Functional characterization of phosphorylation sites in dynamin-related protein 1

Dynamin-related protein 1 is member of the dynamin-family of large GTPases. Similar to the endocytosis motor dynamin, Drp1 uses GTP hydrolysis to power constriction of the outer mitochondrial membrane and ultimately mitochondrial division. Dynamin phosphorylation in its unique C-terminal proline-rich domain interferes with binding of accessory proteins that induce membrane curvature and inhibits clathrin-mediated endocytosis. Evidence within the last few years indicates that Drp1 is also regulated by the phosphorylation/dephosphorylation cycle. Drp1 regulation is complex, in that both inhibitory and activating phosphorylations have been described that lead to, respectively, mitochondrial elongation and shortening. In this chapter, we describe methods for the identification and functional characterization of Drp1 phosphorylation sites. Among these methods is replacement of the endogenous protein by phosphorylation-site mutant Drp1 via combined shRNA and RNAi-resistant cDNA expression from the same plasmid. We also discuss primary astrocyte cultures as a model for regulation of cell death and mitochondrial morphology by Drp1 and present ImageJ macro source code for unbiased quantification of mitochondrial shape changes.