In zip1 and sqhAX3 flies, reduced association of mitochondria with F-actin SB431542 price correlates with increased association of DRP1 with F-actin, observed by coprecipitation of F-actin and DRP1 from total head homogenate using biotinylated-phalloidin ( Figures 8C and 8D). These findings suggest that

myosin II facilitates tethering of mitochondria to F-actin, a connection that is required for mitochondria to interact with DRP1. To determine if myosin II has a general and conserved role in DRP1 localization, we focused on the regulatory light chain, MLC2. We transfected Cos-1 cells with siRNA targeting two independent, nonoverlapping sequences in MLC2. We first confirmed depletion of MLC2 protein by western blot analysis ( Figure S8B). We then assessed mitochondrial morphology and the subcellular localization of DRP1.

In control cells, mitochondria, detected with transfected mitoRFP, are round or slightly tubular and colocalize with DRP1. In MLC2 siRNA-transfected cells, mitochondria are significantly elongated, and DRP1 signal is diffuse ( Figure 8E, Hydroxychloroquine order insets, graph). MLC2 is phosphorylated by myosin light chain kinase (MLCK), which is essential to MLC2 activity ( Watanabe et al., 2007). Treatment of cells with ML-7, a chemical inhibitor of MLCK, recapitulates the effects of MLC2 RNAi on mitochondrial morphology and DRP1 localization ( Figure S8A, insets, graph). These results support a conserved interaction among DRP1, myosin, and actin. Here, we describe a previously unsuspected target for tau neurotoxicity in human neurodegenerative disease: mislocalization of the mitochondrial fission protein DRP1 with subsequent failure of normal mitochondrial dynamics control. Our current data extend a model of the cascade of neurotoxicity triggered by accumulation of human tau. Previous work from our laboratories and others (Ahlijanian et al., 2000; Noble et al., 2005; Steinhilb et al., 2007a, 2007b; Iijima-Ando et al., 2010) places phosphorylation of tau upstream in a sequence of cellular events, including actin stabilization

Rolziracetam (Fulga et al., 2007), which lead to neuronal death. Our new results place tau phosphorylation upstream of altered mitochondrial dynamics (Figure S1) and further indicate that proper regulation of the actin cytoskeleton is critical for localization of DRP1 to mitochondria and subsequent mitochondrial fission. Here, we show a physical interaction between F-actin and DRP1. Further, we find that myosin II is required for both localization of mitochondria to actin and DRP1 to mitochondria (Figures 7 and 8). These results support a model in which DRP1 and mitochondria are recruited to F-actin, followed by actin-based translocation, leading to mitochondrial localization of DRP1 and subsequent mitochondrial fission. Excess actin stabilization inhibits translocation and colocalization of DRP1 and mitochondria, resulting in mitochondrial elongation (Figure S8C).