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, Actin bundle, Dynamin, GTPase, Invadosome
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
Equipment
Software
Procedure
Data analysis
Measure band intensity by ImageJ and subtract the background intensity. The proportion of sedimented actin is interpreted as the F-actin bundling activity in the presence of dynamin and is quantified as pellet fraction intensity in total actin intensity [pellet/(pellet + supernatant)]. Each reaction should be performed at least three times independently for the statistical quantification.
Recipes
Note: For all recipes, use molecular biology grade reagents. Solutions should be prepared with distilled water.
Acknowledgments
This work was supported by Ministry of Science and Technology grant 107-3017-F-002-002 and National Taiwan University grant NTU-CDP-106R7808 to Y. W. Liu. This protocol was derived from our previous work (Chuang et al., 2019).
Competing interests
The authors declare no competing financial interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.