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Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy
通过TIRF显微镜在体外直接观察和定量肌动蛋白的成核和伸长

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Abstract

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, fragmentation and monomer dissociation. Here we present a detailed protocol regarding the visualization and quantification of actin nucleation and filament elongation events by TIRF microscopy in vitro, which is based on the methods previously reported (Liu et al., 2015; Yang et al., 2011) .

Keywords: Actin assembly(肌动蛋白组装), Actin nucleation(肌动蛋白成核), Actin filament elongation(肌动蛋白丝伸长), Single filament dynamics in vitro(体外单丝动力学), TIRF microscopy(TIRF显微镜检查), Oregon-green actin(俄勒冈绿肌动蛋白), Profilin(前纤维蛋白), Formin(成蛋白)

Background

The actin cytoskeleton undergoes constant assembly and disassembly that has been implicated in numerous physiological cellular processes, such as cell division, cell expansion, cytokinesis and maintenance of cell polarity. Understanding how actin dynamics are precisely regulated is a fundamental question in cell biology. Actin as the core component of the actin cytoskeleton can self-assemble into filamentous structure with the diameter of approximately 7 nm in the presence of potassium chloride, adenosine triphosphate, and magnesium. Within cells, however, the actin assembly and disassembly is tightly regulated by different actin-binding proteins (ABPs) to meet the demands of various physiological cellular processes. Reconstitution of how ABPs regulate actin assembly and disassembly as well as the formation of high-order actin structures in vitro may provide insights into the mechanism of action of actin during these physiological cellular processes. In order to achieve this, we need to establish assays to trace the actin assembly and disassembly reaction in vitro. The process of actin assembly and disassembly has been traced by the kinetic pyrenyl-actin assay. However, considering that it is a solution-based bulk assay, it is hard to determine the contribution of individual events to actin polymerization, such as actin nucleation and filament elongation events. Development of total internal reflection fluorescence microscopy (TIRF microscopy, or TIRFM) allows the direct visualization of the dynamics of individual actin filaments and quantification of the associated parameters. In addition, this assay requires the minimal amount of proteins compared to other assays.

Materials and Reagents

  1. Cover glass (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3323 )
  2. 4 x 40 mm double-layer strips
  3. Parafilm (Bemis, catalog number: PM996 )
  4. Microscope slide (Sail brand, catalog number: 7105 )
  5. Capillary paper
  6. Oregon-green (OG) labeled actin (Scott et al., 2011)
  7. Recombinant AtFormin5 and plant AtProfilin5 (Liu et al., 2015)
  8. N-ethylmaleimide-myosin (Amann and Pollard, 2001)
  9. Unlabeled rabbit muscle actin (Kuhn and Pollard, 2005)
  10. EGTA (AMRESCO, catalog number: 0732 )
  11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2393 )
  12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  13. Imidazole (EMD Millipore, catalog number: 814223 )
  14. Tris base (Sigma-Aldrich, catalog number: T3253 )
  15. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  16. ATP (Sigma-Aldrich, catalog number: A6559 )
  17. DTT (Sigma-Aldrich, catalog number: D0632 )
  18. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 )
  19. EDTA (AMRESCO, catalog number: 0322 )
  20. Glucose (Sigma-Aldrich, catalog number: G8270 )
  21. Catalase from bovine liver (Sigma-Aldrich, catalog number: C9322 )
  22. Glucose Oxidase from Aspergillus niger (Sigma-Aldrich, catalog number: G7141 )
  23. Methylcellulose (Sigma-Aldrich, catalog number: M0262 )
  24. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
  25. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  26. 10x KMEI (see Recipes)
  27. 10x ME (see Recipes)
  28. Buffer G (see Recipes)
  29. 1x TIRFM buffer (see Recipes)
  30. HS-TBS (see Recipes)
  31. HS-BSA (see Recipes)
  32. LS-BSA (see Recipes)

Equipment

  1. Alcohol burner
  2. -80 °C freezer
  3. Centrifuge
  4. Spectrophotometer as an accessory in Infinite 200 PRO multimode reader (Tecan Trading)
  5. Olympus IX81 microscope (Olympus, model: IX81) equipped with an inverted TIRF illumination system that has a 100x oil TIRF objective (1.49 numerical aperture)
  6. Argon laser (488 nm excitation)
  7. Photometrics cascade II 512 CCD camera (Photometrics, model: Photometrics cascade II 512 CCD camera)

Software

  1. MicroManager software (https://micro-manager.org/)
  2. ImageJ software (http://rsbweb.nih.gov/ij/; version 1.41)

Procedure

  1. Preparation of TIRFM flow cells
    1. Mount two 4 x 40 mm double-layer strips of Parafilm to the 24 x 60 mm cover glass tightly, leaving the gap for 3-4 mm.
    2. Place a 25 x 76 mm microscope slide on the cover glass perpendicularly to make the flow cell. Press gently.
    3. Heat the flow cell in an alcohol burner for 2-3 sec to ensure that the Parafilm sticks tightly to the cover glass and microscope slide. The chamber volume should be about 40 μl.
    4. See Figure 1 for how to make a flow cell.


      Figure 1. Cartoon for the preparation of flow cells for TIRFM

  2. Preparation of actin monomers
    1. 100 μl OG labeled actin (about 20 μM) is taken out from -80 °C freezer and thawed in ice water. It is then subjected to centrifugation at 200,000 x g for 2 h, at 4 °C. Subsequently, 50 μl supernatant is pipetted out carefully after centrifugation and is kept on ice in dark (see Note 1).
    2. Ten microliter supernatant is diluted into 490 μl buffer G for the determination of actin concentration, using buffer G as the control. Since not all actin monomers are labeled, the concentration of unlabeled or labeled actin is determined by the spectrophotometer associated with an Infinite 200 PRO multimode reader. To determine the concentration of total actin, the absorbance is measured at 290 nm and 491 nm using the extinction coefficient of 26,600 M-1 cm-1 at 290 nm for actin and 77,800 M-1 cm-1 at 491 nm for Oregon-green, respectively, and the concentration of total actin is calculated using the following equation: [total Actin] = (A290 - 0.16991 x A491) x 50 x 106/26,600 μM.
    3. Usually, one actin monomer is labeled by one Oregon-green molecule on Cys-374 (Kuhn and Pollard, 2005), so we can infer the concentration of labeled actin by calculating the concentration of Oregon-green molecules. The concentration of OG is calculated using the following equation: [OG] = A491 x 50 x 106/77, 800 μM. So [OG] is used as [OG-actin]. Usually the concentration of OG-actin is about 5 μM, and unlabeled actin is 1 to 2 μM.
  3. OG actin is mixed with unlabeled actin at equal molar ratio and 1/10 volume of 10x ME is subsequently added into the solution and is kept on ice in dark.
  4. NEM-myosin is diluted to 20 nM in HS-TBS and 40 μl of diluted NEM-myosin is injected into the chamber, incubate 5 min at room temperature in dark.
  5. The flow cell is washed with 40 μl HS-BSA, followed by wash with 40 μl LS-BSA immediately, and incubate for 2 min at room temperature in dark. A small piece of capillary paper is placed on the other side of the chamber by capillarity to help fluid flow out.
  6. The flow cell is washed with 40 μl 1x TIRFM buffer.
  7. Actin monomers (0.75 μM, 50% OG labeled) in 1x TIRFM buffer is injected into the chamber. The growth of single actin filaments is observed under an Olympus IX81 microscope equipped with a 100x oil objective by the TIRF illumination, and images are captured with MicroManager imaging software (Micro-Manager) at 2- or 3-sec intervals. OG is excited by a 488 nm Argon laser, and the time-lapse series images are taken using a Photometrics cascade II 512 CCD camera and saved as a tiff file. The file can be opened and further analyzed by ImageJ software.
  8. Examples
    1. Example 1. Formin5 (FH5) eliminates the inhibitory effect of Profilin5 (PRF5) on spontaneous actin nucleation. Various concentrations of proteins are pre-incubated with actin monomers for 5 min in dark before being injected into the flow cell. See the example in Figure 2.
    2. Example 2. Determination of the effect of PRF4 and PRF5 on actin filament elongation.
      1. Step 1. Actin monomers at 0.75 μM in 1x TIRFM buffer is injected into the chamber. After the actin assembly reaction running for about 10 min, it will generate enough amount of actin filament ends to permit the filament elongation in step 2 (step 8b.ii). Actin filaments are observed under an Olympus IX81 microscope by TIRF illumination. Images are acquired with the sequential-acquire program, which is paused after 10 min. The last frame and time point are marked for further analysis.
      2. Step 2. Equal amount of actin monomers in 1x TIRFM buffer as in step 1 (step 8b.i) is injected into the chamber in the presence or absence of PRFs that are preincubated with actin for 5 min in dark quickly and gently, in order to keep the position of slide. Normally, the injection can be done in one minute. The sequential-acquire program is then restarted to continue the image acquisition for another 10 min. The PRF-actin complex will add onto the ends of existing filaments to promote further elongation. The image series is saved as a tiff file. The time interval between two observations should be taken into consideration when analyzing image series. See the example in Figure 3.

Data analysis

The saved time-lapse image series are opened in ImageJ software and the scale bar is set with 6 pixels as 1 μm. Figure 2 is shown as an example of the visualization and quantification of actin nucleation events. Basically, the nucleation activity is compared by counting the number of actin filaments (> 1 μm) per microscopic field at different time points and plotted (Figure 2). Consistent with the biochemical activity of profilin as an actin monomer sequestering protein, PRF5 inhibits spontaneous polymerization; whereas FH5 can overcome the inhibitory effect of PRF5 on spontaneous actin polymerization via utilizing PRF5-actin complex to accelerate actin polymerization.


Figure 2. Direct visualization of the nucleation of individual actin filaments. A. Time-lapse images of spontaneous actin assembly with or without various ABPs. [Actin], 0.75 μM; [PRF5], 1.5 μM; [AtFH5], 0.9 μM. Bar = 10 μm. See also the entire series in Supplemental Videos 1-3. B. Plot of the number of actin filaments per microscopic field during actin assembly. Values represent mean ± SD, n = 3.

Figure 3 is shown as an example of calculating filament elongation rates at both barbed end and pointed end of actin filaments. The elongation rates of single actin filaments at their ends are calculated by the MultipleKymograph tool in ImageJ software. To calculate the elongation rates of actin filaments, a thin line along the growth path of a filament covering both ends in a time-lapse movie is initially drawn and a Kymograph is subsequently generated. The slope of the leading edges represents the growth rates (μm/sec). The growth rates at both ends (subunits/sec) are subsequently calculated as subunits/sec by assuming that there are 330 subunits per μm filament (Kuhn and Pollard, 2005). As shown in Figure 3, both profilins slow down the elongation rate of single actin filaments at barbed ends, whereas no obvious difference between the effect of PRF4 and PRF5 on filament elongation is observed.


Figure 3. Direct visualization of the elongation of individual actin filaments. A. Time-lapse images of spontaneous actin assembly in the presence or absence of profilins (PRFs). Blue arrows indicate the time point of the addition of actin monomers in the presence or absence of PRFs. [Actin], 0.75 μM; [PRF4], 0.75 μM; [PRF5], 0.75 μM. B. Kymograph analysis of single filament growth in the absence (a) or presence of profilins (b, c). Green and red arrow heads indicate the barbed end and pointed end, respectively. Kymographs of the length (y-axis) of the filaments marked to the left versus time (x-axis, 500 sec). Bar = 5 μm. See also the entire series in Supplemental Videos 4-6. C. Statistics of the filament growth rates at both ends. More than ten individual filaments were used for each analysis. Values represent mean ± SD.

Notes

  1. When pipetting out the supernatant of OG-actin after centrifugation (see step 2a), DO suck from the surface layer of the liquid carefully, DO NOT put pipette deep into the base of the tube because actin monomers at the base are lack of viability.
  2. The 1x TIRFM buffer is very viscous, so DO mix the components thoroughly before use (see Recipes).
  3. Change for fresh 1x TIRFM buffer every 3 h for keeping the reducing circumstance. It is very important for keeping the brightness of fluorescence molecules (Oregon-green).
  4. High laser power or long time exposure will cause the break of actin filaments. The laser power should not be too high, usually 10-20% will be OK for 50 mW laser. Also the exposure time should not be too long, 300-600 msec will be OK (see step 7).

Recipes

  1. 10x KMEI
    10 mM EGTA
    10 mM MgCl2
    500 mM KCl
    100 mM imidazole, pH 7.0
  2. 10x ME
    10 mM EGTA
    1 mM MgCl2
  3. Buffer G
    5 mM Tris-HCl, pH 8.0
    0.2 mM CaCl2
    0.2 mM ATP
    0.2 mM DTT
    0.02% NaN3
  4. 1x TIRFM buffer
    10 mM imidazole, pH 7.0
    50 mM KCl
    1 mM MgCl2
    1 mM EGTA
    50 mM DTT
    0.2 mM ATP
    50 mM CaCl2
    15 mM glucose
    20 μg/ml catalase
    100 μg/ml glucose oxidase
    0.5% methylcellulose
  5. HS-TBS
    50 mM Tris-HCl, pH 7.5
    600 mM NaCl
  6. HS-BSA
    50 mM Tris-HCl, pH 7.5
    600 mM NaCl
    1% BSA
  7. LS-BSA
    50 mM Tris-HCl, pH7.5
    150 mM NaCl
    1% BSA

Acknowledgments

This protocol was adapted from our previously published work (Yang et al., 2011; Liu et al., 2015). We thank the former members from the Huang lab for their efforts on optimizing the protocol and Dr. David Kovar (University of Chicago) for the suggestions on running this assay at the beginning. The research in the Huang lab was supported by grants from Ministry of Science of Technology of China (2013CB945100) and National Natural Science Foundation of China (31671390 and 31471266).

References

  1. Amann, K. J. and Pollard, T. D. (2001). Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy. Proc Natl Acad Sci U S A 98(26): 15009-15013.
  2. Kuhn, J. R. and Pollard, T. D. (2005). Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy. Biophys J 88(2): 1387-1402.
  3. Liu, X., Qu, X., Jiang, Y., Chang, M., Zhang, R., Wu, Y., Fu, Y. and Huang, S. (2015). Profilin regulates apical actin polymerization to control polarized pollen tube growth. Mol Plant 8(12): 1694-1709.
  4. Scott, B. J., Neidt, E. M. and Kovar, D. R. (2011). The functionally distinct fission yeast formins have specific actin-assembly properties. Mol Biol Cell 22(20): 3826-3839.
  5. Yang, W., Ren, S., Zhang, X., Gao, M., Ye, S., Qi, Y., Zheng, Y., Wang, J., Zeng, L., Li, Q., Huang, S. and He, Z. (2011). BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development. Plant Cell 23(2): 661-680.

简介

全内反射荧光(TIRF)显微镜是用于在体外单丝分辨率下可视化肌动蛋白丝的动力学的强大工具。由于各种荧光探针的发展,我们可以轻松监测与肌动蛋白动力学相关的各种事件,包括成核,伸长,捆扎,碎裂和单体解离。在这里,我们提供了一个关于通过TIRF显微镜在体外可视化和定量肌动蛋白成核和细丝伸长事件的详细方案,其基于先前报道的方法(Liu等人, ,2015; Yang等人,2011)。

背景 肌动蛋白细胞骨架经历了许多生理细胞过程,如细胞分裂,细胞扩增,细胞分裂和细胞极性维持等的不断的组装和反汇编。了解肌动蛋白动力学如何被精确地调节是细胞生物学中的一个根本问题。肌动蛋白作为肌动蛋白细胞骨架的核心成分可以在氯化钾,三磷酸腺苷和镁的存在下自组装成直径约7nm的丝状结构。然而,在细胞内,肌动蛋白组装和反汇编被不同的肌动蛋白结合蛋白(ABP)紧密调节以满足各种生理细胞过程的需求。如何重组ABP如何调节肌动蛋白的组装和拆解以及体外高阶肌动蛋白结构的形成可以提供对这些生理细胞过程中肌动蛋白作用机理的见解。为了实现这一点,我们需要建立测定以追踪体外的肌动蛋白组装和分解反应。肌动蛋白组装和反汇编的过程已经通过动力学芘酰肌动蛋白测定法进行了描述。然而,考虑到它是基于溶液的体积分析法,难以确定个体事件对肌动蛋白聚合的贡献,例如肌动蛋白成核和细丝伸长事件。全内反射荧光显微镜(TIRF显微镜或TIRFM)的开发允许直接观察个体肌动蛋白丝的动力学和量化相关参数。此外,与其他测定相比,该测定需要最少量的蛋白质。

关键字:肌动蛋白组装, 肌动蛋白成核, 肌动蛋白丝伸长, 体外单丝动力学, TIRF显微镜检查, 俄勒冈绿肌动蛋白, 前纤维蛋白, 成蛋白

材料和试剂

  1. 盖玻片(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3323)
  2. 4×40mm双层条
  3. 石蜡膜(Bemis,目录号:PM996)
  4. 显微镜幻灯片(帆牌,目录号:7105)
  5. 毛细管纸
  6. 俄勒冈州绿色(OG)标记的肌动蛋白(Scott等人,2011)
  7. 重组AtFormin5和植物AtProfilin5(Liu等人,2015)
  8. N-乙基马来酰亚胺 - 肌球蛋白(Amann和Pollard,2001)
  9. 未标记的兔肌肉肌动蛋白(Kuhn和Pollard,2005)
  10. EGTA(AMRESCO,目录号:0732)
  11. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2393)
  12. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  13. 咪唑(EMD Millipore,目录号:814223)
  14. Tris碱(Sigma-Aldrich,目录号:T3253)
  15. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  16. ATP(Sigma-Aldrich,目录号:A6559)
  17. DTT(Sigma-Aldrich,目录号:D0632)
  18. 叠氮化钠(NaN 3 3)(Sigma-Aldrich,目录号:S2002)
  19. EDTA(AMRESCO,目录号:0322)
  20. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  21. 来自牛肝的过氧化氢酶(Sigma-Aldrich,目录号:C9322)
  22. 来自黑曲霉的葡萄糖氧化酶(Sigma-Aldrich,目录号:G7141)
  23. 甲基纤维素(Sigma-Aldrich,目录号:M0262)
  24. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A1933)
  25. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  26. 10x KMEI(见配方)
  27. 10x ME(见配方)
  28. 缓冲液G(见配方)
  29. 1x TIRFM缓冲区(见配方)
  30. HS-TBS(见配方)
  31. HS-BSA(参见食谱)
  32. LS-BSA(参见食谱)

设备

  1. 酒精燃烧器
  2. -80°C冰箱
  3. 离心机
  4. 分光光度计作为Infinite 200 PRO多模式阅读器(Tecan Trading)的附件
  5. 配备倒置TIRF照明系统的Olympus IX81显微镜(Olympus,型号:IX81),具有100x油TIRF物镜(1.49数值孔径)
  6. 氩激光(488nm激发)
  7. 光度级联II 512 CCD相机(Photometrics,型号:Photometrics cascade II 512 CCD相机)

软件

  1. MicroManager软件( https://micro-manager.org/
  2. ImageJ软件( http://rsbweb.nih.gov/ij/ ;版本1.41)

程序

  1. 制备TIRFM流通池
    1. 将两个4 x 40毫米的Parafilm双层条带紧紧地装在24 x 60毫米的盖玻璃上,留下3-4毫米的间隙。
    2. 将25 x 76 mm的显微镜载玻片垂直放在盖玻片上,制成流通池。轻轻按。
    3. 将酒精燃烧器中的流动池加热2-3秒,以确保石蜡膜紧密地粘到玻璃盖和显微镜载玻片上。腔体积应约为40μl
    4. 参见图1,了解如何制作流通池。


      图1.用于制作TIRFM流动池的卡通

  2. 肌动蛋白单体的制备
    1. 将100μlOG标记的肌动蛋白(约20μM)从-80℃冰箱中取出并在冰水中解冻。然后在4℃下将其在200,000xg下离心2小时。随后,离心后小心吸出50μl上清液,并保存在黑暗的冰上(见注1)
    2. 使用缓冲液G作为对照,将10μl微升上清液稀释至490μl缓冲液G中以测定肌动蛋白浓度。因为不是所有的肌动蛋白单体都被标记,未标记或标记的肌动蛋白的浓度是通过与Infinite 200 PRO多模式阅读器相关联的分光光度计测定的。为了确定总肌动蛋白的浓度,在290nm和491nm处使用在290nm处的激光系数为26,600M cm -1 的肌动蛋白和对于俄勒冈州绿,分别在491nm处为77,800M -1 cm -1,并且使用以下等式计算总肌动蛋白的浓度:[总肌动蛋白] =( 290 <! - SIPO - >为0.16991×A 491 ×50×10 -6 /26,600μM。
    3. 通常,一个肌动蛋白单体由Cys-374上的一个俄克隆 - 绿色分子标记(Kuhn和Pollard,2005),因此我们可以通过计算俄勒冈州 - 绿色分子的浓度来推断标记肌动蛋白的浓度。使用以下等式计算OG的浓度:[OG] = A 491 /77,800μM。所以[OG]被用作[OG-肌动蛋白]。通常OG-肌动蛋白的浓度约为5μM,未标记的肌动蛋白为1〜2μM。
  3. 将OG肌动蛋白与未标记的肌动蛋白以等摩尔比混合,随后将1/10体积的10x ME加入到溶液中并保持在黑暗的冰上。
  4. 在HS-TBS中将NEM-肌球蛋白稀释至20nM,将40μl稀释的NEM-肌球蛋白注射到室中,在室温下在黑暗中孵育5分钟。
  5. 流动池用40μlHS-BSA洗涤,然后立即用40μlLS-BSA洗涤,并在室温下在黑暗中孵育2分钟。一小部分毛细管纸通过毛细作用放置在腔室的另一侧,以帮助流体流出。
  6. 流动池用40μl1x TIRFM缓冲液洗涤。
  7. 将1×TIRFM缓冲液中的肌动蛋白单体(0.75μM,50%OG标记)注入室中。通过TIRF照明在装有100x油物镜的Olympus IX81显微镜下观察到单肌动蛋白丝的生长,并以2或3秒的间隔用MicroManager成像软件(Micro Manager)捕获图像。 OG被488nm氩激光激发,并且使用Photometrics cascade II 512 CCD摄像机拍摄延时系列图像并保存为tiff文件。该文件可以打开并通过ImageJ软件进一步分析。
  8. 例子
    1. 实施例1.Fin5(FH5)消除Profilin5(PRF5)对自发性肌动蛋白成核的抑制作用。将各种浓度的蛋白质在肌动蛋白单体中在暗中预孵育5分钟,然后注入流动池。请参见图2中的示例
    2. 实施例2.测定PRF4和PRF5对肌动蛋白丝长度的影响。
      1. 步骤1.将1xTIRFM缓冲液中的0.75μM的肌动蛋白单体注入腔室。在肌动蛋白组装反应运行约10分钟后,它将产生足够量的肌动蛋白长丝端以允许在步骤2(步骤8b.ii)中的细丝伸长。通过TIRF照明在Olympus IX81显微镜下观察肌动蛋白丝。图像采集顺序采集程序,在10分钟后暂停。最后一帧和时间点被标记为进一步分析。
      2. 步骤2.在步骤1(步骤8b.i)中的1xTIRFM缓冲液中,将等量的肌动蛋白单体在步骤1(步骤8b.i)中的存在或不存在下,在暗处快速且温和地与肌动蛋白预温育5分钟,按顺序保持幻灯片的位置。通常,注射可以在一分钟内完成。然后重新开始顺序采集程序,以继续图像采集另外10分钟。 PRF-肌动蛋白复合物将添加到现有长丝的末端以促进进一步伸长。图像系列保存为tiff文件。分析图像序列时,应考虑两个观察点之间的时间间隔。参见图3中的示例。

数据分析

保存的延时图像系列在ImageJ软件中打开,比例尺设置为6像素为1μm。图2显示为肌动蛋白成核事件的可视化和定量的实例。基本上,通过在不同时间点计数每个微观场的肌动蛋白丝数(>1μm)并绘制(图2)来比较成核活性。与普林林作为肌动蛋白单体螯合蛋白的生化活性一致,PRF5抑制自发聚合;而FH5可以通过利用PRF5-肌动蛋白复合物加速肌动蛋白聚合,克服PRF5对自发肌动蛋白聚合的抑制作用。


图2.单个肌动蛋白丝的成核的直接可视化A.具有或不具有各种ABP的自发肌动蛋白组装的延时图像。 [肌动蛋白],0.75μM; [PRF5],1.5μM; [AtFH5],0.9μM。 Bar =10μm。另请参见补充视频1- 3 。 B.在肌动蛋白组装过程中每个微观场的肌动蛋白丝数量的图。值表示平均值±SD,n = 3
图3示出了在肌动蛋白丝的倒刺末端和尖端两端计算长丝伸长率的实例。单个肌动蛋白丝在其末端的伸长率通过ImageJ软件中的MultipleKymograph工具计算。为了计算肌动蛋白丝的伸长率,最初画出沿着延时电影中覆盖两端的长丝的生长路径的细线,随后产生Kymograph。前缘的斜率表示生长速率(μm/sec)。通过假设每μm细丝有330个亚单位(Kuhn和Pollard,2005),两端的生长速率(亚单位/秒)随后计算为亚单位/秒。如图3所示,两个profilins都减慢了单个肌动蛋白长丝在有倒钩端的伸长率,而PRF4和PRF5对长丝伸长率的影响没有明显的差异。


图3.直接显示单个肌动蛋白丝的伸长率。 A.在存在或不存在profilins(PRF)的情况下自发肌动蛋白组装的延时图像。蓝色箭头表示在存在或不存在PRF的情况下添加肌动蛋白单体的时间点。 [肌动蛋白],0.75μM; [PRF4],0.75μM; [PRF5],0.75μM。 B.在不存在(a)或存在profilins(b,c)的情况下单丝生长的Kymograph分析。绿色和红色箭头分别表示有倒钩的末端和尖端。标记为左侧与时间(x轴,500秒)的细丝的长度(y轴)的扫描图。 Bar =5μm。另请参见补充视频4- 6 。 C.两端长丝生长速率的统计。每次分析使用超过十根单丝。值表示平均值±SD。

笔记

  1. 离心后,移出OG-肌动蛋白的上清液(见步骤2a)后,DO请仔细从表面层吸出液体,不要将移液管深入管中,因为底物的肌动蛋白单体缺乏活力。
  2. 1x TIRFM缓冲液非常粘稠,所以在使用前请彻底混合组分(参见食谱)。
  3. 每3小时更换一次新鲜的TIRFM缓冲液,以保持减少的环境。保持荧光分子的亮度(俄勒冈州绿色)非常重要。
  4. 高激光功率或长时间暴露会导致肌动蛋白丝断裂。激光功率不应太高,通常10-20%对于50mW的激光器是可行的。曝光时间也不要太长,300-600毫秒就可以了(参见步骤7)。

食谱

  1. 10倍KMEI
    10 mM EGTA
    10mM MgCl 2
    500 mM KCl
    100mM咪唑,pH 7.0
  2. 10x ME
    10 mM EGTA
    1mM MgCl 2
  3. 缓冲区G
    5mM Tris-HCl,pH8.0
    0.2mM CaCl 2
    0.2 mM ATP
    0.2 mM DTT
    0.02%NaN 3
  4. 1x TIRFM缓冲区
    10mM咪唑,pH 7.0
    50 mM KCl
    1mM MgCl 2
    1 mM EGTA
    50 mM DTT
    0.2 mM ATP
    50mM CaCl 2
    15 mM葡萄糖
    20μg/ml过氧化氢酶
    100μg/ml葡萄糖氧化酶
    0.5%甲基纤维素
  5. HS-TBS
    50mM Tris-HCl,pH7.5
    600 mM NaCl
  6. HS-BSA
    50mM Tris-HCl,pH7.5
    600 mM NaCl
    1%BSA
  7. LS-BSA
    50mM Tris-HCl,pH7.5
    150 mM NaCl
    1%BSA

致谢

该协议是从我们之前发表的作品(Yang等人,2011; Liu等人,2015)中改编而来的。我们感谢黄实验室的前成员为优化方案所做的努力,以及芝加哥大学医学博士David Kovar先生就开始运行该试验的建议。黄实验室的研究得到了中国科技部(2013CB945100)和国家自然科学基金(31671390和31471266)的资助。

参考文献

  1. Amann,KJ和Pollard,TD(2001)。 Direct使用全内反射荧光显微镜实时观察由Arp2/3复合物介导的肌动蛋白丝分支。 Proc Natl Acad Sci USA 98(26):15009-15013。
  2. Kuhn,JR和Pollard,TD(2005)。 Real通过全内反射荧光显微镜对肌动蛋白丝聚合的时间测量。生物相似物88(2):1387-1402。
  3. Liu,X.,Qu,X.,Jiang,Y.,Chang,M.,Zhang,R.,Wu,Y.,Fu,Y.and Huang,S。(2015)。< a class = profilin调节顶端肌动蛋白聚合以控制极性花粉管生长。 莫尔工厂 8(12):1694-1709。
  4. Scott,BJ,Neidt,EM和Kovar,DR(2011)。  BENT UPPERMOST INTERNODE1编码对肌动蛋白组织和水稻发育至关重要的II类formin FH5。植物细胞 23(2):661-680。
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引用:Jiang, Y. and Huang, S. (2017). Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy. Bio-protocol 7(5): e2146. DOI: 10.21769/BioProtoc.2146.
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