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Physical Removal of the Midbody Remnant from Polarised Epithelial Cells Using Take-Up by Suction Pressure (TUSP)
采用吸压抽吸法(TUSP)物理去除极化上皮细胞中的中间体残留物

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Abstract

In polarised epithelial cells the midbody forms at the apical cell surface during cytokinesis. Once severed, the midbody is inherited by one of the daughter cells remaining tethered to the apical plasma membrane where it participates in non-cytokinetic processes, such as primary ciliogenesis. Here, we describe a novel method to physically remove the midbody remnant from cells and assess the possible effects caused by its loss (Bernabé-Rubio et al., 2016).

Keywords: Epithelial cells(上皮细胞), Midbody remnant(中间体残留物), Primary cilium(初级纤毛), Suction pressure(吸压), Patch-clamp equipment(膜片钳装置)

Background

The midbody or the Flemming body is the central part of the intercellular bridge formed between daughter cells during the final stages of mitosis. The abscission on either side of the bridge by the endosomal sorting complexes required for transport (ESCRT) machinery, results in the physical separation of the two daughter cells (Green et al., 2012). In addition to its known function in the regulation of mitosis, recent studies have begun to elucidate post-mitotic roles for the midbody. Due to its role in the initiation of lumen formation in kidney cells, the midbody has been postulated to serve as a polarity cue (Li et al., 2014). More recently, it has been demonstrated that the midbody remnant is directly involved in primary ciliogenesis by polarised Madin-Darby canine kidney (MDCK) cells (Bernabé-Rubio et al., 2016). It has been also found to have a role in formation of the dorsoventral axis during the development of Caenorhabditis elegans (Singh and Pohl, 2014), and in defining cell fate and differentiation (Kuo et al., 2011). Previous studies have used laser ablation to impair the function of the midbody remnant. When performed in cultured cell lines, however, laser ablation can result in cell death due to damage of the plasma membrane and proximal cytosolic elements. Accordingly, we have designed a gentle procedure, which we have called ‘take-up by suction pressure’ (TUSP). TUSP allows non-deleterious midbody remnant removal from the cell surface of epithelial cells. The fundamental principle is based on using a fine-aperture glass pipette attached to patch-clamp apparatus to physically remove the midbody with applied negative pressure (Figure 1).


Figure 1. Diagram of the TUSP procedure. A. An apical intercellular bridge forms during cytokinesis in polarised epithelial cells. B. After abscission, one of the daughter cells inherits the midbody as a remnant, which will be positioned over the apical cell surface. C-E. By using a glass pipette connected to path-clamp apparatus, the midbody remnant can be removed from cells if suction pressure is applied.

Materials and Reagents

  1. 12 mm glass coverslips #1 (VWR, catalog number: 631-0713 )
  2. Gridded coverslips (optional) (Electron Microscopy Sciences, catalog number: 72265-12 )
  3. Falcon 24-well plates (Corning, catalog number: 353047 )
  4. Permanent marker (Faber-Castell Multimark 1523) (CultPens, catalog number: FC19628 )
  5. 1 mL syringe (BD, catalog number: 303172 )
  6. 25 G 1 ½ needle (BD, catalog number: 305127 )
  7. Epithelial Madin-Darby canine kidney (MDCK II) from ATCC (ATCC, catalog number: CRL2936 )
  8. DNA construct expressing a fluorescent midbody localised protein (e.g., Cherry-tubulin, Addgene, catalog number: 49149 )
  9. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D5796 )
  10. Fetal bovine serum (Sigma-Aldrich, catalog number: F7524 )
  11. Penicillin-streptomycin solution (Sigma-Aldrich, catalog number: P4333 )
  12. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668027 )
  13. Hank’s balanced salt solution (HBSS) without phenol red (Sigma-Aldrich, catalog number: H8264 )
  14. 1 M HEPES solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )

Equipment

  1. Autoclave
  2. Tweezers (Fine Science Tools, catalog number: 11251-20 )
  3. LSM 710 confocal microscope (ZEISS, model: LSM 710 ) or any other inverted confocal microscope with 25x and 40x oil objectives and a numerical aperture of 0.8 and 1.3, respectively
  4. Patch clamp equipment (Axon Instruments)
  5. Microscope BX51 (Olympus, model: BX51 )
  6. Borosilicate glass with filament for pipette fabrication. Outer diameter: 1.5 mm, inner diameter: 0.86 mm, 10 cm length (Linton Instrumentation, catalog number: BF150-86-10 )
  7. CO2 cell culture incubator (Thermo Electron Corporation)
  8. P-97 Flaming/Brown micropipette puller (Sutter Instruments, model: P-97 )
  9. Sutter MP-225 motorised micromanipulators (Sutter Instruments, model: MP-225 )

Software

  1. ImageJ (https://imagej.nih.gov/ij, National Institutes of Health)

Procedure

  1. Using permanent marker, label a round coverslip with a small circle and one extra mark to keep the same orientation of the coverslip when moving between different equipment (Figure 2). Autoclave the coverslips to avoid contaminations.
    Note: Instead of using coverslips labelled with permanent marker, gridded coverslips can be used for subconfluent cell cultures. In confluent fully polarised monolayers, however, the grids can be hard to visualise.


    Figure 2. Round coverslip labelled with permanent marks for generation of cell maps

  2. Place the coverslip into a 24-well plate and seed 200,000 MDCK cells onto the coverslip, which should be oriented so that the unmarked side is on top. Grow the cells in DMEM supplemented with 5% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin.
  3. The following day transfect MDCK cells with a midbody localised protein (e.g., PRC1, MKLP1, or tubulin) genetically fused to a fluorescent protein such as GFP or mCherry. If using stably transfected cells, between 20-60% should be expressing the transgene. Cells can be diluted with untransfected cells to achieve this. It is important to have between 20-60% of cells expressing the transgene in order to have a distinctive cell map allowing easy orientation of the cell map at the later stages. Leave cells to grow for at least two days to allow cells to divide and subsequently generate new midbodies.
    Note: We recommend using Lipofectamine 2000 reagent according to the manufacturers’ instructions for transfection of MDCK cells. In this protocol we use 200,000 cells, 1 µg of DNA, and 1 µl of Lipofectamine 2000 per well.
  4. For the cell map, image the cells situated inside the circle using a confocal microscope at low magnification (Figure 3). Print out the images allowing them to be used for reference in the later steps.
    Note: For the generation of cell maps, we use a 25x objective.


    Figure 3. Generation of a cell map and midbody visualisation. Cells stably expressing GFP-tubulin were used. Cell map is represented on the far left using a 25x objective. The boxed region shows a magnification with a 40x objective, and was used for localising cells exhibiting midbodies. Note that the midbodies are localised on the apical surface. The white arrowheads mark the midbody remnants. Scale bar = 10 µm.

  5. Subsequently, choose some cells to image at higher resolution to document the presence of the midbody remnants before manipulation with patch-clamp apparatus. Cells that are amenable to midbody remnant removal will have the remnants positioned over the apical cell surface (Figure 3; Video 1). Using a pen, make a note of the cells imaged on the printed image.
    Note: Be careful to avoid photobleaching. Move focal plane to the apical surface of the cell monolayer to find midbody remnants.

    Video 1. Z-stack images of MDCK cells stably expressing GFP-tubulin before TUSP. Images are shown from the basolateral to apical membranes (i.e., bottom to the top). The white arrowheads point to the midbody remnants. Note that the remnants are localised at the apical cell surface.

    To play the video, you need to install a newer version of Adobe Flash Player.

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  6. Transfer samples to the patch-clamp apparatus (Figure 4). Fill the chamber of the patch-clamp apparatus with HBSS medium supplemented with 0.5% fetal bovine serum, HEPES 20 mM, pH 7.2-7.5, 50 U/ml penicillin, and 50 µg/ml streptomycin, and place the coverslip into the chamber. To allow easier detection of previously imaged areas, use the marked position on the coverslip to position the sample in the same orientation as was previously used to generate the cell map.


    Figure 4. Patch-clamp apparatus coupled to an epifluorescence microscope

  7. Using the printed cell maps as a reference, identify the cells previously imaged inside the circle under the epifluorescence microscope coupled to the patch-clamp apparatus.
    Note: In this step, use a low magnification objective.
  8. Once the cell map has been correctly oriented, use an objective of higher magnification and move along the XY axis to the position where the cells whose midbody remnants were documented before, are situated. Double-check for the presence of remnants.
    Note: In our epifluorescence microscope this step is performed with a 60x objective. Take a quick look to observe the remnants and turn off the fluorescence channel to avoid photobleaching.
  9. Move the objective to allow the glass pipette to be between the objective and the monolayer.
  10. For fabricating a glass pipette, load a borosilicate glass pipette of 1.5 mm in outer diameter, 0.86 mm in inner diameter, and 10 cm in length into a micropipette puller (Figure 5). Ramp value = 515-535; Pull = 0; Velocity = 15-25; 4 heating cycles.


    Figure 5. Puller used for creating glass pipettes

  11. Once generated, take the glass pipette, and fill it with HBSS medium using a syringe with a 25 G 1 ½ needle.
  12. Fit the glass pipette to the pipette holder, and move it under the objective (Figure 6).


    Figure 6. Glass pipette fitted to the pipette holder of the patch-clamp apparatus should be positioned in between the objective and the sample. The red circle indicates the glass pipette.

  13. Use brightfield illumination to focus on the tip of the glass pipette.
  14. Simultaneously move down the pipette and the objective of the microscope on the z axis until the monolayer comes into the focal plane. Do not let the glass pipette contact the monolayer at this stage.
  15. Once the monolayer is in focus, use fluorescence to detect the midbody remnants (Figure 7; Video 2; Bernabé-Rubio et al., 2016).


    Figure 7. Representative example of cells subjected to TUSP. MDCK cells expressing GFP-tubulin were imaged at real time under an epifluorescence microscope. Green arrows indicate the midbody remnant. Scale bar = 5 μm.

    Video 2. Videomicroscopy of the TUSP procedure. MDCK cells stably expressing GFP-tubulin were subjected to TUSP. Images were captured in real time. Brightfield and fluorescence channels are shown superimposed. The green arrow indicates the position of the midbody remnant.

    To play the video, you need to install a newer version of Adobe Flash Player.

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  16. Select a midbody remnant for removal and slowly move the glass pipette closer to the remnant using the brightfield illumination. Once proximal to each other, keep the brightfield channel and the fluorescence superimposed. Using the mouth to apply negative pressure to the pipette via a tube, draw the midbody remnant from the cell surface (Figures 1 and 7; Video 2; Bernabé-Rubio et al., 2016).
  17. After midbody remnant removal, image the cells under a confocal microscope acquiring different planes of the cell to ensure that removal of the whole midbody has occurred (Figure 8; Video 3).


    Figure 8. Midbody remnants from cells expressing GFP-tubulin were removed by TUSP. Cells exhibiting midbodies are shown before (pre-TUSP) and after TUSP (post-TUSP). Note that the remnants are localised at the apical surface. The white arrowheads mark the midbody remnants. Since cells continuously move, it is often difficult to have several cells in-focus while imaging a cell field. The circle indicates the zone subjected to TUSP. Note that the midbody remnant of the cell exposed to TUSP was removed. Scale bar = 10 µm.

    Video 3. Z-stack images of MDCK cells stably expressing GFP-tubulin before (pre-TUSP) and after TUSP (post-TUSP). Images are shown from the basolateral to apical membranes (i.e., bottom to the top). The white arrowheads point to the midbody remnants. The circle indicates the zone subjected to TUSP. Note that the remnant of the cell subjected to TUSP was completely removed.

    To play the video, you need to install a newer version of Adobe Flash Player.

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  18. Place cells in an incubator at 37 °C in DMEM supplemented with 5% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin under an atmosphere of 5% CO2/95% air.
  19. Various readouts can be used to assess possible biological effects of midbody removal using appropriate experimental setups. For example, we have studied the role of the midbody remnant in primary ciliogenesis using fluorescence microscopy (Bernabé-Rubio et al., 2016).

Data analysis

For qualitative analysis of effects of midbody removal live-cell videomicroscopy can be used. This procedure can also be used to quantitatively address the absence or presence of the cilium 24 h after midbody removal, as used previously (Bernabé-Rubio et al., 2016). Cells should be fixed after 24 h and imaged to detect the presence of the cilium. In this case the data is categorical and as such a chi-squared analysis is appropriate after a minimum of 3 experimental repetitions with controls.

Notes

This protocol is highly reproducible as long as there is no phototoxicity/photobleaching of cells, and cell maps are clearly defined. A short exposure time to light is recommended to reduce phototoxicity/photobleaching.

Recipes

  1. HBSS medium to fill the chamber of the patch-clamp apparatus
    Hank’s balanced salt solution (HBSS) without phenol red
    20 mM HEPEs (pH 7.2-7.5)
    50 U/ml penicillin, 50 µg/ml streptomycin
    0.5% fetal bovine serum
  2. DMEM for MDCK cell culture
    Dulbecco’s modified Eagle’s medium (DMEM)
    50 U/ml penicillin, 50 µg/ml streptomycin
    5% fetal bovine serum

Acknowledgments

We thank Dr. José A. Esteban (Centro de Biología Molecular Severo Ochoa, Madrid, Spain) for the use of his patch-clamp equipment. We express our gratitude to Laura Rangel for taking some of the pictures shown in this protocol, and for recording the video of TUSP procedure. We also thank Minerva Bosch-Fortea for critical reading of the protocol. D.C.G. would like to thank Juan Bonifacino and the Bonifacino Lab for support whilst developing this protocol, and Laura Pellegrini for critically reading the protocol. The expert technical advice of the Optical and Confocal Microscopy Unit of the Centro de Biología Molecular Severo Ochoa is gratefully acknowledged. This work was supported by grant BFU2015-67266-R from the Spanish Ministerio de Economía y Competitividad/Fondo Europeo de Desarrollo Regional (MINECO/FEDER) to M.A. Alonso. M. Bernabé-Rubio is the holder of a fellowship from the Ministerio de Economía y Competitividad.

References

  1. Bernabé-Rubio, M., Andres, G., Casares-Arias, J., Fernandez-Barrera, J., Rangel, L., Reglero-Real, N., Gershlick, D. C., Fernandez, J. J., Millan, J., Correas, I., Miguez, D. G. and Alonso, M. A. (2016). Novel role for the midbody in primary ciliogenesis by polarized epithelial cells. J Cell Biol 214(3): 259-273.
  2. Green, R. A., Paluch, E. and Oegema, K. (2012). Cytokinesis in animal cells. Annu Rev Cell Dev Biol 28: 29-58.
  3. Kuo, T. C., Chen, C. T., Baron, D., Onder, T. T., Loewer, S., Almeida, S., Weismann, C. M., Xu, P., Houghton, J. M. and Gao, F. B., Daley, G. Q. and Doxsey, S. (2011). Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity. Nat Cell Biol 13: 1214-1223.
  4. Li, D., Mangan, A., Cicchini, L., Margolis, B. and Prekeris, R. (2014). FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis. EMBO Rep 15(4): 428-437.
  5. Singh, D. and Pohl, C. (2014). Coupling of rotational cortical flow, asymmetric midbody positioning, and spindle rotation mediates dorsoventral axis formation in C. elegans. Dev Cell 28(3): 253-267.

简介

在极化上皮细胞中,胞质在细胞分裂过程中在顶端细胞表面形成。 一旦切断,中间体被遗留于其中参与非细胞运动过程(例如初级细胞发生)的顶端质膜的其中一个子细胞遗传。 在这里,我们描述了一种从细胞中物理去除中间体残留物并评估其损失所引起的可能影响的新方法(Bernabé-Rubio et al。,2016)。
【背景】中间体或Flemming体是在有丝分裂的最后阶段在子细胞之间形成的细胞间桥的中心部分。通过运输(ESCRT)机器所需的内体分选复合体,桥梁两侧的脱落导致两个子细胞的物理分离(Green等,2012)。除了其在有丝分裂调节中的已知功能外,最近的研究已经开始阐明中间体后有丝分裂后的作用。由于其在肾细胞中腔内形成的启动中的作用,中间体被假定为极性提示(Li等,2014)。最近,已经证明,中间体残留物通过极化的Madin-Darby犬肾(MDCK)细胞(Bernabé-Rubio等,2016)直接参与初级细胞发生。还发现在秀丽隐杆线虫(Singh and Pohl,2014)的发展过程中以及在确定细胞命运和分化过程中,形成背轴的作用(Kuo et al。,2011)。以前的研究使用激光消融来损害中间体残留的功能。然而,当在培养的细胞系中进行时,由于质膜和近端胞质元件的损伤,激光烧蚀可导致细胞死亡。因此,我们设计了一个温和的程序,我们称之为“抽吸压力”(TUSP)。 TUSP允许从上皮细胞的细胞表面去除非有害的中体残留物。基本原理是基于使用连接到膜片钳装置的细孔玻璃移液管,以施加负压物理去除中体(图1)。
图1. TUSP程序图。 A.极化上皮细胞胞质分裂过程中形成顶端细胞间桥。 B.脱落后,其中一个子细胞继承中间体作为残留物,其位于顶端细胞表面。 C-即通过使用连接到路径钳装置的玻璃移液管,如果施加吸入压力,则可以从细胞中除去中间体残留物。

关键字:上皮细胞, 中间体残留物, 初级纤毛, 吸压, 膜片钳装置

材料和试剂

  1. 12毫米玻璃盖玻片#1(VWR,目录号:631-0713)
  2. 网格盖玻片(可选)(电子显微镜科学,目录号:72265-12)
  3. 猎鹰24孔板(康宁,目录号:353047)
  4. 永久标记(Faber-Castell Multimark 1523)(CultPens,目录号:FC19628)
  5. 1 mL注射器(BD,目录号:303172)
  6. 25 G 1½针(BD,目录号:305127)
  7. 来自ATCC的上皮Madin-Darby犬肾(MDCK II)(ATCC,目录号:CRL2936)
  8. 表达荧光中心体定位蛋白(例如,樱桃微管蛋白,Addgene,目录号:49149)的DNA构建体
  9. Dulbecco改良的Eagle's培养基(DMEM)(Sigma-Aldrich,目录号:D5796)
  10. 胎牛血清(Sigma-Aldrich,目录号:F7524)
  11. 青霉素 - 链霉素溶液(Sigma-Aldrich,目录号:P4333)
  12. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM,目录号:11668027)
  13. 汉克的不含酚红的平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H8264)
  14. 1 M HEPES溶液(Thermo Fisher Scientific,Gibco TM,目录号:15630080)

设备

  1. 高压灭菌器
  2. 镊子(精细科学工具,目录号:11251-20)
  3. LSM 710共聚焦显微镜(ZEISS,型号:LSM 710)或任何其他反相共聚焦显微镜,分别为25x和40x油目标,数值孔径分别为0.8和1.3
  4. 贴片钳(Axon Instruments)
  5. 显微镜BX51(Olympus,型号:BX51)
  6. 硼硅酸盐玻璃与细丝移液管制造。外径:1.5mm,内径:0.86mm,长度10cm(Linton Instrumentation,目录号:BF150-86-10)
  7. CO 2细胞培养箱(Thermo Electron Corporation)
  8. P-97火焰/棕色微量吸管拔出器(Sutter Instruments,型号:P-97)
  9. Sutter MP-225电动显微操纵器(Sutter Instruments,型号:MP-225)

软件

  1. ImageJ( https://imagej.nih.gov/ij ,国立研究所的健康)

程序

  1. 使用永久标记,在不同的设备之间移动时,用小圆圈和一个额外的标记标记圆形盖玻片以保持盖玻片的相同取向(图2)。高压灭菌盖子以避免污染。
    注意:不使用标有永久性标记的盖玻片,网格盖玻片可用于亚汇合细胞培养。然而,在汇合的完全极化单层中,网格可能难以可视化。


    图2.圆形盖玻片标有生成细胞图的永久标记

  2. 将盖玻片放入24孔板中,将20万个MDCK细胞种植到盖玻片上,盖玻片应定向,使未标记的一面位于顶部。在补充有5%胎牛血清,50U/ml青霉素和50μg/ml链霉素的DMEM中培养细胞。
  3. 第二天用与GFP或mCherry等荧光蛋白基因融合的中等体定位蛋白(例如,PRC1,MKLP1或微管蛋白)转染MDCK细胞。如果使用稳定转染的细胞,20-60%之间应该表达转基因。可以用未转染的细胞稀释细胞以达到这一目的。具有20-60%的表达转基因的细胞是重要的,以具有独特的细胞图,允许在稍后阶段容易地取向细胞图。使细胞生长至少两天,以使细胞分裂并随后产生新的中等体。
    注意:我们建议根据制造商的说明使用Lipofectamine 2000试剂转染MDCK细胞。在本协议中,我们使用20万个细胞,1μgDNA和1μlLipofectamine 2000 /孔。
  4. 对于细胞图,使用低倍率下的共焦显微镜对位于圆内的细胞进行成像(图3)。打印图像,让它们在以后的步骤中被用来参考。
    注意:为了生成单元格图,我们使用25x的目标。


    图3.细胞图和midbody可视化的生成使用稳定表达GFP-微管蛋白的细胞。细胞图在最左边使用25x物镜表示。盒装区域显示具有40x物镜的放大倍数,并用于定位显示中等体的细胞。注意,中心体位于顶端表面。白色的箭头标记了中部残余物。比例尺= 10μm
  5. 随后,选择一些细胞以更高的分辨率进行成像,记录中间体残留物的存在,然后用膜片钳装置进行手术。适于中间残留移除的细胞将残留物置于顶端细胞表面(图3;视频1)。使用笔,记录打印图像上成像的单元格。
    注意:注意避免光漂白。将焦平面移动到细胞单层的顶端表面,以发现中间残留物。

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    视频1.在TUSP之前稳定表达GFP-微管蛋白的MDCK细胞的Z-叠图像。图像从基底外侧显示为顶端膜(即从底部到顶部)。白色的箭头指向中部残余物。请注意,残留物位于顶端细胞表面。
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  6. 将样品转移到膜片钳装置(图4)。用补充有0.5%胎牛血清,HEPES 20mM,pH 7.2-7.5,50U/ml青霉素和50μg/ml链霉素的HBSS培养基填充膜片钳装置的腔室,并将盖玻片放入室中。为了更容易地检测先前成像的区域,请使用盖玻片上的标记位置以与先前用于生成细胞图的方向相同的方向放置样品。


    图4.耦合到落射荧光显微镜的夹钳装置

  7. 使用印刷细胞图作为参考,识别先前在耦合到膜片钳装置的落射荧光显微镜下圆圈内成像的细胞。
    注意:在此步骤中,使用低倍率目标。
  8. 一旦细胞图被正确定向,使用更高放大倍率的目标,并沿着XY轴移动到之前记录中间体残余的细胞的位置。仔细检查残留物的存在。
    注意:在我们的荧光显微镜下,这个步骤是用60x的目标进行的。快速查看残留物并关闭荧光通道以避免光漂白。
  9. 移动目标以允许玻璃移液管在物镜和单层之间。
  10. 为了制造玻璃移液管,将外径为1.5mm,内径为0.86mm,长度为10cm的硼硅酸盐玻璃移液管装入微量移液管拉拔器(图5)。斜坡值= 515-535;拉= 0;速度= 15-25; 4个加热循环。


    图5.用于创建玻璃移液器的拉拔器

  11. 一旦生成,取玻璃移液管,并用25 G 1½针的注射器将其填充HBSS培养基。
  12. 将玻璃移液器安装到移液器支架上,并将其移动到目标下(图6)。


    图6.装配到膜片钳装置的移液器支架的玻璃移液管应位于物镜和样品之间。红色圆圈表示玻璃移液器。

  13. 使用亮场照明来聚焦玻璃移液器的尖端。
  14. 同时向下移动移液管和显微镜的目标在z轴直到单层进入焦平面。不要让玻璃移液管在此阶段接触单层。
  15. 一旦单层聚焦,使用荧光检测中间残留物(图7; Video 2;Bernabé-Rubio等人,2016)。


    图7.经受TUSP的细胞的代表性实例表达GFP-微管蛋白的MDCK细胞在落射荧光显微镜下实时成像。绿色箭头表示中部残留物。比例尺= 5μm
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    视频2. TUSP程序的视频显微镜。将稳定表达GFP-微管蛋白的MDCK细胞进行TUSP。图像被实时捕获。 Brightfield和荧光通道被叠加显示。绿色箭头表示中间残留物的位置。
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  16. 选择一个中间体残留物进行清除,并使用明场照明将玻璃移液管慢慢移动到残留物。一旦彼此靠近,保持明场通道和荧光叠加。使用口通过管对吸液管施加负压,从细胞表面吸取中间体残留物(图1和7;视频2;Bernabé-Rubio等人,2016)。
  17. 在中间体残留去除后,在共聚焦显微镜下拍摄细胞,获取细胞的不同平面,以确保已经发生整个中间体的去除(图8;视频3)。


    图8.通过TUSP去除表达GFP-微管蛋白的细胞的中间体残留。显示中等体的细胞在(前TUSP)之前和TUSP后(TUSP后)显示。请注意,残留物位于顶端表面。白色的箭头标记了中部残余物。由于细胞连续移动,所以在成像细胞场时,通常难以使多个细胞聚焦。圆圈表示经受TUSP的区域。注意,暴露于TUSP的细胞的中间体残留物被去除。比例尺= 10μm
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    视频3.在前(TUSP前)和TUSP(TUSP后)稳定表达GFP-微管蛋白的MDCK细胞的Z-叠图像从基底外侧向顶膜显示图像(<即,从底部到顶部)。白色的箭头指向中部残余物。圆圈表示经受TUSP的区域。注意,完全去除了经受TUSP的电池的残余物。
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  18. 将细胞置于37℃的培养箱中,在含有5%胎牛血清,50U/ml青霉素和50μg/ml链霉素的DMEM中,在5%CO 2/95%空气。
  19. 可以使用各种读数来使用适当的实验设置来评估中枢去除的可能的生物学效应。例如,我们已经使用荧光显微镜(Bernabé-Rubio等人,2016)研究了中间体残留物在初级细胞发生中的作用。

数据分析

可以使用中位体去除活细胞视频显微镜的影响进行定性分析。如前所述(Bernabé-Rubio等人,2016),该程序也可以用于定量地解决中间体去除后24小时不存在或存在的纤毛。细胞应在24小时后固定,并成像以检测纤毛的存在。在这种情况下,数据是分类的,因此在进行至少3次具有对照的实验重复之后,卡方分析是恰当的。

笔记

只要不存在细胞的光毒性/光漂白,该方案是高度可重复的,并且清楚地定义细胞图。建议短时间的照射时间以减少光毒性/光漂白。

食谱

  1. HBSS介质填充膜片钳装置的腔室
    汉克平衡盐溶液(HBSS),无酚红
    20mM HEPE(pH 7.2-7.5)
    50 U/ml青霉素,50μg/ml链霉素
    0.5%胎牛血清
  2. DMEM用于MDCK细胞培养 Dulbecco改良的Eagle's培养基(DMEM)
    50 U/ml青霉素,50μg/ml链霉素
    5%胎牛血清

致谢

我们感谢JoséA. Esteban博士(西班牙马德里的Centro deBiologíaMolecular Severo Ochoa)使用他的膜片钳设备。我们向Laura Rangel表示感谢,并收录了本协议中显示的一些照片,并记录了TUSP程序的视频。我们还要感谢Minerva Bosch-Fortea对协议的批判性阅读。直升机感谢Juan Bonifacino和Bonifacino实验室的支持,同时制定了该协议,Laura Pellegrini则严格阅读协议。感谢Centro deBiología分子Severo Ochoa的光学和共聚焦显微镜组的专家技术建议,非常感谢。这项工作得到了西班牙经济贸易大学/Fondo Europeo de Desarrollo区域(MINECO/FEDER)给M.A.Aonso的授权BFU2015-67266-R的支持。 Bernabé-Rubio先生是竞争力部长的研究员。

参考文献

  1. BernardéRubio,M.,Andres,G.,Casares-Arias,J.,Fernandez-Barrera,J.,Rangel,L.,Reglero-Real,N.,Gershlick,DC,Fernandez,JJ,Millan,J. ,Correas,I.,Miguez,DG和Alonso,MA(2016)。  通过极化上皮细胞在中枢体的初级细胞发生中的新作用。细胞生物学 214(3):259-273。
  2. Green,RA,Paluch,E.和Oegema,K。(2012)。动物细胞中的细胞因子。 Annu Rev Cell Dev Biol 28:29-58。
  3. 郭伟,TC,Chen,CT,Baron,D.,Onder,TT,Loewer,S.,Almeida,S.,Weismann,CM,Xu,P.,Houghton,JM和Gao,FB,Daley,GQ and Doxsey, S.(2011)。通过逃避自噬的中层积累有助于细胞重编程和致瘤性。 Nat Cell Biol 13:1214-1223。
  4. Li,D.,Mangan,A.,Cicchini,L.,Margolis,B。和Prekeris,R。(2014)。  有丝分裂期间的FIP5磷酸化可以调节顶端的运输和管腔发生。 15(4):428-437。 />
  5. Singh,D.和Pohl,C.(2014)。 28(3):253-267。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bernabé-Rubio, M., Gershlick, D. C. and Alonso, M. A. (2017). Physical Removal of the Midbody Remnant from Polarised Epithelial Cells Using Take-Up by Suction Pressure (TUSP). Bio-protocol 7(8): e2244. DOI: 10.21769/BioProtoc.2244.
  2. Bernabé-Rubio, M., Andres, G., Casares-Arias, J., Fernandez-Barrera, J., Rangel, L., Reglero-Real, N., Gershlick, D. C., Fernandez, J. J., Millan, J., Correas, I., Miguez, D. G. and Alonso, M. A. (2016). Novel roce for the midbody in primary ciliogensis by polarized epithelial cells.J Cell Biol 214(3): 259-273.
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