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Sliding of centrosome-unattached microtubules defines key features of neuronal phenotype

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ABSTRACT

Rao et al. show that during migration, neurons contain a small population of centrosome-unattached microtubules in the leading process that are capable of sliding. Increasing the proportion of centrosome-unattached microtubules alters neuronal morphology, migration path, and microtubule behavior in the leading process.

No MeSH data available.


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Ninein depletion compromises neuronal migration. (A) Schematic depicting the experimental paradigm. Under control conditions, the two red converted zones converge by a distance nearly equal to that of the total migration distance. (B, top) Cerebellar migratory neuron cotransfected with tdEos-tubulin and control siRNA. Minimal MT sliding occurs and soma moves forward. (bottom) Cerebellar migratory neuron cotransfected with tdEos-tubulin and ninein siRNA. Abnormal tortuosity of the leading process and increased MT sliding are observed (regions of yellow indicated by arrowheads [proximal] and arrows [distal]). Cells were imaged for 15 min. (C) Stacked bar graph showing migration rate distribution under control siRNA and ninein siRNA conditions. n = 20 per group. Numeral in parentheses represents the mean migration rate per category. (D) Bar graph depicting the total migration distance after treatment with control siRNA or ninein siRNA. ***, P < 0.0001 (n = 20 per group). (E) Bar graph of migration synchrony under control and ninein-depleted conditions. Total migration distance was divided by the change in zone distance. ***, P < 0.0001 (n = 20 per group). See also Video 2. Bars, 15 µm. Data are represented as mean ± SEM.
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fig3: Ninein depletion compromises neuronal migration. (A) Schematic depicting the experimental paradigm. Under control conditions, the two red converted zones converge by a distance nearly equal to that of the total migration distance. (B, top) Cerebellar migratory neuron cotransfected with tdEos-tubulin and control siRNA. Minimal MT sliding occurs and soma moves forward. (bottom) Cerebellar migratory neuron cotransfected with tdEos-tubulin and ninein siRNA. Abnormal tortuosity of the leading process and increased MT sliding are observed (regions of yellow indicated by arrowheads [proximal] and arrows [distal]). Cells were imaged for 15 min. (C) Stacked bar graph showing migration rate distribution under control siRNA and ninein siRNA conditions. n = 20 per group. Numeral in parentheses represents the mean migration rate per category. (D) Bar graph depicting the total migration distance after treatment with control siRNA or ninein siRNA. ***, P < 0.0001 (n = 20 per group). (E) Bar graph of migration synchrony under control and ninein-depleted conditions. Total migration distance was divided by the change in zone distance. ***, P < 0.0001 (n = 20 per group). See also Video 2. Bars, 15 µm. Data are represented as mean ± SEM.

Mentions: We would anticipate that greater numbers of centrosome-unattached MTs would lead to greater mobility (i.e., more sliding) of MTs in the leading process of these neurons. The increase in CAMSAP particles in the leading process after partial ninein depletion was our first indication of enhanced MT sliding because more free minus ends of MTs in the leading process would be consistent with greater movement of MTs from the soma into the leading process. To more directly assay for MT sliding, tandem-dimer Eos (tdEos)–tagged tubulin was expressed overnight in neurons into which either control or ninein siRNA had been introduced 2 d before. tdEos is a tag that fluoresces green until it is photoconverted by exposure to 405 nm light, after which it fluoresces red. Two distinct regions of the cell were converted, one in the soma and proximal region of the leading process and another located in the middle one third of the leading process (Fig. 3 A). Once converted, dual-channel live-cell imaging of the neurons was performed. For this set of experiments, the distance was measured between the two converted zones (dz), as well as the total distance (dt) of migration, and the two were compared to determine whether coordination existed between these two parameters as well as to confirm that expression of tdEos-tubulin does not affect migration. After ninein depletion, when red and green images were merged, a greater amount of MT movement was observed in the leading process, as indicated by bright yellow (yellow represents the comingling of red and green signal) regions flanking the proximal and distal borders of the red converted zones in the ninein-depleted neuron. This was not observed in the control neuron (Fig. 3 B and Video 2). Several neurons treated with ninein siRNA showed an elongated leading process that was also notably tortuous along its length, a phenomenon that was never observed in neurons treated with control siRNA (Video 2). We speculate that this tortuosity of the leading process is caused by buckling of centrosome-unattached MTs, with an individual MT buckling because of an abrupt interruption in a bout of sliding.


Sliding of centrosome-unattached microtubules defines key features of neuronal phenotype
Ninein depletion compromises neuronal migration. (A) Schematic depicting the experimental paradigm. Under control conditions, the two red converted zones converge by a distance nearly equal to that of the total migration distance. (B, top) Cerebellar migratory neuron cotransfected with tdEos-tubulin and control siRNA. Minimal MT sliding occurs and soma moves forward. (bottom) Cerebellar migratory neuron cotransfected with tdEos-tubulin and ninein siRNA. Abnormal tortuosity of the leading process and increased MT sliding are observed (regions of yellow indicated by arrowheads [proximal] and arrows [distal]). Cells were imaged for 15 min. (C) Stacked bar graph showing migration rate distribution under control siRNA and ninein siRNA conditions. n = 20 per group. Numeral in parentheses represents the mean migration rate per category. (D) Bar graph depicting the total migration distance after treatment with control siRNA or ninein siRNA. ***, P < 0.0001 (n = 20 per group). (E) Bar graph of migration synchrony under control and ninein-depleted conditions. Total migration distance was divided by the change in zone distance. ***, P < 0.0001 (n = 20 per group). See also Video 2. Bars, 15 µm. Data are represented as mean ± SEM.
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Related In: Results  -  Collection

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fig3: Ninein depletion compromises neuronal migration. (A) Schematic depicting the experimental paradigm. Under control conditions, the two red converted zones converge by a distance nearly equal to that of the total migration distance. (B, top) Cerebellar migratory neuron cotransfected with tdEos-tubulin and control siRNA. Minimal MT sliding occurs and soma moves forward. (bottom) Cerebellar migratory neuron cotransfected with tdEos-tubulin and ninein siRNA. Abnormal tortuosity of the leading process and increased MT sliding are observed (regions of yellow indicated by arrowheads [proximal] and arrows [distal]). Cells were imaged for 15 min. (C) Stacked bar graph showing migration rate distribution under control siRNA and ninein siRNA conditions. n = 20 per group. Numeral in parentheses represents the mean migration rate per category. (D) Bar graph depicting the total migration distance after treatment with control siRNA or ninein siRNA. ***, P < 0.0001 (n = 20 per group). (E) Bar graph of migration synchrony under control and ninein-depleted conditions. Total migration distance was divided by the change in zone distance. ***, P < 0.0001 (n = 20 per group). See also Video 2. Bars, 15 µm. Data are represented as mean ± SEM.
Mentions: We would anticipate that greater numbers of centrosome-unattached MTs would lead to greater mobility (i.e., more sliding) of MTs in the leading process of these neurons. The increase in CAMSAP particles in the leading process after partial ninein depletion was our first indication of enhanced MT sliding because more free minus ends of MTs in the leading process would be consistent with greater movement of MTs from the soma into the leading process. To more directly assay for MT sliding, tandem-dimer Eos (tdEos)–tagged tubulin was expressed overnight in neurons into which either control or ninein siRNA had been introduced 2 d before. tdEos is a tag that fluoresces green until it is photoconverted by exposure to 405 nm light, after which it fluoresces red. Two distinct regions of the cell were converted, one in the soma and proximal region of the leading process and another located in the middle one third of the leading process (Fig. 3 A). Once converted, dual-channel live-cell imaging of the neurons was performed. For this set of experiments, the distance was measured between the two converted zones (dz), as well as the total distance (dt) of migration, and the two were compared to determine whether coordination existed between these two parameters as well as to confirm that expression of tdEos-tubulin does not affect migration. After ninein depletion, when red and green images were merged, a greater amount of MT movement was observed in the leading process, as indicated by bright yellow (yellow represents the comingling of red and green signal) regions flanking the proximal and distal borders of the red converted zones in the ninein-depleted neuron. This was not observed in the control neuron (Fig. 3 B and Video 2). Several neurons treated with ninein siRNA showed an elongated leading process that was also notably tortuous along its length, a phenomenon that was never observed in neurons treated with control siRNA (Video 2). We speculate that this tortuosity of the leading process is caused by buckling of centrosome-unattached MTs, with an individual MT buckling because of an abrupt interruption in a bout of sliding.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Rao et al. show that during migration, neurons contain a small population of centrosome-unattached microtubules in the leading process that are capable of sliding. Increasing the proportion of centrosome-unattached microtubules alters neuronal morphology, migration path, and microtubule behavior in the leading process.

No MeSH data available.


Related in: MedlinePlus