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Mitochondrial movement and positioning in axons: the role of growth factor signaling

Sonita R. Chada and Peter J. Hollenbeck^*

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA

View larger version (30K): [in a new window]   Fig. 1. Mitochondria accumulate at sites of nerve growth factor (NGF) stimulation. Phase-contrast micrographs (A,C) and epifluorescent images of mitochondria stained with rhodamine 123 (R123) in living neurons (B,D) were used to compare the distribution of mitochondria in the axon adjacent to an NGF-coated bead (A,B) and a heat-denatured NGF-coated bead (C,D). Mitochondria accumulate at sites of bead–axon contact within 1 h. No mitochondrial accumulation is observed using the control heat-denatured NGF-coated beads. Scale bar, 10 µm.

View larger version (18K): [in a new window]   Fig. 2. Local nerve growth factor (NGF) activation regulates mitochondrial transport and distribution in neurons. Histograms of mitochondrial rhodamine 123 (R123) fluorescence intensities in the 10 µm region immediately adjacent to an NGF-coated bead vs the 10 µm control regions located 100 µm away in either direction along the axon from the point of bead contact. The mitochondrial fluorescence intensity is significantly higher (shown by the asterisk) in the 10 µm region of the axon adjacent to an NGF-coated bead than in similar proximal and distal non-bead control regions (N=10; paired t-test). No difference in mitochondrial densities was observed when using the control heat-denatured NGF-coated beads (N=8; paired t-test) or with a high background concentration of NGF. Adding 10 ng ml-1 NGF to the culture medium, which is above the Kd of the high affinity TrkA receptor (approximately 1 ng ml-1) but below the Kd of the lower affinity p75 receptor (approximately 40 ng ml-1), significantly reduced the mitochondrial response to NGF-coated beads (N=8; paired t-test). The mitochondrial accumulation was also blocked in the presence of 90 ng ml-1 NGF (N=9; paired t-test).

View larger version (37K): [in a new window]   Fig. 3. Nerve growth factor (NGF)-coated beads do not affect the cytoskeleton. Primary cultured DRG neurons were double immunostained for microtubules (MTs) and F-actin (MFs). In all panels, the positions of the NGF-coated beads prior to fixation are indicated by circles. The regions of the axons in contact with NGF-coated beads have MT arrays and MF density essentially identical to control regions of the axon not contacting a bead. Scale bar, 10 µm

View larger version (15K): [in a new window]   Fig. 4. LY294002 inhibits mitochondrial accumulation at sites of nerve growth factor (NGF) stimulation. LY294002 treatment prevented the accumulation of mitochondria in the region of NGF stimulation (N=12; paired t-test). Dimethylsulfoxide (DMSO; vehicle) alone did not affect the mitochondrial response to NGF stimulation. The density of mitochondria is 3-fold higher in the 10 µm region of the axon adjacent to the NGF-coated bead than in similar proximal and distal non-bead control regions (N=9; paired t-test). * represents statistical significance at P0.001.