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Apr 2019
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Measurement of Mitotic Spindle Angle and Mitotic Cell Distance in Fixed Tissue of Drosophila Larval Brains
果蝇幼虫固定脑组织中有丝分裂纺锤体角度和有丝分裂细胞距离测定   

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Abstract

The positioning and the cleavage plane orientation of mitotic cells in pseudostratified epithelia (PSE) must be tightly regulated since failures in any of these processes might have fatal consequences during development. Here we present a simple method to determine the spindle orientation as well as the positioning of neuroepithelial mitotic cells within the Outer Proliferation Center (OPC) of Drosophila larval brains.

Keywords: Mitosis (有丝分裂), Spindle orientation (纺锤体方向), Interkinetic nuclear migration (细胞核动态迁移), Pseudostratified Epithelia (假复层上皮), Neuroepithelial cells (神经上皮细胞), Proliferation (增殖), Drosophila (果蝇)

Background

Pseudostratified epithelia (PSE) are epithelia in which the interphase nuclei of the epithelial cells dynamically position at different depths within the epithelial monolayer, while mitotic nuclei localize at the most apical side of the epithelium. PSE are usually highly proliferative tissues acting as organ precursors in many organisms, including the building blocks of the epiblast in the gastrulating mouse embryo, the pancreatic buds and different areas of the central nervous system in vertebrates, as well as the imaginal discs and the optic lobe anlage in Drosophila (Meyer et al., 2011; Ichikawa et al., 2013; Strzyz et al., 2016).

Almost a century ago, Sauer observed that in PSE nuclei moved to the apical surface to divide (Sauer, 1935). The apical migration of nuclei is linked to the cell cycle progression, occurring at G2 phase, and it is known as Interkinetic Nuclear Migration (INM) or Pre-mitotic Rapid Apical Migration (PRAM). Thus, INM is responsible for the spatial organization of proliferating cells in PSE and the localization of mitotic nuclei at the PSE basal side is frequently linked to failures in INM (Tsuda et al., 2009). The apical “mitotic zone” serves as a niche for signaling and spatial control of mitotic entry, and it has been recently shown that apical daughter cells reintegrate better into the PSE (reviewed in Norden, 2017).

Better known is the control of the mitotic spindle orientation, which will determine tissue architecture or cell fate specification in the context of symmetric or asymmetric cell divisions, respectively. As failures in both processes are implicated in tumorigenesis, spindle misorientation might promote or, at least, facilitate tumor development (Pease and Tirnauer 2011; Noatynska et al., 2012, Bergstralh and St Johnston 2014). Epithelial cells tend to divide with their mitotic spindle parallel to the plane of the epithelial sheet. The significance of tightly regulating this process during early development, when epithelia are in a high proliferative phase, is manifest by the existence of several neurological diseases associated with mitotic spindle orientation failures, such as microcephaly, lissencephaly and Huntington disease (Noatynska et al., 2012).

Several protocols for 3D automated measurements of mitotic spindles have been recently published (Juschkeet al., 2014; Lázaro-Diéguez et al., 2014). Although these protocols are very reliable as they eliminate any bias due to hand processing of the images, they are established to work in vitro, in cell culture. In Franco and Carmena (2019), we have developed a simple and reliable method to determine the angle of the mitotic spindle and the positioning of the mitotic nucleus of neuroepithelial cells in fixed whole brains of Drosophila third instar larvae, making possible to visualize in vivo, the morphogenetic consequences of failures in any of these processes. Since planar divisions and INM are considered hallmarks of some neural progenitors, these measurements can be an effective way of studying the architecture and organization of a developing neuroepithelium.

Materials and Reagents

  1. Dissection wells (Winlab, catalog number: 190029500)
  2. Dissection forceps (Fine Science Tools, Dumont, catalog number: 55)
  3. Glass slides and covers 
  4. Adhesive tape
  5. Yellow and white pipette tips
  6. Ice bucket
  7. Drosophila early third instar larvae (60 h after larval hatching ALH)
  8. BSA (Albumin from bovine serum, Sigma-Aldrich, catalog number: A3059)
  9. Primaries antibodies against a spindle pole protein, for example an anti-γTubulin (Mouse monoclonal anti-γTubulin, Sigma-Aldrich, catalog number: T5326), and against Phospho-Histone 3 (PH3) to determine metaphasic nuclei (Rabbit polyclonal anti-PH3 (Ser10), Millipore, catalog number: 06-570)
  10. Secondary antibodies coupled to Alexa Fluor 488 (Goat anti-Mouse IgG, Alexa Fluor Plus 488, Invitrogen, catalog number: A32723) and 555 (Goat anti-Rabbit IgG, Alexa Fluor Plus 555, Invitrogen, catalog number: A32732)
  11. Na2HPO4
  12. KCl
  13. NaCl
  14. Triton X-100 (Sigma-Aldrich, catalog number: T8787)
  15. PFA 16% solution (EM grade, Electron Microscopy Sciences, catalog number: 15710)
  16. Phalloidin linked to a fluorophore (Alexa Fluor 633 phalloidin, Invitrogen, catalog number: A22284), used to determine the cellular and tissue morphology
  17. Vectashield Mounting medium (Linaris, catalog number: H-1000)
  18. Phosphate-buffered saline (PBS) (see Recipes)
  19. PBT (see Recipes)
  20. Blocking Reagent (see Recipes)
  21. 4% Paraformaldehyde (PFA) in PBT-0.1T (see Recipes)

Equipment

  1. Orbital shaker
  2. Confocal microscope
  3. Autoclave

Software

  1. Lite Leika
  2. Image J
  3. Excel

Procedure

  1. Stain larval brains according to the following protocol
    Note: All the washes and incubations are done on an orbital shaker at slow motion.
    1. Roughly dissect about 15 brains per genotype in cold PBS. Try not to prolong more than 20 min the dissection time to preserve the quality of the material. Keep the material already dissected on ice-cold PBS.
    2. Fix by incubation in 4% PFA in PBT-0.1T for 20 min with gentle agitation.
    3. Rinse with PBT by replacing the solution 3 times and wash by gentle agitation for another 3 x 15 min each. During these washes, clean the brains of other pieces of tissue sticking to it.
    4. Incubate brains in PBT + 0.1% BSA for at least 1 h at Room Temperature (RT).
    5. Incubate brains with the corresponding primary antibodies at the right dilution in blocking reagent overnight at 4 °C.
    6. Rinse with PBT by replacing the solution 3 times and wash for another 3 x 15 min each.
    7. Incubate with secondary antibodies at the right dilution in blocking reagent for 2 h at RT in the dark.
    8. Rinse with PBT by replacing the solution 3 times and wash for another 3 x 15 min each.
    9. Wash with PBS for 10 min at RT.
    10. After removing the PBS, add Vectashield to the well and leave at 4 °C overnight (this incubation facilitates the mounting step).
    11. Mount brains onto glass slides using the bridge method illustrated in Figure 1, preserving their three-dimensional configuration.


      Figure 1. Bridge method for mounting Drosophila brains

    12. Glue two pieces of adhesive tape leaving a thin space (approx. 5 mm) in between as shown in Figure 1.
    13. Add the brains in the smallest possible volume of Vectashield with a cut yellow pipette tip.
    14. Remove as much as possible the excess of Vectashield with a cut white pipette tip.
    15. Arrange the brains with the ventral side facing up.
    16. Add a cover glass and fix it with 2 pieces of adhesive tape.
    17. Fill the space under the bridge with Vectashield.

  2. Imaging acquisition
    Fluorescent images were recorded using a Leica upright DMR microscope with an HCX Plan Apochromat 63x/1.32-0.6 NA oil confocal scanning objective. 2 x Zoom with a distance between focal planes in each Z-stack of 0.8 μm was used.

Data analysis

Open the Z-stack with ImageJ (shown in Figure 2)


Figure 2. A Z-stack of Drosophila early third instar larval brain hemisphere. PH3, γ-Tubulin and Phalloidin staining are shown in red, green and blue, respectively.
Note: It is important to analyze only the portion of neuroepithelium where the cells look more perpendicular to the longitudinal neuroepithelial plane.


  1. Spindle angle measurement
    1. Select the “angle” tool and draw a line linking both centrosomes, press the mouse button and draw an angle respect to a second line running parallel to the apical surface of the neuroepithelium, as it is shown in Figure 3. Then press <m> and a window with the measurement results will appear.


      Figure 3. Measurement of the mitotic spindle angle

      Note: These measurements frequently require correction in the xy plane to allow that both spindle poles are present in a single Z-plane. To avoid overcorrection, only mitosis with centrosomes in the same or in two consecutive Z planes are considered for quantification.

    2. Collect all the measurements.
    3. Save the results as an excel file.
    4. Open the document in Excel. Copy the column with the angle results and paste it in a new column (Column A in the example shown in Figure 4). Arrange the data in order (Column B in the example) and proceed with the following steps. Step 1: Transform the values above 90 degrees by applying: y = 180 - x (Column C in the example), where x is the value obtained in the measurement. Step 2: Divide all the data into categories (every 15 degrees, for example).


      Figure 4. Excel sheet workup for data analysis of the mitotic spindle orientation

      With the data arranged in this way, perform different statistic tests to see whether two populations have the same distribution of mitotic angles respect to the plane. It is recommendable to understand well and define properly the behavior of your control, as well as to define whether your data follow a Parametric distribution or not.
      Note: Angles respect to the apical surface equal or minor than 30 degrees are considered wild type.

  2. Measurement of mitotic cell positioning
    1. Select the “straight line” tool and, in each mitotic cell, draw two lines: one from the apical surface to the middle of the metaphasic plate (and press <m>), and another from the apical to the basal side of the epithelium where the mitotic cell is located (and press <m>). These lines correspond to “a” and “b”, respectively, in Figure 5.


      Figure 5. Measurement of mitotic cells relative positioning within the neuroepithelium

    2. Collect all the measurements. 
    3. Save the results as an excel file. 
    4. Open the document in Excel. 
    5. Calculate (Column D in the example in Figure 6) the Relative Position (%) of the mitotic plate respect to the apical side of the epithelium for every cell, by applying:



      where “a” is the distance from the apical surface to the mitotic plate and “b” is the whole width of the epithelia at this position (see also Figure 5).

    6. Order all the data and see how the mitotic nuclei arrange along the apico-basal axis of the NE.


      Figure 6. Excel sheet workup for data analysis of the relative mitotic cell distance

    7. With the data arranged in this way, represent the distribution of the mitotic cells along the apico-basal axis of the neuroepithelium. Then, perform different statistic tests to analyze possible differences between two samples.

Having measured both parameters (mitotic spindle orientation and mitotic cell distance) for a given genotype, correlations to see whether one parameter has an effect on the other can be performed. For example, in Franco and Carmena (2019), we determined that 80% of the cells in the control divide in the most apical part (0-40%) of the NE. Even though we could not find a straight correlation between failures in positioning and spindle orientation, we could conclude that basal mitosis was more prone to show defects in mitotic spindle alignment.

Recipes

  1. PBS
    10 mM Na2HPO4
    2.68 mM KCl
    140 mM NaCl
    pH 7.4
    Sterilize by autoclaving
  2. PBT
    PBS with 0.3% Triton
  3. Blocking Reagent
    PBT with 0.1% BSA
  4. 4% Paraformaldehyde (PFA) in PBT-0.1T
    Dilute 16% PFA in PBS with 0.1333 % Triton

Acknowledgments

Our lab was supported by the Spanish grants from the Ministry of Economy and Competitiveness (MINECO) BFU2012-33020 and BFU2015-64251, and by FEDER (European Regional Development Fund). The Instituto de Neurociencias in Alicante is a “Severo Ochoa” Center of Excellence. The original research paper where this protocol was used is Franco and Carmena, 2019.

Competing interests

The authors declare no competing interests.

References

  1. Bergstralh, D. T. and St Johnston, D. (2014). Spindle orientation: what if it goes wrong? Semin Cell Dev Biol 34: 140-145.
  2. Franco, M. and Carmena, A. (2019). Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells. J Cell Biol 218(4): 1200-1217.
  3. Ichikawa, T., Nakazato, K., Keller, P. J., Kajiura-Kobayashi, H., Stelzer, E. H., Mochizuki, A. and Nonaka, S. (2013). Live imaging of whole mouse embryos during gastrulation: migration analyses of epiblast and mesodermal cells. PLoS One 8(7): e64506.
  4. Juschke, C., Xie, Y., Postiglione, M. P. and Knoblich, J. A. (2014). Analysis and modeling of mitotic spindle orientations in three dimensions. Proc Natl Acad Sci U S A 111(3): 1014-1019.
  5. Lazaro-Dieguez, F., Ispolatov, I. and Musch, A. (2015). Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum. Mol Biol Cell 26(7): 1286-1295.
  6. Meyer, E. J., Ikmi, A. and Gibson, M. C. (2011). Interkinetic nuclear migration is a broadly conserved feature of cell division in pseudostratified epithelia. Curr Biol 21(6): 485-491.
  7. Noatynska, A., Gotta, M. and Meraldi, P. (2012). Mitotic spindle (DIS)orientation and DISease: cause or consequence? J Cell Biol 199(7): 1025-1035.
  8. Norden, C. (2017). Pseudostratified epithelia-cell biology, diversity and roles in organ formation at a glace. J Cell Sci 130(11): 1859-1863.
  9. Pease, J. C. and Tirnauer, J. S. (2011). Mitotic spindle misorientation in cancer--out of alignment and into the fire. J Cell Sci 124(Pt 7): 1007-1016.
  10. Sauer, F. C. (1935). Mitosis in the neural tube. J Comp Neurol 62(2): 337-405. 
  11. Strzyz, P. J., Matejcic, M. and Norden, C. (2016). Heterogeneity, cell biology and tissue mechanics of pseudostratified epithelia: coordination of cell divisions and growth in tightly packed tissues. Int Rev Cell Mol Biol 325: 89-118.
  12. Tsuda, S., Kitagawa, T., Takashima, S., Asakawa, S., Shimizu, N., Mitani, H., Shima, A., Tsutsumi, M., Hori, H., Naruse, K., Ishikawa, Y. and Takeda, H. (2010). FAK-mediated extracellular signals are essential for interkinetic nuclear migration and planar divisions in the neuroepithelium. J Cell Sci 123(Pt 3): 484-496.

简介

伪分层上皮细胞(PSE)中有丝分裂细胞的定位和切割平面取向必须受到严格调节,因为任何这些过程中的失败都可能在发育过程中造成致命后果。在这里,我们提出了一种简单的方法来确定果蝇幼虫大脑外扩散中心(OPC)内的纺锤体方向以及神经上皮有丝分裂细胞的位置。
【背景】伪分层上皮(PSE)是上皮细胞的相间核动态定位在上皮单层内不同深度的上皮细胞,而有丝分裂核位于上皮的最顶端。 PSE通常是高度增殖的组织,在许多生物体中均充当器官前体,包括在胃肠道中的小鼠胚胎中的上皮细胞,脊椎动物的胰腺芽和中枢神经系统的不同区域,以及假想盘和视神经果蝇中的叶裂(Meyer等人,2011; Ichikawa等人,2013; Strzyz等人。,2016年)。

大约一个世纪前,绍尔(Sauer)观察到,在PSE中,原子核移至顶端表面分裂(Sauer,1935)。核的顶峰迁移与发生在G2期的细胞周期进程相关,这被称为动代核迁移(INM)或有丝分裂前快速顶迁移(PRAM)。因此,INM负责PSE中增殖细胞的空间组织,而有丝分裂核在PSE基底侧的定位通常与INM的失败有关(Tsuda et al。,2009)。根尖的“有丝分裂区”充当有丝分裂进入的信号传导和空间控制的利基,并且最近显示,根尖的子细胞可以更好地重新整合到PSE中(综述于Norden,2017年)。

众所周知,对有丝分裂纺锤体定向的控制将分别在对称或不对称细胞分裂的背景下确定组织结构或细胞命运规范。由于这两个过程的失败都与肿瘤发生有关,因此纺锤体方向错误可能会促进或至少促进肿瘤的发展(Pease和Tirnauer,2011年; Noatynska等,2012年,Bergestralh和St Johnston,2014年)。上皮细胞倾向于分裂,其有丝分裂纺锤体平行于上皮层的平面。上皮细胞处于高增殖期时,在早期发育过程中严格调节这一过程的重要性体现在与有丝分裂纺锤体定向失败相关的几种神经系统疾病的存在,例如小头畸形,小脑畸形和亨廷顿病(Noatynska et等,2012)。

最近发布了几种用于有丝分裂纺锤体3D自动测量的协议(Juschke 等人,,2014;Lázaro-Diéguez等人,,2014)。尽管这些协议非常可靠,因为它们消除了由于手工处理图像而引起的任何偏见,但它们被确立为在细胞培养中体外起作用。在Franco和Carmena(2019)中,我们开发了一种简单可靠的方法来确定果蝇第三龄幼虫固定全脑中有丝分裂纺锤体的角度和神经上皮细胞的有丝分裂核的位置,从而可以可视化体内在任何这些过程中失效的形态发生后果。由于平面划分和INM被认为是某些神经祖细胞的标志,因此这些测量结果可能是研究发育中的神经上皮细胞的结构和组织的有效方法。

关键字:有丝分裂, 纺锤体方向, 细胞核动态迁移, 假复层上皮, 神经上皮细胞, 增殖, 果蝇

材料和试剂

  1. 解剖孔(Winlab,目录号:190029500)
  2. 解剖钳(Fine Science Tools,Dumont,目录号:55)
  3. 载玻片和盖
  4. 胶带
  5. 黄色和白色移液器技巧
  6. 冰桶
  7. 果蝇初三龄幼虫(幼虫孵化后ALH 60小时)
  8. BSA(牛血清白蛋白,Sigma-Aldrich,目录号:A3059)
  9. 针对纺锤体极点蛋白(例如抗γTubulin(小鼠单克隆抗γTubulin,Sigma-Aldrich,目录号:T5326))和抗磷酸化组蛋白3(PH3)的初级抗体,以确定中期核(兔多克隆抗PH3) (Ser10),Millipore,货号:06-570)
  10. 与Alexa Fluor 488(山羊抗小鼠IgG,Alexa Fluor Plus 488,Invitrogen,目录号:A32723)和555(山羊抗兔IgG,Alexa Fluor Plus 555,Invitrogen,目录号:A32732)偶联的二抗
  11. Na 2 HPO 4
  12. 氯化钾
  13. 氯化钠
  14. 海卫一X-100(西格玛奥德里奇,目录号:T8787)
  15. PFA 16%溶液(EM级,电子显微镜科学,目录号:15710)
  16. 与荧光团相连的鬼笔环肽(Alexa Fluor 633鬼笔环肽,Invitrogen,目录号:A22284),用于确定细胞和组织的形态
  17. Vectashield安装介质(Linaris,目录号:H-1000)
  18. 磷酸盐缓冲盐水(PBS)(请参阅食谱)
  19. PBT(请参阅食谱)
  20. 封闭试剂(请参见配方)
  21. PBT-0.1T中的4%多聚甲醛(PFA)(请参阅食谱)

设备

  1. 轨道振动器
  2. 共聚焦显微镜
  3. 高压灭菌器

软件

  1. 莱特·莱卡(Lite Leika)
  2. 图片J
  3. Excel

程序

  1. 根据以下方案对幼虫脑进行染色
    注意:所有清洗和孵育都是在定轨摇床上缓慢进行的。
    1. 在冷PBS中,每个基因型大约解剖15个大脑。尽量不要延长解剖时间超过20分钟,以保持材料的质量。使材料已经在冰冷的PBS上解剖。
    2. 通过在PBT-0.1T中的4%PFA中温和搅拌孵育20分钟来固定。
    3. 更换溶液3次,用PBT冲洗,然后再轻轻搅拌3 x 15分钟,以洗涤。在这些洗涤过程中,清洁粘附在其上的其他组织的大脑。
    4. 在室温(RT)下,将大脑在PBT + 0.1%BSA中孵育至少1小时。
    5. 在适当的稀释度下,将脑与相应的一抗在封闭剂中于4°C孵育过夜。
    6. 更换溶液3次,用PBT冲洗,然后每次清洗3 x 15分钟。
    7. 在黑暗中与适度稀释的二抗在封闭剂中于室温孵育2小时。
    8. 更换溶液3次,用PBT冲洗,然后每次清洗3 x 15分钟。
    9. 在室温用PBS洗涤10分钟。
    10. 除去PBS后,将Vectashield添加至孔中,并在4°C下放置过夜(此孵育有助于安装步骤)。
    11. 使用图1所示的桥接方法将大脑固定在载玻片上,保留其三维结构。


      图1.安装桥的方法 果蝇 大脑

    12. 粘贴两片胶带,在它们之间留出一个狭窄的空间(大约5毫米),如图1所示。
    13. 用切好的黄色移液器吸头将大脑放入最小体积的Vectashield中。
    14. 用切开的白色移液器吸头除去多余的Vectashield。
    15. 安排大脑,使腹侧朝上。
    16. 加盖玻璃并用2条胶带固定。
    17. 用Vectashield填充桥下的空间。

  2. 影像采集
    使用具有HCX Plan Apochromat 63x / 1.32-0.6 NA油共聚焦扫描物镜的Leica立式DMR显微镜记录荧光图像。使用每个z堆栈中焦平面之间的距离为0.8μm的2倍缩放。

数据分析

使用ImageJ打开Z堆栈 (如图2所示)


图2. Z堆 果蝇 早期三龄幼虫脑半球。 PH3,γ-微管蛋白和鬼笔环肽染色分别以红色,绿色和蓝色显示。
注意:重要的是仅分析神经上皮细胞看起来与纵向神经上皮平面更垂直的部分。

  1. 主轴角度测量
    1. 选择“角度”工具并绘制一条连接两个中心体的线,按鼠标按钮并相对于平行于神经上皮顶表面的第二条线绘制一个角度,如图3所示。然后按&lt; m&gt; ;将会出现一个带有测量结果的窗口。


      图3.有丝分裂纺锤体角的测量

      注意:这些测量通常需要在xy平面中进行校正,以使两个主轴都位于单个Z平面中。为避免过度校正,仅考虑在相同或两个连续Z平面中具有中心体的有丝分裂进行定量。

    2. 收集所有测量值。
    3. 将结果另存为excel文件。
    4. 在Excel中打开文档。复制带有角度结果的列,并将其粘贴到新列中(图4中示例中的列A)。按顺序排列数据(示例中的B列),然后执行以下步骤。第1步:通过应用以下公式变换90度以上的值:y = 180-x(在示例中为C列),其中x是在测量中获得的值。第2步:将所有数据划分为类别(例如,每15度)。


      图4.用于对有丝分裂纺锤体定向进行数据分析的Excel工作表

      使用以这种方式排列的数据,执行不同的统计检验以查看两个总体相对于平面是否具有相同的有丝分裂角分布。建议您很好地理解并正确定义控件的行为,以及定义数据是否遵循参数分布。
      注意:相对于顶面的角度等于或小于30度被认为是野生型。

  2. 有丝分裂细胞定位的测量
    1. 选择“直线”工具,并在每个有丝分裂细胞中绘制两条线:一条从心尖表面到中性板的中间(并按&lt; m&gt;),另一条从心尖的顶点到基端。上皮细胞位于有丝分裂细胞所在的位置(并按&lt; m&gt;)。这些行分别对应于图5中的“ a”和“ b”。


      图5.测量神经上皮内有丝分裂细胞的相对位置

    2. 收集所有测量值。
    3. 将结果另存为excel文件。
    4. 在Excel中打开文档。
    5. 计算每个细胞的有丝分裂板相对于上皮顶侧的相对位置(%,在图6的示例中为D列),方法如下:



      其中“ a”是从根尖表面到有丝分裂板的距离,“ b”是在该位置上皮的整个宽度(另请参见图5)。

    6. 对所有数据进行排序,看看有丝分裂核如何沿NE的apico-基轴排列。


      图6.用于对有丝分裂相对细胞距离进行数据分析的Excel工作表

    7. 以这种方式排列的数据表示有丝分裂细胞沿神经上皮的apico-基底轴的分布。然后,执行不同的统计检验以分析两个样本之间的可能差异。

测量了给定基因型的两个参数(有丝分裂纺锤体定向和有丝分裂细胞距离)后,就可以进行相关性以确定一个参数是否对另一个有影响。例如,在Franco和Carmena(2019)中,我们确定对照组中80%的细胞分裂为NE的最顶端部分(0-40%)。即使我们找不到定位失败与主轴定向之间的直接关系,我们也可以得出结论,基础有丝分裂更倾向于显示有丝分裂主轴对齐的缺陷。

菜谱

  1. PBS
    10 mM Na 2 HPO 4
    2.68 mM氯化钾
    140毫米氯化钠
    pH 7.4
    通过高压灭菌消毒
  2. PBT
    含0.3%Triton的PBS
  3. 封闭剂
    含0.1%BSA的PBT
  4. PBT-0.1T中的4%多聚甲醛(PFA)
    用0.1333%Triton稀释PBS中的16%PFA

致谢

我们的实验室得到了经济与竞争力部(MINECO)的西班牙资助BFU2012-33020和BFU2015-64251,以及FEDER(欧洲区域发展基金)的支持。阿利坎特的神经科学研究所是“ Severo Ochoa”卓越中心。使用此协议的原始研究论文是Franco和Carmena,2019。

利益争夺

作者宣称没有利益冲突。

参考文献

  1. Bergstralh,D.T.和St Johnston,D.(2014)。 主轴定位:如果出现问题怎么办? Semin Cell Dev Biol < / em> 34:140-145。
  2. Franco,M.和Carmena,A.(2019年)。 Eph信号控制神经上皮细胞中的有丝分裂纺锤体定向和细胞增殖。 J Cell Biol 218(4):1200-1217。
  3. Ichikawa,T.,Nakazato,K.,Keller,P.J.,Kajiura-Kobayashi,H.,Stelzer,E.H.,Mochizuki,A.和Nonaka,S.(2013)。 全胃胚化过程中整个小鼠胚胎的实时成像:表皮和中胚层细胞的迁移分析。 PLoS One 8(7):e64506。
  4. Juschke,C.,Xie,Y.,Postiglione,M.P.和Knoblich,J.A.(2014)。 在三个维度上对有丝分裂纺锤体定向进行分析和建模。 Proc Natl Acad美国科学(Sci USA) 111(3):1014-1019。
  5. Lazaro-Dieguez,F.,Ispolatov,I.和Musch,A.(2015年)。 细胞形状会影响有丝分裂纺锤体相对于基质的定位。 Mol Biol Cell 26(7):1286-1295。
  6. Meyer,E。J.,Ikmi,A。和Gibson,M。C.(2011)。 动态核迁移是伪分层上皮细胞分裂的一个广泛保守的特征。 Curr Biol 21(6):485-491。
  7. Noatynska,A.,Gotta,M.和Meraldi,P.(2012)。 有丝分裂纺锤体(DIS)的定位和疾病:原因还是后果? J细胞生物学 199(7):1025-1035。
  8. Norden,C.(2017年)。 假性分层的上皮细胞生物学,多样性及其在器官形成过程中的作用。 J细胞科学(Sci)130(11):1859-1863。
  9. Pease,J.C.和Tirnauer,J.S.(2011)。 癌症中的有丝分裂纺锤取向错误-失去对准并着火了。 J Cell Sci 124(Pt 7):1007-1016。
  10. 绍尔·FC(1935年)。 神经管中的有丝分裂。 J Comp Neurol 62(2):337-405。
  11. Strzyz,P.J.,Matejcic,M.和Norden,C.(2016年)。 假复层上皮细胞的异质性,细胞生物学和组织力学:紧密包装组织中细胞分裂和生长的协调 Int Rev Cell Mol Biol 325:89-118。
  12. 津田,北田川,高岛,浅川,南清水,三谷,H.,Shima,A.,Tsutsumi,M.,Hori,H.,成濑,K., Ishikawa,Y.和Takeda,H.(2010)。 FAK介导的细胞外信号对于动力学上的核迁移和神经上皮的平面分裂至关重要。 J Cell Sci 123(Pt 3):484-496。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Franco, M. and Carmena, A. (2019). Measurement of Mitotic Spindle Angle and Mitotic Cell Distance in Fixed Tissue of Drosophila Larval Brains. Bio-protocol 9(22): e3432. DOI: 10.21769/BioProtoc.3432.
  2. Franco, M. and Carmena, A. (2019). Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells. J Cell Biol 218(4): 1200-1217.
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