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High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport.

Yi JY, Ori-McKenney KM, McKenney RJ, Vershinin M, Gross SP, Vallee RB - J. Cell Biol. (2011)

Bottom Line: The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities.Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs.Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Cell Biology, Columbia University, New York, NY 10027, USA.

ABSTRACT
The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities. We used a novel approach to overcome these challenges, combining acute in vivo inhibition with automated high temporal and spatial resolution particle tracking. Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs. Acute LIS1 inhibition or LIS1 RNA interference had little effect on lysosomes/late endosomes but severely inhibited axonal transport of large, but not small, vesicular structures. Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo. Our data also reveal a specific but cell type-restricted role for LIS1 in large vesicular transport and provide the first quantitative support for a general role for LIS1 in high-load dynein functions.

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Differential lysosomal motility inhibition phenotypes induced by NudE/L and LIS1 inhibition in neurons. Lysos/LEs in rat cortical neurons showed mostly retrograde transport in uninjected and IgG controls, whereas severe inhibition was observed with 74.1 Ab, NudE/L antibody, and LIS1 antibody–injected cells. (A) Kymographs showing cells 1–6 min after injection. Arrest of several large particles is observed by 6 min in LIS1 antibody–injected cells. (B) Time-lapse images of the same LIS1 antibody–injected cell showing large lysosome arresting at the axonal kink (yellow arrow). Boxed areas show LysoTracker red versus dextran fluorescence versus phase-contrast images of the region shown. (C and D) Long-duration videos of the same LIS1 antibody–injected axon and a NudE/L antibody–injected axon. Note the near-complete arrest of motility in the LIS1 antibody–injected case by 20 min as opposed to continued bidirectional motility in the NudE/L antibody–injected case. Note the severe arrest (yellow arrow) as well as the directional reversal (green arrow) in the first 20 min after injection using anti-NudE/L antibody but the extensive bidirectional movement persistent beyond 20 min (orange arrows in C). Lysos/LEs in LIS1 antibody–injected cells showed progressive linear arrest with distance from the cell body (dotted line) during the first 21 min after injection, which is consistent with the diffusion of the antibody along the axon. Although larger particles remained immobile, smaller particles showed rapid transport (orange arrows in D) and could bypass the larger particles (dotted circles). (E) In control cells, lysos/LEs moved mostly in retrograde direction. Kymographs were generated from as near the cell body as imaging could be performed to up to 140 µm away.
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fig3: Differential lysosomal motility inhibition phenotypes induced by NudE/L and LIS1 inhibition in neurons. Lysos/LEs in rat cortical neurons showed mostly retrograde transport in uninjected and IgG controls, whereas severe inhibition was observed with 74.1 Ab, NudE/L antibody, and LIS1 antibody–injected cells. (A) Kymographs showing cells 1–6 min after injection. Arrest of several large particles is observed by 6 min in LIS1 antibody–injected cells. (B) Time-lapse images of the same LIS1 antibody–injected cell showing large lysosome arresting at the axonal kink (yellow arrow). Boxed areas show LysoTracker red versus dextran fluorescence versus phase-contrast images of the region shown. (C and D) Long-duration videos of the same LIS1 antibody–injected axon and a NudE/L antibody–injected axon. Note the near-complete arrest of motility in the LIS1 antibody–injected case by 20 min as opposed to continued bidirectional motility in the NudE/L antibody–injected case. Note the severe arrest (yellow arrow) as well as the directional reversal (green arrow) in the first 20 min after injection using anti-NudE/L antibody but the extensive bidirectional movement persistent beyond 20 min (orange arrows in C). Lysos/LEs in LIS1 antibody–injected cells showed progressive linear arrest with distance from the cell body (dotted line) during the first 21 min after injection, which is consistent with the diffusion of the antibody along the axon. Although larger particles remained immobile, smaller particles showed rapid transport (orange arrows in D) and could bypass the larger particles (dotted circles). (E) In control cells, lysos/LEs moved mostly in retrograde direction. Kymographs were generated from as near the cell body as imaging could be performed to up to 140 µm away.

Mentions: Lysos/LEs in uninjected rat cortical neurons showed predominantly retrograde transport (Fig. 3 A and Video 7), which was strongly inhibited by the 74.1 (Fig. 3 A and Video 8) and anti-NudE/L antibodies (Figs. 3 [A and C] and S3 C and Video 9). The effect was not as immediate as in nonneuronal cells, probably in part reflecting the greater distance required for antibody diffusion along the axon. Plus-end transport persisted at some level in most cells throughout the initial 10-min observation period (unpublished data) in contrast to the effects observed in COS-7 cells, suggesting less plus–minus coupling in neurons. The end result of the 74.1 injection was a substantial shift to plus-end movements. However, in cells injected with anti-NudE/L antibody, bidirectional movement of small particles persisted (Fig. 3 C, orange arrows).


High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport.

Yi JY, Ori-McKenney KM, McKenney RJ, Vershinin M, Gross SP, Vallee RB - J. Cell Biol. (2011)

Differential lysosomal motility inhibition phenotypes induced by NudE/L and LIS1 inhibition in neurons. Lysos/LEs in rat cortical neurons showed mostly retrograde transport in uninjected and IgG controls, whereas severe inhibition was observed with 74.1 Ab, NudE/L antibody, and LIS1 antibody–injected cells. (A) Kymographs showing cells 1–6 min after injection. Arrest of several large particles is observed by 6 min in LIS1 antibody–injected cells. (B) Time-lapse images of the same LIS1 antibody–injected cell showing large lysosome arresting at the axonal kink (yellow arrow). Boxed areas show LysoTracker red versus dextran fluorescence versus phase-contrast images of the region shown. (C and D) Long-duration videos of the same LIS1 antibody–injected axon and a NudE/L antibody–injected axon. Note the near-complete arrest of motility in the LIS1 antibody–injected case by 20 min as opposed to continued bidirectional motility in the NudE/L antibody–injected case. Note the severe arrest (yellow arrow) as well as the directional reversal (green arrow) in the first 20 min after injection using anti-NudE/L antibody but the extensive bidirectional movement persistent beyond 20 min (orange arrows in C). Lysos/LEs in LIS1 antibody–injected cells showed progressive linear arrest with distance from the cell body (dotted line) during the first 21 min after injection, which is consistent with the diffusion of the antibody along the axon. Although larger particles remained immobile, smaller particles showed rapid transport (orange arrows in D) and could bypass the larger particles (dotted circles). (E) In control cells, lysos/LEs moved mostly in retrograde direction. Kymographs were generated from as near the cell body as imaging could be performed to up to 140 µm away.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3198168&req=5

fig3: Differential lysosomal motility inhibition phenotypes induced by NudE/L and LIS1 inhibition in neurons. Lysos/LEs in rat cortical neurons showed mostly retrograde transport in uninjected and IgG controls, whereas severe inhibition was observed with 74.1 Ab, NudE/L antibody, and LIS1 antibody–injected cells. (A) Kymographs showing cells 1–6 min after injection. Arrest of several large particles is observed by 6 min in LIS1 antibody–injected cells. (B) Time-lapse images of the same LIS1 antibody–injected cell showing large lysosome arresting at the axonal kink (yellow arrow). Boxed areas show LysoTracker red versus dextran fluorescence versus phase-contrast images of the region shown. (C and D) Long-duration videos of the same LIS1 antibody–injected axon and a NudE/L antibody–injected axon. Note the near-complete arrest of motility in the LIS1 antibody–injected case by 20 min as opposed to continued bidirectional motility in the NudE/L antibody–injected case. Note the severe arrest (yellow arrow) as well as the directional reversal (green arrow) in the first 20 min after injection using anti-NudE/L antibody but the extensive bidirectional movement persistent beyond 20 min (orange arrows in C). Lysos/LEs in LIS1 antibody–injected cells showed progressive linear arrest with distance from the cell body (dotted line) during the first 21 min after injection, which is consistent with the diffusion of the antibody along the axon. Although larger particles remained immobile, smaller particles showed rapid transport (orange arrows in D) and could bypass the larger particles (dotted circles). (E) In control cells, lysos/LEs moved mostly in retrograde direction. Kymographs were generated from as near the cell body as imaging could be performed to up to 140 µm away.
Mentions: Lysos/LEs in uninjected rat cortical neurons showed predominantly retrograde transport (Fig. 3 A and Video 7), which was strongly inhibited by the 74.1 (Fig. 3 A and Video 8) and anti-NudE/L antibodies (Figs. 3 [A and C] and S3 C and Video 9). The effect was not as immediate as in nonneuronal cells, probably in part reflecting the greater distance required for antibody diffusion along the axon. Plus-end transport persisted at some level in most cells throughout the initial 10-min observation period (unpublished data) in contrast to the effects observed in COS-7 cells, suggesting less plus–minus coupling in neurons. The end result of the 74.1 injection was a substantial shift to plus-end movements. However, in cells injected with anti-NudE/L antibody, bidirectional movement of small particles persisted (Fig. 3 C, orange arrows).

Bottom Line: The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities.Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs.Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Cell Biology, Columbia University, New York, NY 10027, USA.

ABSTRACT
The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities. We used a novel approach to overcome these challenges, combining acute in vivo inhibition with automated high temporal and spatial resolution particle tracking. Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs. Acute LIS1 inhibition or LIS1 RNA interference had little effect on lysosomes/late endosomes but severely inhibited axonal transport of large, but not small, vesicular structures. Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo. Our data also reveal a specific but cell type-restricted role for LIS1 in large vesicular transport and provide the first quantitative support for a general role for LIS1 in high-load dynein functions.

Show MeSH
Related in: MedlinePlus