Abstract
While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.
Highlights
Mitochondrial fission and fusion create a dynamic network that is necessary for mitochondrial distribution and homeostasis
We developed a live-cell assay to measure mitochondrial fission rate, in which peripheral cellular regions are imaged over 10 min for dynamics of a fluorescent mitochondrial marker
Merging is a reversible process, and can be followed by splitting of the merged punctum (Figure 2—figure supplement 1B, Video 4). The combination of these results suggests that a population of Drp1 is in rapid equilibrium between the cytosol and the outer mitochondrial membrane (OMM), and that one mechanism for assembly of larger Drp1 oligomers is through merging of mitochondrially-bound oligomers
Summary
Mitochondrial fission and fusion create a dynamic network that is necessary for mitochondrial distribution and homeostasis. Fission is necessary to distribute mitochondria between daughter cells. Fission and transport maintains mitochondrial distribution throughout the length of these highly polarized cells. Drp exists in a number of oligomeric states, including dimers, tetramers, and higher-order oligomers (Frohlich et al, 2013; Macdonald et al, 2014). Drp can bind to the OMM through several factors, including the mitochondrial-specific lipid cardiolipin (Macdonald et al, 2014; Stepanyants et al, 2015; BustilloZabalbeitia et al, 2014; Ugarte-Uribe et al, 2014); and several ‘receptors’ on the OMM, such as Mff, MiD49, and MiD51 (Richter et al, 2015)
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