F-actin Bundle Sedimentation Assay
F-肌动蛋白束沉淀法
(*contributed equally to this work) 发布: 2019年11月05日第9卷第21期 DOI: 10.21769/BioProtoc.3419 浏览次数: 5993
评审: David PaulGuillaume BompardEsteban Paredes-Osses
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参见作者原研究论文
The authors used this protocol in:
Mar 2019
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Abstract
Understanding the molecular mechanism governing the higher-order regulation of actin dynamics requires chemically-defined and quantitative assays. Recently, the membrane remodeling large GTPase, dynamin, has been identified as a new actin cross-linking molecule. Dynamin regulates actin cytoskeleton through binding to, self-assembling around, and aligning them into actin bundles. Here we utilize dynamin as an example and present a simple protocol to analyze the actin bundling activity in vitro. This protocol details the method for F-actin reconstitution as well as quantitative and qualitative analyses for actin bundling activity of dynamins. Measurement of the actin bundling activity of other actin-binding proteins may also be applied to this protocol with appropriate adjustments depending on the protein of interest.
Keywords: Actin crosslinker (肌动蛋白交联剂)
Actin bundle (肌动蛋白束)
Dynamin (动力蛋白)
GTPase (GTP酶)
Invadosome (侵袭体)
Background
Dynamin is a membrane remodeling GTPase best-known for catalyzing membrane fission during clathrin-mediated endocytosis to release vesicles from the plasma membrane (McMahon and Boucrot, 2011; Schmid and Frolov, 2011; Antonny et al., 2016). In addition to its indispensable role for membrane fission, dynamin is also localized to actin-rich structures, such as lamellilpodia, dorsal membrane ruffles, and invadosomes, where it regulates actin reorganization via interacting with other actin polymerization factors (Ferguson and De Camilli, 2012). It has been discovered that the neuronal isoform of dynamin, dynamin-1 (Dyn1), could directly interact with actin filaments and facilitate the rate of actin polymerization (Gu et al., 2010). Subsequently, the ubiquitously expressed dynamin isoform, dynamin-2 (Dyn2), was also proved to be equipped with actin bundling activity and to affect the structure and stiffness of the protrusive actin-based structure, invadosome (Chuang et al., 2019). Here we detail the simple method to quantitatively analyze the actin bundling activity of Dyn1 and Dyn2. Dynamin may be substituted with other actin-binding and crosslinking proteins with proper adjustments of protein concentration, incubation time, or buffer composition in order to test their actin bundling activity.
Materials and Reagents
- 1.5 ml clear microcentrifuge tubes (Gunster biotech, catalog number: MB-E17-A)
- Rabbit muscle G-actin (Cytoskeleton, catalog number: AKL99-C)
- Recombinant human Dyn1 and Dyn2 proteins were expressed and purified as described previously (Liu et al., 2011; Chin et al., 2015)
- Protein Ladder (ARROWTEC, catalog number: PM2500)
- Tris (AppliChem, catalog number: A1086)
- CaCl2 (AppliChem, catalog number: A1873)
- ATP (Jena Bioscience, catalog number: NU-1010)
- DTT (AppliChem, catalog number: A2948)
- KCl (AppliChem, catalog number: A1362)
- MgCl2 (AppliChem, catalog number: 131396)
- SDS, 20% (w/v) solution (Bio Basic Canada Inc., catalog number: SD8119)
- Glycine (AppliChem, catalog number: A1067)
- Methanol (J.T.Baker, catalog number: 9093-68)
- Coomassie Brilliant blue R-250 (AppliChem, catalog number: A1092)
- Acetic acid (AppliChem, catalog number: 131008)
- Alexa Fluor 488 phalloidin (optional, Invitrogen, catalog number: A12379)
- Sucrose (AppliChem, catalog number: 131621)
- Glycerol (AppliChem, catalog number: 141339)
- Bromophenol blue (AppliChem, catalog number: A3640)
- HEPES (AppliChem, catalog number: A1069)
- EDTA (AppliChem, catalog number: A1104)
- BSA (AppliChem, catalog number: A1391)
- Methylcellulose (Sigma-Aldrich, catalog number: M0387)
- Liquid nitrogen
- Dextran
- HCl
- Glucose
- 4x SDS-PAGE sample buffer (see Recipes)
- Coomassie Brilliant Blue solution (see Recipes)
- General actin buffer (see Recipes)
- 10x actin polymerization buffer (see Recipes)
- Imaging buffer (optional, see Recipes)
- SDS running buffer (see Recipes)
- Destain buffer (see Recipes)
Equipment
- P2 and P200 Micropipettes
- -80 °C freezer
- High Speed Micro Refrigerated Centrifuge (KUBOTA, model: 3700)
- Biohazard-proof Rotor (KUBOTA, model: AF-2724A)
- Fluorescence confocal microscope equipped with 63x, 1.4-NA oil-immersion objective (ZEISS, LSM 700)
- Electrophoresis power supply (Major Science MP-300V power supplies or similar)
- SDS-PAGE electrophoresis system
- Scanner (EPSON Perfection V600 Photo)
Software
- ImageJ image processing and analysis software (National Institutes of Health Image, https://imagej.nih.gov/ij/)
Procedure
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文章信息
版权信息
© 2019 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Lin, S., Chuang, M. and Liu, Y. (2019). F-actin Bundle Sedimentation Assay. Bio-protocol 9(21): e3419. DOI: 10.21769/BioProtoc.3419.
- Chuang, M. C., Lin, S. S., Ohniwa, R. L., Lee, G. H., Su, Y. A., Chang, Y. C., Tang, M. J. and Liu, Y. W. (2019). Tks5 and Dynamin-2 enhance actin bundle rigidity in invadosomes to promote myoblast fusion. J Cell Biol 218(5): 1670-1685.
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分类
癌症生物学 > 侵袭和转移
生物化学 > 蛋白质 > 结构
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