Figure 5.
Figure 5. Virtual photoactivation paradigm to evaluate contribution of actin hotspots/trails to slow transport. (A) Simulation of photoactivation imaging paradigm. Initially, all actin is in monomeric state (top). After equilibration of the system, axonal actin within a 15-µm region is virtually “activated” (middle; green arrowheads represent photoactivated actin). Subsequently, the fluorescent actin either diffuses or translocates after incorporation into trails (bottom; see Materials and methods for more details). (B) Left: Labeled (green dotted line) and unlabeled (black solid line) actin concentration profiles along the axon shortly after photoactivation. Right: Expected outcomes of photoactivation simulations: For no net transport, the center of the labeled G-actin distribution is expected to be at its initial position (red line). For net anterograde or retrograde transport, the center is expected to move anterogradely (blue line) or retrogradely (cyan line), respectively. (C) Simulated intensity center shift for equal anterograde and retrograde trail nucleation rates. The center of fluorescent G-actin (green points) fluctuated about its initial position x = 0 as indicated by the red line. Results shown were averaged over 100 simulation runs. (D) Simulated intensity center shift when only anterograde trails were nucleated. The center of fluorescent G-actin (green points) moved anterogradely at a rate of 0.0135 µm/s (1.17 mm/d). Results were averaged over 1,000 simulation runs and fitted to a straight line (red line). (E) Simulated intensity center shift when only retrograde trails were nucleated. The center of fluorescent G-actin (green points) moved retrogradely at a rate of −0.009 µm/s (−0.78 mm/d). Results were averaged over 1,000 simulation runs and fitted to a straight line (red line). (F) Simulated intensity center shift for 58/42% bias of anterograde–retrograde trail frequencies. Center of fluorescent G-actin (green points) moved anterogradely at a rate of 0.0045 µm/s (0.39 mm/d). Results were averaged over 1,000 simulation runs and fitted to a straight line (red line). (G) Individual runs of simulated pulse-chase experiment (without averaging). Left: 1,000 individual simulation runs (gray lines) and the averaged (red line) intensity center shift. Right: 50 individual runs of the pulse-chase experiment.

Virtual photoactivation paradigm to evaluate contribution of actin hotspots/trails to slow transport. (A) Simulation of photoactivation imaging paradigm. Initially, all actin is in monomeric state (top). After equilibration of the system, axonal actin within a 15-µm region is virtually “activated” (middle; green arrowheads represent photoactivated actin). Subsequently, the fluorescent actin either diffuses or translocates after incorporation into trails (bottom; see Materials and methods for more details). (B) Left: Labeled (green dotted line) and unlabeled (black solid line) actin concentration profiles along the axon shortly after photoactivation. Right: Expected outcomes of photoactivation simulations: For no net transport, the center of the labeled G-actin distribution is expected to be at its initial position (red line). For net anterograde or retrograde transport, the center is expected to move anterogradely (blue line) or retrogradely (cyan line), respectively. (C) Simulated intensity center shift for equal anterograde and retrograde trail nucleation rates. The center of fluorescent G-actin (green points) fluctuated about its initial position x = 0 as indicated by the red line. Results shown were averaged over 100 simulation runs. (D) Simulated intensity center shift when only anterograde trails were nucleated. The center of fluorescent G-actin (green points) moved anterogradely at a rate of 0.0135 µm/s (1.17 mm/d). Results were averaged over 1,000 simulation runs and fitted to a straight line (red line). (E) Simulated intensity center shift when only retrograde trails were nucleated. The center of fluorescent G-actin (green points) moved retrogradely at a rate of −0.009 µm/s (−0.78 mm/d). Results were averaged over 1,000 simulation runs and fitted to a straight line (red line). (F) Simulated intensity center shift for 58/42% bias of anterograde–retrograde trail frequencies. Center of fluorescent G-actin (green points) moved anterogradely at a rate of 0.0045 µm/s (0.39 mm/d). Results were averaged over 1,000 simulation runs and fitted to a straight line (red line). (G) Individual runs of simulated pulse-chase experiment (without averaging). Left: 1,000 individual simulation runs (gray lines) and the averaged (red line) intensity center shift. Right: 50 individual runs of the pulse-chase experiment.

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