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
发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2146 浏览次数: 10027
评审: Marisa RosaPablo Bolanos-VillegasStefanie Rosa
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参见作者原研究论文
The authors used this protocol in:
Dec 2015
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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
- Cover glass (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3323 )
- 4 x 40 mm double-layer strips
- Parafilm (Bemis, catalog number: PM996 )
- Microscope slide (Sail brand, catalog number: 7105 )
- Capillary paper
- Oregon-green (OG) labeled actin (Scott et al., 2011)
- Recombinant AtFormin5 and plant AtProfilin5 (Liu et al., 2015)
- N-ethylmaleimide-myosin (Amann and Pollard, 2001)
- Unlabeled rabbit muscle actin (Kuhn and Pollard, 2005)
- EGTA (AMRESCO, catalog number: 0732 )
- Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2393 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
- Imidazole (EMD Millipore, catalog number: 814223 )
- Tris base (Sigma-Aldrich, catalog number: T3253 )
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
- ATP (Sigma-Aldrich, catalog number: A6559 )
- DTT (Sigma-Aldrich, catalog number: D0632 )
- Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 )
- EDTA (AMRESCO, catalog number: 0322 )
- Glucose (Sigma-Aldrich, catalog number: G8270 )
- Catalase from bovine liver (Sigma-Aldrich, catalog number: C9322 )
- Glucose Oxidase from Aspergillus niger (Sigma-Aldrich, catalog number: G7141 )
- Methylcellulose (Sigma-Aldrich, catalog number: M0262 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- 10x KMEI (see Recipes)
- 10x ME (see Recipes)
- Buffer G (see Recipes)
- 1x TIRFM buffer (see Recipes)
- HS-TBS (see Recipes)
- HS-BSA (see Recipes)
- LS-BSA (see Recipes)
Equipment
- Alcohol burner
- -80 °C freezer
- Centrifuge
- Spectrophotometer as an accessory in Infinite 200 PRO multimode reader (Tecan Trading)
- Olympus IX81 microscope (Olympus, model: IX81) equipped with an inverted TIRF illumination system that has a 100x oil TIRF objective (1.49 numerical aperture)
- Argon laser (488 nm excitation)
- Photometrics cascade II 512 CCD camera (Photometrics, model: Photometrics cascade II 512 CCD camera)
Software
- MicroManager software (https://micro-manager.org/)
- ImageJ software (http://rsbweb.nih.gov/ij/; version 1.41)
Procedure
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物生物化学 > 蛋白质 > 活性
生物化学 > 蛋白质 > 标记
生物化学 > 蛋白质 > 活性
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