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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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High-resolution lyso/LE particle–tracking analysis in COS-7 cells. High-temporal resolution recordings (∼17 frames/s) of lyso/LE motion were processed for analysis. (A) Examples of lysosomal tracks from an uninjected cell showing normal bidirectional lyso/LE motility, a 74.1 Ab–injected cell showing long plus-end–directed travel, and LIS1 DN–injected cells showing normal minus-end–directed transport and oscillatory movement (also see Fig. S2 B). (B–G) Effect of 74.1 and DN LIS1 injection on COS-7 cell lyso/LE motility. Run length (B), percentage of motility (C), and MSD (D) are shown. (E) Net displacement/particle (= [Σ-plus runs (nm) − Σ-minus runs (nm)]/number of particles). (F) Average particle velocity is shown. All error bars represent SEM. *, P < 0.05. (G) Sample size for particle-tracking analysis.
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fig2: High-resolution lyso/LE particle–tracking analysis in COS-7 cells. High-temporal resolution recordings (∼17 frames/s) of lyso/LE motion were processed for analysis. (A) Examples of lysosomal tracks from an uninjected cell showing normal bidirectional lyso/LE motility, a 74.1 Ab–injected cell showing long plus-end–directed travel, and LIS1 DN–injected cells showing normal minus-end–directed transport and oscillatory movement (also see Fig. S2 B). (B–G) Effect of 74.1 and DN LIS1 injection on COS-7 cell lyso/LE motility. Run length (B), percentage of motility (C), and MSD (D) are shown. (E) Net displacement/particle (= [Σ-plus runs (nm) − Σ-minus runs (nm)]/number of particles). (F) Average particle velocity is shown. All error bars represent SEM. *, P < 0.05. (G) Sample size for particle-tracking analysis.

Mentions: To test the effects of acute dynein inhibition on subcellular cargos, we injected several purified function-blocking reagents into live COS-7 cells. Immediately after injection of a dynein function-blocking monoclonal antibody (74.1 Ab), the majority of LysoTracker-positive lysosomes/late endosomes (LEs; lysos/LEs) redistributed en masse toward the cell periphery. Rapid long-range centrifugal movements were initially evident (Figs. 1 A and S1 C; see Video 1 vs. Video 2 for IgG control), although by 10 min, the fraction of stationary particles had increased (∼60% at 10 min vs. ∼30% at 1 min; Figs. 1 A and 2 D). A similar pattern of rapid dispersal followed by an overall reduction in motility was observed for another lyso/LE marker, GFP-NPC1 (25 cells showing dispersal phenotype/25 total), and markers for early endosomes (25/25), Golgi elements (30/30), and fluorescently labeled adenovirus (40/40; Figs. 1 B and S1 E). We observed similar effects on lysos/LEs using the 70.1 anti-dynein antibody (18/18) and a function-blocking anti-NudE/NudEL (NudE/L) antibody (19/19 cells; Stehman et al., 2007) as well as the DN dynactin CC1 fragment (5/5; Fig. 1 C). No apparent effect was observed using an antibody to the dynactin p150Glued cytoskeleton-associated protein–Gly microtubule-binding domain (seven cells showing no dispersal phenotype/seven total), a function-blocking anti-LIS1 N-terminal antibody (5/5; Faulkner et al., 2000), or a DN N-terminal LIS1 fragment (28/28; Tai et al., 2002) expressed in Escherichia coli (Figs. 1 D and S1 D). The latter results were despite severe effects of the antibody on mitotic progression (Faulkner et al., 2000). We also found overnight incubation of cells injected with the N-terminal fragment to cause arrest in mitosis (27/35 cells; Fig. S1 A). Similar effects of 74.1 Ab on lysos/LEs were obtained in HeLa-M, A549, and GFP-Lamp1–expressing COS-7 cells (Fig. S1 B).


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)

High-resolution lyso/LE particle–tracking analysis in COS-7 cells. High-temporal resolution recordings (∼17 frames/s) of lyso/LE motion were processed for analysis. (A) Examples of lysosomal tracks from an uninjected cell showing normal bidirectional lyso/LE motility, a 74.1 Ab–injected cell showing long plus-end–directed travel, and LIS1 DN–injected cells showing normal minus-end–directed transport and oscillatory movement (also see Fig. S2 B). (B–G) Effect of 74.1 and DN LIS1 injection on COS-7 cell lyso/LE motility. Run length (B), percentage of motility (C), and MSD (D) are shown. (E) Net displacement/particle (= [Σ-plus runs (nm) − Σ-minus runs (nm)]/number of particles). (F) Average particle velocity is shown. All error bars represent SEM. *, P < 0.05. (G) Sample size for particle-tracking analysis.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: High-resolution lyso/LE particle–tracking analysis in COS-7 cells. High-temporal resolution recordings (∼17 frames/s) of lyso/LE motion were processed for analysis. (A) Examples of lysosomal tracks from an uninjected cell showing normal bidirectional lyso/LE motility, a 74.1 Ab–injected cell showing long plus-end–directed travel, and LIS1 DN–injected cells showing normal minus-end–directed transport and oscillatory movement (also see Fig. S2 B). (B–G) Effect of 74.1 and DN LIS1 injection on COS-7 cell lyso/LE motility. Run length (B), percentage of motility (C), and MSD (D) are shown. (E) Net displacement/particle (= [Σ-plus runs (nm) − Σ-minus runs (nm)]/number of particles). (F) Average particle velocity is shown. All error bars represent SEM. *, P < 0.05. (G) Sample size for particle-tracking analysis.
Mentions: To test the effects of acute dynein inhibition on subcellular cargos, we injected several purified function-blocking reagents into live COS-7 cells. Immediately after injection of a dynein function-blocking monoclonal antibody (74.1 Ab), the majority of LysoTracker-positive lysosomes/late endosomes (LEs; lysos/LEs) redistributed en masse toward the cell periphery. Rapid long-range centrifugal movements were initially evident (Figs. 1 A and S1 C; see Video 1 vs. Video 2 for IgG control), although by 10 min, the fraction of stationary particles had increased (∼60% at 10 min vs. ∼30% at 1 min; Figs. 1 A and 2 D). A similar pattern of rapid dispersal followed by an overall reduction in motility was observed for another lyso/LE marker, GFP-NPC1 (25 cells showing dispersal phenotype/25 total), and markers for early endosomes (25/25), Golgi elements (30/30), and fluorescently labeled adenovirus (40/40; Figs. 1 B and S1 E). We observed similar effects on lysos/LEs using the 70.1 anti-dynein antibody (18/18) and a function-blocking anti-NudE/NudEL (NudE/L) antibody (19/19 cells; Stehman et al., 2007) as well as the DN dynactin CC1 fragment (5/5; Fig. 1 C). No apparent effect was observed using an antibody to the dynactin p150Glued cytoskeleton-associated protein–Gly microtubule-binding domain (seven cells showing no dispersal phenotype/seven total), a function-blocking anti-LIS1 N-terminal antibody (5/5; Faulkner et al., 2000), or a DN N-terminal LIS1 fragment (28/28; Tai et al., 2002) expressed in Escherichia coli (Figs. 1 D and S1 D). The latter results were despite severe effects of the antibody on mitotic progression (Faulkner et al., 2000). We also found overnight incubation of cells injected with the N-terminal fragment to cause arrest in mitosis (27/35 cells; Fig. S1 A). Similar effects of 74.1 Ab on lysos/LEs were obtained in HeLa-M, A549, and GFP-Lamp1–expressing COS-7 cells (Fig. S1 B).

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