A close look at axonal transport: Cargos slow down when crossing stationary organelles

Abstract

The bidirectional transport of cargos along the thin axon is fundamental for the structure, function and survival of neurons. Defective axonal transport has been linked to the mechanism of neurodegenerative diseases. In this paper, we study the effect of the local axonal environment to cargo transport behavior in neurons. Using dual-color fluorescence imaging in microfluidic neuronal devices, we quantify the transport dynamics of cargos when crossing stationary organelles such as non-moving endosomes and stationary mitochondria in the axon. We show that the axonal cargos tend to slow down, or pause transiently within the vicinity of stationary organelles. The slow-down effect is observed in both retrograde and anterograde transport directions of three different cargos (TrkA, lysosomes and TrkB). Our results agree with the hypothesis that bulky axonal structures can pose as steric hindrance for axonal transport. However, the results do not rule out the possibility that cellular mechanisms causing stationary organelles are also responsible for the delay in moving cargos at the same locations.

Keywords: Axonal swelling; Axonal transport; Fluorescence imaging; Pseudo-TIRF microscopy; Roadblocks; Transport dynamics.

Figure 1

Moving TrkA cargos slow down when crossing stationary TrkA markers in DRG axons. (A) Kymograph of TrkA trajectories in DRG neurons transfected with TrkA-mCherry. Horizontal lines (red arrows) indicate the stationary TrkA in the axon. Yellow arrowheads show the regions where the moving TrkA cargos clearly slow down when crossing the stationary TrkA. (B) The process of quantifying TrkA transport behavior when crossing stationary markers. The trajectory of a retrograde TrkA cargo (red dots) is overlapped onto the positions of the stationary TrkA (white horizontal lines) in the same axon. Inset shows enlarged image of a crossing event (gray rectangle in the original kymograph), where the speed of the cargo right before the stationary TrkA region (v_before, yellow) is compared to the speed of the cargo inside the stationary TrkA region (v_inside, green). (C) Comparison of the distribution of Δv (v_before−v_inside) for retrogradely moving TrkA cargos when crossing stationary TrkA sites (n=351) vs. when crossing control sites that are absent of stationary TrkA (n=1811). (D) TrkA transport behavior when crossing stationary TrkA markers is categorized into three groups – slow, fast and unchanged based on Δv (v_before−v_inside) values. Data were cumulated from 7 independent experiments. The cargo behavior is normalized to the total number of crossing events. Bootstrapping (10 sets of 300 randomly selected events) was performed to calculate the standard deviation. Error bars represents standard deviation. Statistical significance was assessed using Student’s t-test, p<0.001. Horizontal bar, 5s. Vertical bar, 5 µm.

Figure 2

Moving TrkA cargos slow down when crossing stationary mitochondria in DRG axons. DRG neurons are co-transfected with TrkA-mCherry and Mito-YFP. (A) A representative mitochondrial kymograph. Horizontal lines indicate the stationary mitochondria in the axon. (B) The kymograph of TrkA cargos in the same axon. Shadows of the four mitochondria in the axon can also be seen on TrkA kymograph. Red arrows show the positions of mitochondriain both the mitochondrial and TrkA kymographs. Yellow arrowheads indicate the regions where TrkA cargos clearly slow down when crossing the stationary mitochondria in the axon. Horizontal bar, 5s.Vertical bar, 5µm. (C–D) Quantification of TrkA transport behavior in the retrograde direction (C) and the anterograde direction (D). Data were cumulated from 7 independent experiments. Bootstrapping (10 sets of 300 randomly selected events) was performed to calculate the standard deviation. Error bars represents standard deviation. Statistical significance was assessed using Student’s t-test, p<0.001. (E–F) The distribution of pause duration for TrkA cargos at mitochondrial sites compared to control sites that are not associated with mitochondria. Data were cumulated from 7 independent experiments.

Figure 3

Transport behavior of lysosomes and TrkB when crossing stationary mitochondria. (A) Representative kymograph showing transport behavior of lysosomes in DRG neurons. Red arrows indicate stationary mitochondrial traces in the kymograph. Yellow arrows show the regions where the cargos clearly slow down at mitochondrial sites. (B–C) Transport behavior of lysosomes in DRG neurons. Data were cumulated from 3 independent cultures. (D) Representative kymograph showing transport behavior of TrkB in hippocampal neurons. (E–F) Behavior of TrkB transport in hippocampal neurons. Data were cumulated from 4 independent cultures. Error bars show standard deviation. Bootstrapping (10 sets of 200 randomly selected events) was performed to calculate the standard deviation. Statistical significance was assessed using Student’s t-test, p<0.001. Horizontal bar, 5s.Vertical bar, 5 µm.