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Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs
透性化细胞中肌动蛋白逆流:横向弧的肌球蛋白II激发的向性运动   

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本实验方案简略版
Nature Cell Biology
Apr 2015

Abstract

Numerous biological functions such as cytokinesis, changes in cell shape and cell migration require actomyosin-driven cellular contractility. However, the detailed mechanism of how contractile forces drive cellular processes are difficult to decipher due to the complexity of the intracellular environment. In particular, the mesoscopic description of the myosin II-dependent actin retrograde flow in cell lamellum is missing. Here, we describe a methodology for detergent extraction of cell, which preserves integrity of the actin cytoskeleton. This semi-in vitro cell model allows for the observation, using light microscopy, and quantification of changes in the actin cytoskeleton resulting from the activation of cellular contractility upon addition of ATP. This assay also allows for the evaluation of the effects of actin-associated proteins and other related factors in the modulation of the actin contractile activities. Here, we demonstrate the retrograde flow of a well-known actin-based structures- transverse arcs, which are myosin IIA-containing structures that emerge at the boundary between lamellipodium-lamellum and move centripetally in myosin II-dependent fashion.

Keywords: Actin fibers (肌动蛋白纤维), Actin flow (肌动蛋白流), Actomyosin contractility (actomyosin contractility), Micropatterning (micropatterning), Triton-insoluble cytoskeleton (细胞骨架的Triton不溶于)

Materials and Reagents

  1. 35-mm ibidi’s hydrophobic uncoated μ-dishes for cell culture (ibidi GmbH, catalog number: 80131 )
  2. 1 x 1 cm polydimethylsiloxane (PDMS) stamp containing microfeatures of circles (area, 1,800 μm2; center-to-center distance, 100 μm)
    Note: For a detailed protocol on preparation of PDMS stamps and micro-contact printing see Théry and Piel (2009) and Tee et al., (2015).
  3. NuncTM Cell Culture Treated Flasks with Filter Caps (Thermo Fisher Scientific, catalog number: 136196 )
  4. Human foreskin fibroblast (ATCC, catalog number: SCRC-1041 )
  5. Growth medium: Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Thermo Fisher Scientific, catalog number: 11965-092 ), supplemented with
    1. 10% fetal bovine serum (Thermo Fisher Scientific, catalog number: 10438-026 )
    2. 1 mM sodium pyruvate (Thermo Fisher Scientific, catalog number: 11360-070 )
    3. 10 U/ml penicillin and streptomycin (Thermo Fisher Scientific, catalog number: 15140148 )
  6. TrypLETM Express Enzyme (Thermo Fisher Scientific, catalog number: 12604013 )
  7. PBS (1x), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010-023 )
  8. Fibronectin (Merck Millipore Corporation, catalog number: 341635 )
  9. Imidazole (Sigma-Aldrich, catalog number: I15513 )
  10. KCl (First BASE Laboratories Sdn Bhd, catalog number: BIO-1300 )
  11. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  12. EDTA (First BASE Laboratories Sdn Bhd, catalog number: BIO-1050 )
  13. Ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  14. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  15. TritonTM X-100 (Sigma-Aldrich, catalog number: X100 )
  16. Polye (ethylene glycol) MW35,000 (PEG) (Sigma-Aldrich, catalog number: 81310 )
  17. Protease inhibitors cocktail (for use with mammalian cell and tissue extracts, DMSO solution) (Sigma-Aldrich, catalog number: P8340 )
  18. N, N-Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: 227056 )
  19. Methanol (Thermo Fisher Scientific, catalog number: M/4000/17 )
  20. Sterile Milli-Q water
  21. Alexa Fluor® 488 Phalloidin (Thermo Fisher Scientific, catalog number: A12379 ) (see Recipes)
  22. Dark phalloidin [Phalloidin from Amanita phalloides (≥90%)] (Sigma-Aldrich, catalog number: P2141 ) (see Recipes)
  23. Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A6419 ) (see Recipes)
  24. Extraction buffer A (see Recipes)
  25. Extraction buffer B (see Recipes)
  26. Staining solution (see Recipes)
  27. Contractility buffer (see Recipes)

Equipment

  1. Confocal microscope equipped with 100x oil immersion objective
  2. 37 °C on-stage incubation chamber

Software

  1. Image J software [National Institutes of Health (NIH)] (http://imagej.nih.gov/ij/)

Procedure

  1. Substrate patterning and cell seeding
    1. Micro-contact print circular islands of extracellular matrix protein fibronectin onto 35-mm ibidi’s hydrophobic uncoated μ-dishes to confine cells spreading to an isotropic shape. Circle area of 1,800 μm2 was chosen to ensure cells would spread over the entire island and consistently assemble actin transverse arcs. For a detailed protocol on micro-contact printing see Tee et al. (2015).
    2. Culture human foreskin fibroblasts (HFF) in growth medium in an incubator at 37 °C with 5% CO2 in air atmosphere.
    3. To dissociate cells, begin by aspirating culture medium, rinse cells once with 1x PBS and then add trypsin solution to culture flask and incubate for 5 min at 37 °C.
    4. Resuspend trypsinized cells in growth medium.
    5. Transfer cells into a centrifuge tube and centrifuge at 800 x g for 3 min.
    6. Remove supernatant, resuspend cell pellet in growth medium and then centrifuge at 800 x g for 3 min.
    7. Remove supernatant, resuspend cell pellet in growth medium and then perform cell count.
    8. Seed 5 x 104 cells in 1 ml growth medium onto the 35-mm printed dish from step A1.
    9. Incubate dish for 10 min at 37 °C for cells attachment onto fibronectin circular islands.
    10. Aspirate culture medium and rinse once with 1x PBS to remove unattached cells.
    11. Add 1 ml of growth medium and return dish to incubator at 37 °C with 5% CO2.
    12. Incubate cells for 5-7 h prior to cell permabilization.

  2. Cell permeabilization
    1. Aspirate the culture medium from dish.
    2. Permeabilize cells by gently adding 1 ml of extraction buffer A.
    3. Incubate in extraction buffer A for 10 min at room temperature on bench top.
    4. Gently aspirate extraction buffer A.
    5. Gently wash permeabilized cells with 2 ml of extraction buffer B.
    6. Incubate in extraction buffer B for 1-3 min on bench top.
    7. Gently aspirate extraction buffer B.
    8. Repeat steps B5-7 three times.

  3. Actin cytoskeleton contractility assay
    1. Stain actin cytoskeleton in cells in 1 ml of staining solution for 10-30 min at room temperature on bench top in the dark.
    2. [Optional step] Image the AlexaFluor-488-phalloidin labeled actin cytoskeleton in staining solution under 100x objective for 10-30 min at 37 °C with a 2 min time interval and a 5-10 μm Z-section (step-size, 0.35 μm). Actin cytoskeleton should remain inactive prior to induction of contractility with ATP.
    3. Aspirate staining solution.
    4. Add 1 ml of contractility buffer.
    5. Image the AlexaFluor-488-phalloidin labeled actin cytoskeleton in cells under 100x objective for 30-60 min at 37 °C with a 2 min time interval and a 5-10 μm Z-section (step-size 0.35 μm).

  4. Kymograph analysis using Image J
    1. Download freeware Image J from http://imagej.nih.gov/ij/.
    2. Open time-series Z-stack image (XYZT) in Image J. Perform a Z-projection (click Image → Stacks → Z project), and choose Max Intensity to add up the brightest pixels from each frame. Use the time-series maximum intensity projection image for step D3.
    3. Draw a line along the region of interest - actin transverse arcs between a pair of radial fibers, using the ‘Straight Line’ tool in Image J. In the example Figure 1, a line is drawn from the cell edge towards the cell center.
    4. Use the Image J function “Reslice” (click Image → Stacks → Reslice [/]) to generate a kymograph of the time-series stack for the white line using the settings-output spacing 3 pixels, slice count 1, rotate 90 degrees and avoid interpolation. Briefly, each time point gives an intensity line profile, in y-axis, averaged over a 3-pixel width along the drawn line. These line profiles are stacked side by side along the x-axis for all time points, so we get a single image of a distance over time plot in the y- and x-axis respectively (see Figure 1C).
    5. In the example Figure 1, velocity of the centripetally moving actin transverse arcs can be measured from the slope of the green intensity line (μm min-1) (e.g., in the condition with addition of ATP only). The slope of the line in the kymograph is proportional to velocity. In addition, if the line in the kymograph is parallel to the x-axis (e.g., in the conditions without ATP, with addition of AMP-PNP and addition of ATP together with blebbistatin), this means there is no movement over time.

Representative data


Figure 1. Semi-in vitro detergent-permeabilized cell system. Human foreskin fibroblasts were spread on micro-contact printed circular fibronectin island for 5 h. Actin cytoskeleton (labeled by AlexaFluor-488-phalloidin) organized into a radially symmetrical system with actin radial fibers (RFs) oriented perpendicular to the cell edge and actin transverse arcs (magenta arrowheads) arranged perpendicular to the radial fibers. (A). Actin cytoskeleton remained inactive in contractility buffer without ATP. No movements of the transverse arcs were observed. Yellow arrow points to a radial fiber (RF). (B). Centripetal movement of transverse arcs along the radial fibers was seen in 2 mM ATP containing contractility buffer. Magenta arrowheads indicate the positions of transverse arcs. Magenta dotted lines indicate the initial positions of transverse arcs. Kymograph analysis in C is performed along the white lines indicated. Scale bar, 10 μm. (C). Kymograph analysis of transverse arcs in various experimental conditions: (i) without ATP, (ii) addition of 2 mM ATP, (iii) addition of 2 mM AMP-PNP and (iv) addition of 2 mM ATP together with 100 μM of blebbistatin. Centripetal movement of transverse arcs in permeabilized cells is ATP- and myosin-dependent since movement was only seen following the addition of ATP but not in the conditions with AMP-PNP, a non-hydrolyzable ATP analog, nor in the presence of blebbistatin, a myosin II inhibitor. Vertical scale bar, 2 μm.

Notes

  1. ATP-induced cell contractility was first reported in water-glycerol extracted cell model by Hoffmann-Berling (1954), see Thery and Piel (2009).
  2. For an example of actin cytoskeleton contractility assay performed in cells on non-micropatterned glass substrate, see Tint et al. (1991).
  3. Do not freeze-thaw aliquots of ATP stock solution.
  4. 2-Mercaptoethanol, phalloidin and protease inhibitors cocktail are to be added fresh each time.
  5. Addition of PEG and phalloidin to extraction buffer A serve to stabilize the cytoskeleton during extraction.
  6. Incubation in staining solution and fluorescently-labeled actin cytoskeleton should be kept away from light to minimize bleaching.
  7. Other fluorescently-conjugated phalloidin can also be used to label the actin cytoskeleton.
  8. If fluorescent labeling of the actin cytoskeleton is insufficient or excessive, adjust the duration of incubation or dilution of phalloidin in staining solution accordingly.
  9. If bleaching during image acquisition is significant, add AlexaFluor-488-phalloidin (1:250-500) into the contractility buffer. If the image gets progressively brighter during imaging, reduce the amount of AlexaFluor-488-phalloidin used in the contractility buffer.
  10. To minimize bleaching during image acquisition, consider increasing camera gain and sensitivity, reducing exposure time and laser intensity and lengthening the time interval between each frame.
  11. In the event where there is insufficient contrast between the fluorescently-labeled actin cytoskeleton and the background fluorescence from AlexaFluor-488-phalloidin present in the solution during imaging, (i) do not introduce additional fluorescently-tagged phalloidin in contractility buffer and (ii) remove staining solution and wash twice or more times with extraction buffer B prior to imaging control condition in the absence of ATP.
  12. If drug treatment is needed, drug can be added together with the staining solution prior to induction of ATP-mediated contractility and maintained in the contractility buffer.
  13. Cell permeabilization can be attempted in cells expressing fluorescently-tagged proteins. In an initial trial, fluorescently-tagged actin markers such as LifeAct-GFP (Riedl et al., 2008) and tdTomato-F-tractin (Johnson and Schell, 2009) are undetectable or weakly seen following cell permeabilization, while fluorescently-tagged protein such as GFP-myosin regulatory light chain and GFP-α-actinin are visible after cell permeabilization. Fluorescence signal retention of other fluorescently-tagged proteins remains to be evaluated.

Recipes

  1. AlexaFluor-488-phalloidin
    Reconstitute in 1.5 ml methanol as per manufacturer instruction
    Stored at -20 °C
  2. Dark phalloidin
    Reconstitute to 500 μM in ice-cold DMF
    Aliquot and stored at -20 °C
  3. ATP
    Reconstitute to 500 mM in ice-cold sterile Milli-Q water
    Aliquot and stored at -80 °C (see also ‘Notes’ for tips on handling)
  4. Extraction buffer A
    50 mM imidazole (pH 6.8)
    50 mM KCl
    0.5 mM MgCl2
    0.1 mM EDTA
    1 mM EGTA
    1 mM 2-Mercaptoethanol (see also ‘Notes’ for tips on handling)
    0.1% Triton-X100
    4% PEG MW35000
    250 nM dark phalloidin (see also ‘Notes’ for tips on handling)
    2 ul ml-1 protease inhibitors cocktail (see also ‘Notes’ for tips on handling)
  5. Extraction buffer B
    50 mM imidazole (pH 6.8)
    50 mM KCl
    0.5 mM MgCl2
    0.1 mM EDTA
    1 mM EGTA
    1 mM 2-Mercaptoethanol
    250 nM dark phalloidin
    2 μl ml-1 protease inhibitors cocktail
  6. Staining solution
    Extraction buffer B, supplement with AlexaFluor-488-phalloidin (1:250 dilution)
  7. Contractility buffer
    Extraction buffer B, supplement with 2 mM ATP

Acknowledgments

This protocol was adapted from the previously reported in Tint et al. (1991). This work was supported by the National Research Foundation Singapore, Ministry of Education of Singapore, Grant R-714-006-006-271, and administrated by the National University of Singapore.

References

  1. Hoffmann-Berling, H. (1954). Adenosintriphosphat als Betriebsstoff von Zellbewegungen. Biochim Biophys Acta 14 (2): 182-194.
  2. Johnson, H. W. and Schell, M. J. (2009). Neuronal IP3 3-kinase is an F-actin-bundling protein: role in dendritic targeting and regulation of spine morphology. Mol Biol Cell 20(24): 5166-5180.
  3. Riedl, J., Crevenna, A. H., Kessenbrock, K., Yu, J. H., Neukirchen, D., Bista, M., Bradke, F., Jenne, D., Holak, T. A., Werb, Z., Sixt, M. and Wedlich-Soldner, R. (2008). Lifeact: a versatile marker to visualize F-actin. Nat Methods 5(7): 605-607.
  4. Tee, Y. H., Shemesh, T., Thiagarajan, V., Hariadi, R. F., Anderson, K. L., Page, C., Volkmann, N., Hanein, D., Sivaramakrishnan, S., Kozlov, M. M. and Bershadsky, A. D. (2015). Cellular chirality arising from the self-organization of the actin cytoskeleton. Nat Cell Biol 17(4): 445-457.
  5. Tint, I. S., Hollenbeck, P. J., Verkhovsky, A. B., Surgucheva, I. G. and Bershadsky, A. D. (1991). Evidence that intermediate filament reorganization is induced by ATP-dependent contraction of the actomyosin cortex in permeabilized fibroblasts. J Cell Sci 98 (Pt 3): 375-384.
  6. Thery, M. and Piel, M. (2009). Adhesive micropatterns for cells: a microcontact printing protocol. Cold Spring Harb Protoc 2009(7): pdb prot5255.

简介

许多生物学功能如细胞分裂,细胞形状和细胞迁移的变化需要肌动蛋白驱动的细胞收缩性。然而,由于细胞内环境的复杂性,收缩力如何驱动细胞过程的详细机制难以解读。特别是,细胞层中的肌球蛋白II依赖性肌动蛋白逆行流的介观描述缺失。在这里,我们描述了洗涤剂提取的细胞,保留肌动蛋白细胞骨架的完整性的方法。这种半体外细胞模型允许使用光学显微镜观察和定量在添加ATP时由于细胞收缩性的活化而导致的肌动蛋白细胞骨架中的变化。该测定还允许评估肌动蛋白相关蛋白和其他相关因子在肌动蛋白收缩活性的调节中的作用。在这里,我们证明逆行流动的着名的基于肌动蛋白的结构 - 横向弧,这是肌球蛋白IIA含有结构出现在lamellipodium-lamellum之间的边界和肌球蛋白II依赖的方式向心移动。

关键字:肌动蛋白纤维, 肌动蛋白流, actomyosin contractility, micropatterning, 细胞骨架的Triton不溶于

材料和试剂

  1. 35-mm ibidi的用于细胞培养的疏水性未涂覆的μ-皿(ibidi GmbH,目录号:80131)
  2. 包含圆形微观特征(面积,1,800μm<2μm,中心到中心距离,100μm)的1×1cm聚二甲基硅氧烷(PDMS)印模
    注意:有关PDMS邮票和微接触印刷准备的详细协议,请参见Théry和Piel(2009年)和Tee等人(2015年)。
  3. 具有过滤器盖的Nunc TM细胞培养处理的培养瓶(Thermo Fisher Scientific,目录号:136196)
  4. 人类包皮成纤维细胞(ATCC,目录号:SCRC-1041)
  5. 生长培养基:Dulbecco改良Eagle培养基(DMEM)高葡萄糖(Thermo Fisher Scientific,目录号:11965-092),补充有
    1. 10%胎牛血清(Thermo Fisher Scientific,目录号:10438-026)
    2. 1mM丙酮酸钠(Thermo Fisher Scientific,目录号:11360-070)
    3. 10U/ml青霉素和链霉素(Thermo Fisher Scientific,目录号:15140148)
  6. TrypLE TM Express Enzyme(Thermo Fisher Scientific,目录号:12604013)
  7. PBS(1x),pH 7.4(Thermo Fisher Scientific,目录号:10010-023)
  8. 纤连蛋白(Merck Millipore Corporation,目录号:341635)
  9. 咪唑(Sigma-Aldrich,目录号:I15513)
  10. KCl(First BASE Laboratories Sdn Bhd,目录号:BIO-1300)
  11. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  12. EDTA(First BASE Laboratories Sdn Bhd,目录号:BIO-1050)
  13. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  14. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  15. TritonX-100(Sigma-Aldrich,目录号:X100)
  16. 聚(亚乙基二醇)MW35,000(PEG)(Sigma-Aldrich,目录号:81310)
  17. 蛋白酶抑制剂混合物(用于哺乳动物细胞和组织提取物,DMSO溶液)(Sigma-Aldrich,目录号:P8340)
  18. N,N-二甲基甲酰胺(DMF)(Sigma-Aldrich,目录号:227056)
  19. 甲醇(Thermo Fisher Scientific,目录号:M/4000/17)
  20. 无菌Milli-Q水
  21. Alexa Fluor 488鬼笔环肽(Thermo Fisher Scientific,目录号:A12379)(参见配方)
  22. 黑暗鬼笔idin [伞形毒蕈Phalloides中的鬼笔环肽(≥90%)](Sigma-Aldrich,目录号:P2141)(参见配方)
  23. 腺苷5'-三磷酸二钠盐水合物(ATP)(Sigma-Aldrich,目录号:A6419)(参见配方)
  24. 提取缓冲液A(参见配方)
  25. 提取缓冲液B(参见配方)
  26. 染色溶液(见配方)
  27. 收缩缓冲液(参见配方)

设备

  1. 具有100x油浸物镜的共聚焦显微镜
  2. 37℃的阶段孵育室

软件

  1. Image J软件[National Institutes of Health(NIH)]( http://imagej.nih.gov/ij/

程序

  1. 基底图案化和细胞接种
    1. 微接触打印细胞外基质蛋白的圆形岛 纤连蛋白到35-mm ibidi的疏水未涂覆的μ-皿上进行限制 细胞扩散成各向同性形状。圆面积为1,800μm2/s 以确保细胞扩散到整个岛上 一致地组装肌动蛋白横向弧。有关详细协议 微接触印刷见Tee等人(2015)。
    2. 在培养箱中在37℃,5%CO 2空气气氛下培养人包皮成纤维细胞(HFF)在生长培养基中。
    3. 为了分离细胞,从抽吸培养基开始,冲洗细胞 ?一次用1x PBS,然后加入胰蛋白酶溶液到培养瓶中 在37℃孵育5分钟
    4. 将生长培养基中的胰蛋白酶化细胞重悬
    5. 将细胞转移到离心管中并以800×g离心3分钟
    6. 除去上清液,将细胞沉淀重悬于生长培养基中,然后以800×g离心3分钟。
    7. 取出上清液,将细胞沉淀重悬在生长培养基中,然后进行细胞计数
    8. 在1ml生长培养基中将5×10 4个细胞接种到来自步骤A1的35mm印刷的皿上。
    9. 孵育10分钟,在37℃的细胞附着到纤连蛋白环状岛上
    10. 吸出培养基,用1x PBS冲洗一次,以去除未附着的细胞
    11. 加入1ml生长培养基,并在37℃,5%CO 2下将培养皿放回培养箱。
    12. 在细胞永久化之前孵育细胞5-7小时。

  2. 细胞透化
    1. 从培养皿中吸出培养基。
    2. 通过轻轻加入1ml提取缓冲液A使细胞通透
    3. 在提取缓冲液A中在室温下在台面上孵育10分钟
    4. 轻轻吸出萃取缓冲液A.
    5. 用2ml提取缓冲液B轻轻冲洗透化细胞
    6. 在提取缓冲液B中在台面上孵育1-3分钟
    7. 轻轻吸出萃取缓冲液B.
    8. 重复步骤B5-7三次。

  3. 肌动蛋白细胞骨架收缩性测定
    1. 将细胞中的肌动蛋白细胞骨架在1ml染色溶液中在室温下在黑暗中的台面上染色10-30分钟。
    2. [可选步骤]图像AlexaFluor-488-phalloidin标记的肌动蛋白 细胞骨架在100x物镜染色溶液中10-30分钟 37℃,2分钟时间间隔和5-10μmZ-切片(步长, 0.35μm)。肌动蛋白细胞骨架应在诱导前保持无活性 的与ATP的收缩性
    3. 吸出染色溶液
    4. 加入1ml收缩缓冲液。
    5. 图像AlexaFluor-488-鬼笔环肽标记的肌动蛋白细胞骨架 细胞在100x物镜下在37℃下2分钟时间30-60分钟 间隔和5-10μmZ截面(步长0.35μm)。

  4. 使用Image J的Kymograph分析
    1. http://imagej.nih.gov/ij/下载免费图片J。
    2. 打开 时间序列图像J中的Z-堆叠图像(XYZT)。执行Z投影 (单击图像→堆叠→Z项目),然后选择最大强度累加 每帧的最亮像素。使用时间序列最大值 用于步骤D3的强度投影图像
    3. 沿着画一条线 感兴趣区域 - 肌动蛋白横向弧在一对径向之间 纤维,使用图像J中的"直线"工具 ?1,从单元格边缘向单元格中心绘制一条线。
    4. 使用Image J函数"Reslice"(单击图像→堆栈→Reslice [/]) 以产生白线的时间序列堆叠的计时器 使用设置输出间隔3像素,切片计数1,旋转90 度并避免插值。简而言之,每个时间点给出 强度线轮廓,沿y轴在3像素宽度上平均 绘制线。这些线轮廓沿着所有时间点的 x轴并排堆叠,所以我们得到一个距离的单个图像 时间图分别在 y 和 x 轴(见图1C)。
    5. 在 例子图1,向心运动肌动蛋白的速度 横向弧可以从绿色强度的斜率测量 线(μmmin -1 -1)(例如在仅添加ATP的条件下)。的 kymograph中线的斜率与速度成正比。在 另外,如果在没有ATP的条件下,在kymograph中的线与 x 轴平行(例如),添加AMP-PNP和添加 ATP与blebbistatin),这意味着没有移动 时间。

代表数据


图1.半裸的去污剂透化细胞系统。将人包皮成纤维细胞铺在微接触印刷的圆形纤连蛋白岛上5小时。肌动蛋白细胞骨架(由AlexaFluor-488-鬼笔环肽标记)组织成具有垂直于细胞边缘定向的肌动蛋白径向纤维(RF)和垂直于径向纤维排列的肌动蛋白横向弧(品红色箭头)的径向对称系统。 (一个)。肌动蛋白细胞骨架在没有ATP的收缩性缓冲液中保持无活性。没有观察到横向弧的运动。黄色箭头指向径向光纤(RF)。 (B)。在含有2mM ATP的收缩性缓冲液中观察到沿着径向纤维的横向弧的向心运动。洋红色箭头表示横向弧的位置。洋红色虚线表示横向弧的初始位置。沿着所示的白线进行C中的Kymograph分析。比例尺,10μm。 (C)。在各种实验条件下的横向电弧的Kymograph分析:(i)无ATP,(ii)加入2mM ATP,(iii)加入2mM AMP-PNP和(iv)加入2mM ATP与100μM肌球蛋白抑制剂。在透化细胞中横向弧的向心运动是ATP-和肌球蛋白依赖性的,因为运动仅在加入ATP之后才观察到,但在具有AMP-PNP,不可水解的ATP类似物的条件下,而不在肌球蛋白抑制剂肌球蛋白II抑制剂。垂直比例尺,2μm。

笔记

  1. ATP诱导的细胞收缩性首先在Hoffmann-Berling(1954)的水 - 甘油提取细胞模型中报道,参见Thery和Piel(2009)。
  2. 对于在非微图案化玻璃基底上的细胞中进行的肌动蛋白细胞骨架收缩性测定的实例,参见Tint等人(1991)。
  3. 不要冻融ATP原液的等分试样。
  4. 每次新鲜加入2-巯基乙醇,鬼笔环肽和蛋白酶抑制剂混合物
  5. 向提取缓冲液A中加入PEG和鬼笔环肽用于在提取期间稳定细胞骨架
  6. 在染色溶液和荧光标记的肌动蛋白细胞骨架孵育应避光,以尽量减少漂白。
  7. 其他荧光缀合的鬼笔环肽也可以用于标记肌动蛋白细胞骨架
  8. 如果肌动蛋白细胞骨架的荧光标记不足或过度,则相应地调整染色溶液中孵育或稀释鬼笔环肽的持续时间。
  9. 如果在图像采集期间漂白是显着的,将AlexaFluor-488-鬼笔环肽(1:250-500)添加到收缩性缓冲液中。如果图像在成像期间逐渐变亮,减少收缩性缓冲液中使用的AlexaFluor-488-phalloidin的量。
  10. 为了尽量减少图像采集期间的漂白,请考虑增加相机增益和灵敏度,减少曝光时间和激光强度,并延长每帧之间的时间间隔。
  11. 在成像期间荧光标记的肌动蛋白细胞骨架和来自溶液中存在的AlexaFluor-488-鬼笔环肽的背景荧光之间没有足够的对比度的情况下,(i)不在收缩性缓冲液中引入另外的荧光标记的鬼笔环肽,和(ii)除去染色溶液,并在没有ATP的成像对照条件下用提取缓冲液B洗涤两次或更多次
  12. 如果需要药物治疗,可以在诱导ATP介导的收缩之前将药物与染色溶液一起加入并保持在收缩性缓冲液中。
  13. 可以在表达荧光标记的蛋白质的细胞中尝试细胞透化。在初始试验中,荧光标记的肌动蛋白标记如LifeAct-GFP(Riedl等人,2008)和tdTomato-F-tractin(Johnson和Schell,2009)在细胞后是不可检测的或弱的透化,而荧光标记蛋白如GFP-肌球蛋白调节轻链和GFP-α-辅肌动蛋白在细胞透化后可见。其他荧光标记蛋白的荧光信号保留仍有待评估。

食谱

  1. AlexaFluor-488-phalloidin
    按照制造商说明书
    在1.5ml甲醇中复溶 储存于-20°C
  2. 黑色鬼伞素
    在冰冷的DMF中重构至500μM 等分并储存在-20°C
  3. ATP
    在冰冷无菌Milli-Q水中重构至500mM 等分并储存在-80°C(另见"注意事项"处理提示)
  4. 提取缓冲液A
    50mM咪唑(pH6.8) 50 mM KCl
    0.5mM MgCl 2 sub 0.1mM EDTA
    1 mM EGTA
    1 mM 2-巯基乙醇(另见"注意事项"处理提示)
    0.1%Triton-X100 4%PEG MW35000
    250 nM黑暗鬼笔环肽(另见"注意事项"处理提示)
    2 ul ml -1 蛋白酶抑??制剂混合物(另见"注意事项"处理提示)
  5. 提取缓冲液B
    50mM咪唑(pH6.8) 50 mM KCl
    0.5mM MgCl 2 sub 0.1mM EDTA
    1 mM EGTA
    1mM 2-巯基乙醇 250nM黑色鬼伞素 2μlml蛋白酶抑制剂混合物
  6. 染色溶液
    提取缓冲液B,补充AlexaFluor-488-鬼笔环肽(1:250稀释)
  7. 收缩性缓冲区
    提取缓冲液B,补充2mM ATP

致谢

该协议改编自先前在Tint等人(1991)中报道的。这项工作由新加坡国家研究基金会,新加坡教育部,Grant R-714-006-006-271,由新加坡国立大学管理支持。

参考文献

  1. Hoffmann-Berling,H。(1954)。 Adenosintriphosphat als Betriebsstoff von Zellbewegungen。 Biochim Biophys Acta (2):182-194
  2. Johnson,H.W。和Schell,M.J。(2009)。 神经元IP3 3激酶是一种F-肌动蛋白结合蛋白:在树突靶向和调节中的作用脊柱形态。 Mol Biol Cell 20(24):5166-5180
  3. Riedl,J.,Crevenna,AH,Kessenbrock,K.,Yu,JH,Neukirchen,D.,Bista,M.,Bradke,F.,Jenne,D.,Holak,TA,Werb,Z.,Sixt,M和Wedlich-Soldner,R。(2008)。 Lifeact:用于可视化F-肌动蛋白的通用标记。 Nat Methods 5(7):605-607。
  4. Tee,YH,Shemesh,T.,Thiagarajan,V.,Hariadi,RF,Anderson,KL,Page,C.,Volkmann,N.,Hanein,D.,Sivaramakrishnan,S.,Kozlov,MM和Bershadsky,AD 2015)。 由肌动蛋白细胞骨架的自组织引起的细胞手性。 Cell Biol 17(4):445-457
  5. Tint,I.S.,Hollenbeck,P.J.,Verkhovsky,A.B.,Surgucheva,I.G.and Bershadsky,A.D。(1991)。 中透性纤维重组是由渗透性成纤维细胞中肌动球蛋白皮质ATP依赖性收缩诱导的证据。/a> J Cell Sci 98(Pt 3):375-384
  6. Thery,M。和Piel,M。(2009)。 细胞粘附微图案:微接触印刷协议 Cold Spring Harb Protoc 2009(7):pdb prot5255。
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引用:Tee, Y. H. and Bershadsky, A. D. (2016). Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs. Bio-protocol 6(5): e1743. DOI: 10.21769/BioProtoc.1743.
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