FIGURE 4.

Activity and the expression levels of Rab32 regulate the activity of Drp1. A, phosphorylation of Drp1 on serine 656 depends on the activity of Rab32. HeLa control cells, cells overexpressing Rab32, and Rab32 mutants as indicated were lysed, and lysates were analyzed by Western blot for the presence of Drp1 phosphorylated on serine 656. Amounts were normalized with the signals for total Drp1 and actin, and Drp1 phosphorylated on serine 656 was quantified (n = 3). *, p < 0.05. B, Rab32 does not influence Drp1 targeting. Membranes from HeLa control cells, cells overexpressing Rab32 and Rab32 mutants, or cells where Rab32 has been knocked down as indicated were fractionated into low and high speed pellets and the cytosol, which were analyzed by Western blot for Drp1. HM, heavy membranes; LM, light membranes; Cyt, cytosol. C, Rab32 activity and expression levels influence mitochondrial membrane dynamics. Top row, expression of Rab32 FLAG T39N leads to the perinuclear clustering of mitochondria as described previously (34). MitoTracker-loaded HeLa cells were transfected with Rab32FLAG T39N, and transfected cells were identified by their positive FLAG signal (left). Bottom row, cells were transfected and processed as in the top row but incubated for 2 h with 10 μm H89. Scale bar, 25 μm. D, Rab32 knockdown leads to an increase in fragmented mitochondria. HeLa cells transfected with scrambled (scr) siRNA (data not shown) or siRNA for Rab32 were loaded with MitoTracker. 10 randomly selected images showing about 50 cells each were quantified for the percentage of cells with fragmented mitochondria (right). p = 0.025. Scale bar, 25 μm.