Supplemental Material

This article contains the following supporting material:

• Movie 1 - GFP-mitochondria in cultured embryonic neurons.
  Cells from embryos homozygous for mitoGFP and the D42 Gal4 driver were cultured on coverslips. Two time-lapse confocal movies are shown of mitochondria in neurites extending to the right from neuroblast cell bodies. The images were collected at 2.2 sec intervals and are played back at 8 frames/sec.

• Movie 2 (Figure 2) - Mitochondria in the motor axons of a wild-type larval segmental nerve.
  A larva homozygous for mitoGFP and the D42 Gal4 driver was dissected to expose segmental nerves and mounted in a perfusion chamber. GFP-Mitochondria were photobleached in the center section, then the movement of new fluorescent mitochondria into the bleached zone was imaged at 1 frame/sec with a Biorad MRC600 confocal microscope at ~22°C. The images are played back at 15 frames/sec.

• Movie 3 (Figure 4) - Comparison of GFP-mitochondria in the motor axons of wild-type and dynein mutant segmental nerves.
  The wild-type nerve from Video 2 is stacked above a Dhc64C^6-10/ Dhc64C^4-19 mutant nerve. Both were photobleached and imaged as described in the legend of Video 2.

• Movie 4 - Mitochondria in and around an axon swelling.
  Larvae homozygous for a temperature sensitive Khc allele, Khc^1ts, were grown for 4 days at restrictive temperature (29°C) to allow the formation of axonal swellings, then were dissected and observed at permissive temperature (~22°C). Nerves were imaged at 1 frame every 2.2 sec. Playback is at 15 frames/sec.

• Movie 5 - Comparison of the behavior of mitochondria in the motor axons of wild-type and kinesin-1 mutant larvae.
  The lower panel shows a time-lapse movie of mitochondria in a larva carrying Khc⁶/ Khc²⁷, which causes a partial loss of Khc function. The upper panel shows a wild-type nerve (Video 2) for comparison. When the first anterograde mitochondrion in the mutant nerve reaches the center of the bleached zone it circulates in what was probably a small axonal swelling. Both nerves were imaged at 1 frame/sec and are played back at 15 frames/sec.

• Figure 1 - Co-localization of mitoGFP and a mitochondrial dye in cultured embryonic neuroblasts.
  Cells from disrupted embryos homozygous for D42 mitoGFP were cultured on coverslips, fixed and stained with Mito-Tracker Red. Micrographs are shown of a neuroblast with a long neurite extending to the right. A merge shows that the mitoGFP and MitoTracker Red fluorescence patterns are identical.

• Figure 2 - Fractionation of adult cytoplasm by differential sedimentation.
  Adult flies carrying UAS-mitoGFP and the D42-Gal4 driver were homogenized, then debris and nuclei were sedimented at low speed. The supernatant (S1) was then subjected to another round of sedimentation at higher speed and the last pellet was resuspended (P2) for further fractionation by equilibrium density sedimentation (Figure 8 in Results). The fractions shown were separated by SDS-PAGE on a 15% acrylamide gel. Western blots were stained simultaneously with anti-Khc, anti-cystein string protein, which associates with vesicles (CSP), anti-GFP (mitoGFP) and anti-cytochrome C (CytC). The positions of molecular weight standards (MW) are noted on the left.

• Figure 3 - Co-localization of mitochondria, Dhc and Khc in larval segmental nerves.
  Larvae homozygous for D42 and mitoGFP were dissected and co-stained with anti-Khc and anti-Dhc antibodies. Single confocal optical sections are shown. A) Dhc antibodies gave a diffuse plus punctate distribution. B) Khc antibodies showed a more diffuse distribution. C) The GFP signal from mitochondria consistently overlapped with Dhc and Khc as seen in the merged image (D).

• Table 1
• Table 2