time. The main advantage of this technique is that different spatial components of the cytokinetic machinery can be analyzed over long periods of time with minimal toxicity to the cells.Though this method can provide insight into cytokinesis and fission yeast, it can also be applied to budding yeast and other fungi. To begin this procedure, pour three to four milliliters of melted media laced with Vitamin C onto a glass-bottomed culture dish. Once the media has solidified, use a sharp scalpel to gently raise the media slab from the culture dish, and place a pipette tip between the media slab and the culture dish to prevent the slab from falling back into the dish.Next load two to five microliters of re-suspended cell culture between the media slab and the glass bottom of the culture dish. Then very gently remove the pipette tip and place the media slab back in its original position on the culture dish. Incubate the cells in the culture dish for 30 minutes to an hour in the dark at the temperature the microscopy will be performed.When the cells are ready for imaging, place a drop of wild on the outer side of the glass on the culture dish. Place the culture dish on an inverted microscope. Focus on the medial plane of the cells, and ensure that they are well-spaced and not too crowded.Make sure to start image acquisition of the cells in a suitable stage of the cell cycle. In this experiment, it is late G2.Next, program the image acquisition software. Set the exposure time for DIC to 100 milliseconds.For GFP and RFP filters, use exposure times of 75 milliseconds. Set the laser power to 50%but note that this will vary depending upon the individual experiment. To capture images in three dimensions, program the software to acquire Z series.Use of a maximum step size of 4 micrometers and a distance of 3.0 micrometers around the central focus point of the cells for the capture of 16 Z frames per time point for the total Z distance of 6 micrometers. These parameters can be altered in accordance with the thickness of the cell. Set the program to take images every two minutes.Select the exposure time based on the requirements of the experiment. In this example, 75 milliseconds is used. Program the software to stop acquisition after 90 minutes.And then start the image acquisition. ImageJ software is used to analyze the image. To begin, open the image series for each of the wavelengths.Select a cell to be studied. Double click on the line option of the toolbar to open another window to adjust the line width. Adjust the line width to correspond to the cell width which is about 40 to 50 pixels for a wild type cell.Draw a line along the long axis of the cell of interest. Click on analyze then tools then ROI manager and then click on add. This will add the selected line to the ROI window for later use.Do not close this window. Click on the image of interest then select edit, then selection, then straighten. Check process entire stack to straighten the cell horizontally.Click on image then transform then rotate 90 degrees right to straighten the image vertically. Click on image then stacks then make montage. This will open a window for assigning the number of rows and columns for the stack, the scale factor for the image size, the first and last slice or frame for the montage, and the frame increment for the montage.Check label slices and hit enter. As a window with a montage of the cell of interest is shown, open the image series for the other wavelength. In the ROI manager, click on the line identifier to select the same cell on the second image file.Repeat the previous steps to open a montage of the second image. On the image with the spindle pole body marker, Sad1-mCherry, look for the separation of the spindle pole body marker, and mark that time point as time zero. Follow the signal of the ring protein Rlc1-tomato on the image over time, and look for the signal that appears as a distinct line as opposed to the patches of Rlc1-tomato.This time point marks completion of actomyosin ring assembly or the start of the maturation phase. Next scroll through the movie over time to determine when the Rlc1-tomato ring starts to decrease in size. This is marked as the end of maturation phase or onset of ring constriction.Follow the Rlc1-tomato signal over time throughout constriction until it appears as a dot in the middle of the cell axis. This is marked as the end of ring constriction or end of septum ingression. Following the montage of DIC images, determine when abscission is completed.Record the time point when the cells physically separate. Begin this analysis by selecting a cell with a visible actomyosin ring. Double click on the line option of the ImageJ toolbar.Adjust the line width to correspond to the ring thickness about 15 to 20 pixels. Draw a line along the ring, taking care to ensure the entire thickness of the ring is included. Click on analyze, then tools, then ROI Manager, and click on add.This will add the select line to the ROI window for later use. Do not close this window. Click on the image of interest and select edit, then selection, then straighten.Check process entire stack to straighten the ring horizontally. Reset the intensity of the brightest plane in the straighten Z stack using image, then adjust, then brightness and contrast, then reset. Click on the tab SGK then 3D project to open a dialogue box.Change the projection method to brightest point and slice spacing to two to three pixels. Finally, change the axis of rotation to X or Y-axis depending upon the desired viewing angle. Click interpolate and click OK to generate a 3D projection of the image.Use the scroll bar beneath the image to rotate the image. Next, open the image series for the other wavelength. Click on the line identifier in the ROI manager to select the same ring on the second image file.Repeat the previous steps to open a 3D ring in the image with the other wavelength. With both 3D image rings open, click on image, then color, then merge channels. Select each image from the dropdown menu for the corresponding color desired and click OK.Compare the localization of different markers with regards to the localization at the cytokinetic ring or ingressing membrane.Fission yeast cells expressed in the spindle pole body marker, Sad1-mCherry and ring marker Rlc1-GFP were imaged during cytokinesis. Spindle pole body separation occurred at 17 minutes, designated as time zero. Rlc1-GFP appears at the division site four minutes before spindle pole body separation.After spindle pole body separation, ring maturation starts at ten minutes. Ring constriction begins at 31 minutes, and ends at 53 minutes. Finally, cell abscission occurs at 80 minutes after spindle pole body separation.The timeline of cytokinetic events determined in the previous images can be plotted in a graph with reference to mitosis as determined by the distance between the spindle pole body markers. Analysis of the ring marker Rlc1-tomato and the septum membrane marker Bgs1-GFP at the division site throughout cytokinesis reveal that the ring assembled before Bgs1-GFP recruitment. As the ring constricts, Bgs1-GFP also localizes to the ingressing membrane adjacent to the constricting ring.Finally, Rlc1-tomato signal dissipates after ring constriction while Bgs1-GFP appears as a disk, remaining within the membrane barrier. The efficiency of cytokinetic events can be determined by the distribution of proteins along the ring. Here Cdc15-GFP distribution along the ring is even on the left image but uneven on the right image.Once mastered, this technique can be done in two to three hours if performed properly. While attempting this procedure, it's important to remember to choose cells with an appropriate field, ideally containing six to eight well-spaced cells in late G2 or early M phase, with no visible impurities in the agar. After watching this video, you should have a good understanding of how to spatiotemporally analyze protein localization during cytokinesis.

Research

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