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First published online 18 May 2004
doi: 10.1242/jcs.01130

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Axonal mitochondrial transport and potential are correlated

Department of Biological Sciences, Room 713 Fairchild Building, Columbia University, New York, NY 10027, USA

View larger version (53K): [in a new window]   Fig. 2. Mitochondrion transported by anterograde and retrograde transport dock in the middle of the gap between stationary mitochondria. (A) Examples of mitochondria pausing during fast axonal transport. (B) Normalized distribution of mitochondrial stops relative to the two closest stationary mitochondria (n=58 stopping events). (C) Histogram analysis of the relative position of the stops in relation to the two closest mitochondria (n=58 stopping events). (D) Circular histogram of stopping events. Line with arrowhead shows the sum (R) and net angle of the vectors. (E) Cumulative probabilities of the vector sums of 58 random events generated by Monte Carlo simulation. From this, we estimate the probability of the observed clustering shown in (C) to be P<0.036.

View larger version (51K): [in a new window]   Fig. 1. Mitochondria are uniformly distributed along the axon. (A) Distribution of Mitotracker-labeled mitochondria after 1 minute from a time-lapse movie. (B) Kymograph of the movie: horizontal bar=10 µm, vertical bar=1 minute. (C) Color-inverted image of (A) with height stretched by a factor of five in order to resolve the individual mitochondria better. (A-D) Brackets show a cluster of four mitochondria that can be resolved by analysis of the kymograph but cannot be resolved in the still picture. (D) Estimated centers of individual mitochondria, used for statistical analysis. (E) Intensity plot of the mitochondria, with arrows showing divisions between peaks. (F) Sum of the area for each of the peaks shown in (E). (G) Peak size vs estimated number of mitochondria. (H) Frequency distribution from the 2 analysis of the goodness of fit of the predicted Poisson distribution vs the observed distribution.

View larger version (69K): [in a new window]   Fig. 3. Antimycin, an inhibitor of the electron transport chain, doubles retrograde transport. (A) Kymograph of distribution and movements of Mitotracker-labeled mitochondria. Transport of the mitochondria was observed for 5-10 minutes for the pre-drug, early drug, late drug and washout application conditions as delineated by the horizontal green lines. The position and time of drug application (100 µM antimycin and Lucifer Yellow in DMEM) is shown in blue. The length of the axon was also divided into three sections with reference to the position of drug application (cell-body side, CB; position of drug application, drug; growth-cone side, GC). (B) Hand-drawn traces of anterograde (blue) and retrograde (red) movements used to quantify the mitochondrial transport. (C-F) Normalized anterograde and retrograde transport. Error bars are the normalized 95% confidence intervals (n=5 experiments). (D) There is significant transient increase in retrograde transport but no effect on anterograde transport. (E) After 5 minutes, retrograde transport returns to baseline levels.

View larger version (71K): [in a new window]   Fig. 4. Application of 2-DG, a competitive inhibitor of glycolysis, has a minor effect on mitochondrial transport. (A) Kymograph of distribution and movements of Mitotracker-labeled mitochondria. The position and time of drug application (200 mM 2-DG, and Lucifer Yellow in DMEM) is shown in blue. (B) Hand-drawn traces of anterograde (blue) and retrograde (red) movements used to quantify the mitochondrial transport. (C-F) Normalized anterograde and retrograde transport. Error bars are the normalized 95% confidence intervals (n=5 experiments). (D) Decreases in anterograde and retrograde transport start but do not reach significant levels. (E) After 5 minutes of drug application both anterograde and retrograde transport are modestly (P<0.05) decreased for each direction of transport past the point of drug application. (F) Anterograde and retrograde transport after drug application is stopped.

View larger version (69K): [in a new window]   Fig. 5. Local application of mannitol, an ionic control for 2-DG, suggests that inhibition of transport by 2-DG is not due to inhibition of glycolysis. (A) Kymograph of distribution and movements of Mitotracker-labeled mitochondria. The position and time of drug application (200 mM mannitol and Lucifer Yellow in DMEM) is shown in blue. (B) Hand-drawn traces of anterograde (blue) and retrograde (red) mediated movements used to quantify the mitochondrial transport. (C-F) Normalized anterograde and retrograde transport. Error bars are the normalized 95% confidence intervals (n=5 experiments). (D-E) After 5 minutes of drug application, both anterograde and retrograde transport are significantly (P<0.05) decreased for each direction of transport past the point of drug application. (F) Anterograde and retrograde transport increase after drug application is stopped.

View larger version (72K): [in a new window]   Fig. 6. The mitochondrial uncoupler CCCP decreases both kinesin- and dynein-mediated transport but does not lead to a transient rise in retrograde transport. (A) Kymograph of distribution and movements of Mitotracker-labeled mitochondria. The position and time of drug application (1 mM CCCP and Lucifer Yellow in DMEM) is shown in blue. (B) Hand-drawn traces of anterograde (blue) and retrograde (red) mediated movements used to quantify the mitochondrial movements. (C-F) Normalized anterograde and retrograde mediated transport. Error bars are the normalized 95% confidence intervals (n=5 experiments). (D,E) There is significant decrease in both anterograde and retrograde mediated transport. (F) After washout, transport recovers.

View larger version (99K): [in a new window]   Fig. 7. The direction of mitochondrial transport is correlated with mitochondrial potential as assayed with JC-1 and Mitotracker FM-green. (A) Hand-drawn traces of anterograde and retrograde mitochondrial movements from the middle of an axon. Red and green lines represent the movements of mitochondria with a correspondingly high or low ratio of red/green staining. The arrow and triangle point to two mitochondrial traces that are exceptions to the correlation between potential and direction of transport. The vertical bar equals 1 minute and the horizontal bar equals 10 µm. (B) Color-inverted kymograph of red channel showing movements of mitochondria with high potential as assayed by JC-1. The circle indicates a mitochondrion undergoing a decrease in potential. (C) Color-inverted kymograph of green channel showing movements of JC-1/Mitotracker-FM-green-stained mitochondria. (D) The first frame of the time-lapse sequence shows the mixed polarity of mitochondria along the axon. (E) Overlay of red and green channels. (F) The last frame of the time-lapse sequence. (G) Hand-drawn traces of mitochondrial movements close to the growth cone. (H) Color-inverted kymograph of red channel. The double circle illustrates accumulation of mitochondria with high potential at the growth cone. (I) Color-inverted kymograph of green channel. (J) The first frame of the time-lapse sequence. (K) Overlay of red and green channels. (L) The last frame of the time-lapse sequence.

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