Figure 1. Transgenic expression of GTPase mutant dMiro proteins. A, Amino acid sequence of the P loop in the N-terminal (top) and C-terminal GTPase domain (bottom) of human Miro1 (hMiro1), yeast Miro (pGem1), and dMiro. The point mutations (T25N, T460N, A20V, K455V) introduced into the GTPase domains of myc-tagged dMiro (RC-transcript, FlyBase) are highlighted in red. B, C, Normal myc-dMiro (control) and mutant myc-dMiroA20V, myc-dMiroT25N, myc-dMiroK455V, and myc-dMiroT460N were pan-neuronally expressed in dmiro-null mutants (−/−, B) or (C) overexpressed (OE) in otherwise wild-type animals (w1118) using an elav-Gal4 driver. Viability was assayed daily by counting freshly hatched adult flies. Data represent means ± SEM of three independent matings. Only significant differences between control and mutant genotypes are indicated by asterisks (p < 0.05, N > 3, n > 100; 1-way ANOVA, Tukey's post test). D, E, Protein expression levels of myc-tagged GTPase-mutant dMiro proteins that were transgenically expressed in dmiro-null mutant (−/−) neurons. Immunoblots of larval brain protein extracts were stained with anti-Myc (D) or anti-dMiro (E) antibodies. β-Tubulin was used as loading control to normalize mutant protein levels to control (dMiro, 1.0; dMiroA20V, 0.9; dMiroT25N, 0.8; dMiroK455V, 1.1; dMiroT460N, 0.9; n = 3). Genotypes are indicated. F, Coimmunolocalization of the mitochondrial marker mitoGFP (green, top row) with myc-tagged dMiro proteins (red, middle row) in MN cell bodies of third-instar larvae. Wild-type control (WT) neurons lack transgenic expression of myc-dMiro. Transgenes encoding dMiroA20V, dMiroT25N, dMiroK455V, dMiroT460N, and normal dMiro were coexpressed with a transgene encoding mitoGFP in motor neurons of dmiro-null mutants with an Ok6-Gal4 driver. Scale bar, 2 μm.