Controls for rapalog motility experiments and perimitochondrial actin recruitment. (A) Representative example neurites showing rapalog recruits mCherry-FRB to mitochondria in the presence of vehicle or AntA. Neurons were transfected with mito-GFP-FKBP and mCherry-FRB and pretreated for 1 h with either 4 nM AntA or vehicle before addition of 1 µM rapalog as in Fig. 2. (B) Representative images of hippocampal neurons that were fixed and stained with Alexa Fluor 488 phalloidin conjugate and Hoechst. Either control neurons or neurons pretreated with 4 nM AntA were exposed to 5 µM LatA. Note the persistence of a LatA-resistant pool of actin after pretreatment with AntA. (C) Quantification of filamentous actin from the average Alexa Fluor 488 phalloidin intensity of 60× imaging fields of neurons treated as in B. (D) Quantification of mitochondrial motility in hippocampal neurons pretreated for 1 h with 4 nM AntA and then 30 min with both 5 uM LatA and 4 nM AntA. Once arrested by treatment with AntA, LatA could not reverse the arrest. (E) Time-lapse series of fibroblasts treated for 1 h with 40 nM AntA and imaged as in Fig. 3 A. The arrowhead indicates a mitochondrion that appears to lack perimitochondrial actin and moves, while nearby mitochondria do not. (F) Representative images of rat embryonic fibroblasts transduced with GFP-F-tractin and mito-mRaspberry from the quantification in Fig. 3 D. Cells cultured in galactose were treated with 40 nM AntA or vehicle. One hour after drug treatment, cells either had glucose or galactose added to a concentration of 25 mM, or no further addition. Cells were imaged after 30 min. P values for C were calculated by two-tailed, unpaired t tests with Welch’s correction. A blocked one-way ANOVA was performed with Tukey’s multiple comparison correction for D. Select comparisons are shown for D. For supplemental images, 3A scale bars = 10 µm. The scale bar in 3E is 5 µm. For supplemental images, 3B and 3F scale bars = 20 μm.