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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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Knockdown of NudE and LIS1 resembles acute inhibitions. (A) Lysos/LEs in rat hippocampal neurons moved mostly in the retrograde direction in wild-type and scrambled RNAi controls, but an increase in bidirectionally moving lysosomes and severe immobility of larger lysosomal particles (≥1 µm in diameter) were apparent in NudE RNAi and LIS1 RNAi cells, respectively. (A–C) Consistent with the NudE/L antibody–injected case, knockdown of NudE caused an increase in bidirectionally moving lysos/LEs (A and B), whereas knockdown of LIS1 protein more severely interfered with mobility of larger particles than smaller ones, which is consistent with the LIS1 antibody–injected case (A–C). Stationary, anterograde/retrograde, and bidirectional particles were defined as particles that displaced /ΔX/ < 1 µm, >5 µm in one direction, and >3 µm in both directions, respectively. The error bars represent SD (*, P < 0.001). The number of particles analyzed are as follows: control (small), n = 60; control (large), n = 55; LIS1 RNAi (small), n = 40; LIS1 RNAi (large), n = 66; NudE RNAi (small), n = 46; and NudE RNAi (large), n = 38 (using 10 axons per condition).
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fig4: Knockdown of NudE and LIS1 resembles acute inhibitions. (A) Lysos/LEs in rat hippocampal neurons moved mostly in the retrograde direction in wild-type and scrambled RNAi controls, but an increase in bidirectionally moving lysosomes and severe immobility of larger lysosomal particles (≥1 µm in diameter) were apparent in NudE RNAi and LIS1 RNAi cells, respectively. (A–C) Consistent with the NudE/L antibody–injected case, knockdown of NudE caused an increase in bidirectionally moving lysos/LEs (A and B), whereas knockdown of LIS1 protein more severely interfered with mobility of larger particles than smaller ones, which is consistent with the LIS1 antibody–injected case (A–C). Stationary, anterograde/retrograde, and bidirectional particles were defined as particles that displaced /ΔX/ < 1 µm, >5 µm in one direction, and >3 µm in both directions, respectively. The error bars represent SD (*, P < 0.001). The number of particles analyzed are as follows: control (small), n = 60; control (large), n = 55; LIS1 RNAi (small), n = 40; LIS1 RNAi (large), n = 66; NudE RNAi (small), n = 46; and NudE RNAi (large), n = 38 (using 10 axons per condition).

Mentions: As a control for these observations and to increase sample size, we also performed NudE and LIS1 RNAi in hippocampal neurons. The results were strikingly similar to those from antibody injection. Again, NudE RNAi caused a pronounced shift from retrograde to bidirectional movement (Fig. 4, A and B), whereas LIS1 RNAi caused substantial particle arrest. Nonetheless, as for LIS1 antibody injection, neurons subjected to LIS1 RNAi showed persistent transport of small vesicles (Fig. 4, A–C).


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)

Knockdown of NudE and LIS1 resembles acute inhibitions. (A) Lysos/LEs in rat hippocampal neurons moved mostly in the retrograde direction in wild-type and scrambled RNAi controls, but an increase in bidirectionally moving lysosomes and severe immobility of larger lysosomal particles (≥1 µm in diameter) were apparent in NudE RNAi and LIS1 RNAi cells, respectively. (A–C) Consistent with the NudE/L antibody–injected case, knockdown of NudE caused an increase in bidirectionally moving lysos/LEs (A and B), whereas knockdown of LIS1 protein more severely interfered with mobility of larger particles than smaller ones, which is consistent with the LIS1 antibody–injected case (A–C). Stationary, anterograde/retrograde, and bidirectional particles were defined as particles that displaced /ΔX/ < 1 µm, >5 µm in one direction, and >3 µm in both directions, respectively. The error bars represent SD (*, P < 0.001). The number of particles analyzed are as follows: control (small), n = 60; control (large), n = 55; LIS1 RNAi (small), n = 40; LIS1 RNAi (large), n = 66; NudE RNAi (small), n = 46; and NudE RNAi (large), n = 38 (using 10 axons per condition).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Knockdown of NudE and LIS1 resembles acute inhibitions. (A) Lysos/LEs in rat hippocampal neurons moved mostly in the retrograde direction in wild-type and scrambled RNAi controls, but an increase in bidirectionally moving lysosomes and severe immobility of larger lysosomal particles (≥1 µm in diameter) were apparent in NudE RNAi and LIS1 RNAi cells, respectively. (A–C) Consistent with the NudE/L antibody–injected case, knockdown of NudE caused an increase in bidirectionally moving lysos/LEs (A and B), whereas knockdown of LIS1 protein more severely interfered with mobility of larger particles than smaller ones, which is consistent with the LIS1 antibody–injected case (A–C). Stationary, anterograde/retrograde, and bidirectional particles were defined as particles that displaced /ΔX/ < 1 µm, >5 µm in one direction, and >3 µm in both directions, respectively. The error bars represent SD (*, P < 0.001). The number of particles analyzed are as follows: control (small), n = 60; control (large), n = 55; LIS1 RNAi (small), n = 40; LIS1 RNAi (large), n = 66; NudE RNAi (small), n = 46; and NudE RNAi (large), n = 38 (using 10 axons per condition).
Mentions: As a control for these observations and to increase sample size, we also performed NudE and LIS1 RNAi in hippocampal neurons. The results were strikingly similar to those from antibody injection. Again, NudE RNAi caused a pronounced shift from retrograde to bidirectional movement (Fig. 4, A and B), whereas LIS1 RNAi caused substantial particle arrest. Nonetheless, as for LIS1 antibody injection, neurons subjected to LIS1 RNAi showed persistent transport of small vesicles (Fig. 4, A–C).

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