Dynamin Superfamily GTPases Methods and Protocols

This volume provides methods and protocols on dynamin superfamily GTPases. Chapters are divided into three parts; detailing expression, purification, initial biochemical steps, undescribed analytical techniques, and biophysical and cellular characterization of DSPs in membrane remodeling, fission and fusion. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Dynamin Superfamily GTPases: Methods and Protocols to ensure successful results in the further study of this vital field.

PART I: Yeast DSPs – Isolation and Biochemical Characterization

1. Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae

2. Purification of the Dynamin-related Protein Vps1 using Mammalian and Bacterial Expression Systems

Natalia V. Varlakhanova and Marijn G. J. Ford

PART II: Mammalian DSPs – Isolation and Biochemical Characterization

3. A Single Common Protocol for the Expression and Purification of Soluble Mammalian DSPs from Escherichia coli

Natalia Stepanyants, Patrick J. Macdonald, Pooja Madan Mohan, and Rajesh Ramachandran

4. Affinity Purification and Functional Characterization of Dynamin-related Protein 1

Ryan W. Clinton, Brianna L. Bauer, and Jason A. Mears

5. Purification and Characterization of MxB

Frances Joan D. Alvarez and Peijun Zhang

6. Purification of Farnesylated hGBP1 and Characterization of Its Polymerization and Membrane Binding

Linda Sistemich and Christian Herrmann

7. Microscale Thermophoresis (MST) as a Tool for Measuring Dynamin Superfamily Protein (DSP)-lipid Interactions

Nikhil Bharambe and Rajesh Ramachandran

8. Nucleotide-dependent Dimerization and Conformational Switching of Atlastin

John P. O’Donnell, Carolyn M. Kelly, and Holger Sondermann

9. Analysis of Mitochondrial Membrane Fusion GTPase OPA1 Expressed by the Silkworm Expression System

Tadato Ban and Naotada Ishihara

10. Cell-free Analysis of Mitochondrial Fusion by Fluorescence Microscopy

Nyssa Becker Samanas and Suzanne Hoppins

11. Electrophysiological Methods For Detection Of Membrane Leakage and Hemifission by Dynamin 1

Pavel V. Bashkirov, Ksenia V. Chekashkina, Anna V. Shnyrova, and Vadim A. Frolov

12. Integrating Optical and Electrochemical Approaches to Assess the Actions of Dynamin at the Fusion Pore

Katherine A. Smith, Emily R. Prantzalos, and Arun Anantharam

13. Cellular Assays for Measuring Dynamin Activity in Muscle Cells

Jessica Laiman and Ya-Wen Liu

14. Measuring Drp1 Activity in Mitochondrial Fission in vivo

Di Hu and Xin Qi

15. Quantifying Drp1-mediated Mitochondrial Fission by Immunostaining in Fixed Cells

Di Hu and Xin Qi

16. Imaging Dynamin-related Protein 1 (Drp1)-mediated Mitochondrial Fission in Living Cells

Felipe Montecinos-Franjola and Rajesh Ramachandran