Figure 5.
Depletion of Msps strongly perturbs the ability of centrosomes to organize and nucleate microtubules in the absence of γ-tubulin complexes. (A–E) Fluorescence images of Drosophila brain cells in either prophase or prometaphase from either wild-type (A), cnn,grip71,grip163 (B), cnn,grip71,grip163,msps (C), cnn,grip71,grip163,tacc (D), or cnn,grip71,grip163,mei38-RNAi cell (E) immunostained for alpha-tubulin (microtubules, green), mitotic DNA (blue), and Asl (centrioles, magenta). Note that some cells lacking Cnn have abnormal numbers of centrosomes due to centrosome segregation problems during cell division (Conduit et al., 2010). (F) Graph showing the percentage of prophase cells in which microtubules are associated with at least one centrosome within the various genotypes, as indicated. The number of cells analyzed (n) is indicated. Datasets were compared to the cnn,grip71,grip163 dataset using one-way Chi-squared tests. Note there is a no significant reduction between wild-type and cnn,grip71,grip163 mutant cells in the proportion of cells displaying centrosome-associated microtubules, but there are significant reductions between cnn,grip71,grip163 mutant cells and cells that are also depleted for either Msps, TACC, or mei-38, indicating that Msps, TACC, and mei-38 have a role in γ-TuRC-independent microtubule nucleation. (G and H) Graphs of raw data (G) and log transformed data (H) showing the distance of centrosomes from spindle poles during prometaphase in the different genotypes, as indicated. The percentage of centrosomes that were farther than 1 μm from the spindle poles is indicated above each dataset in the graph in G. Increased distance from the spindle pole is indicative of a failure to properly organize microtubules. Kruskal-Wallis tests were used to compare the distribution of the cnn,grip71,grip163 dataset with those of the other genotypes. Each datapoint represents an individual centrosome. N numbers are: WT 62, cnn,grip71,grip163 72, cnn,grip71,grip163,msps 133, cnn,grip71,grip163,tacc 139, and cnn,grip71,grip163,mei38-RNAi 134. Note that there is a significant difference only between cnn,grip71,grip163 and cnn,grip71,grip163,msps mutant cells, indicating that Msps is particularly important for microtubule organization at centrosomes in the absence of γ-TuRCs. (I and J) Parts of a whole graph (I) and images (J) represent analyses of centrosomal aster types in cells fixed and immunostained for alpha-tubulin (microtubules, green), mitotic DNA (blue), and Asl (centrioles, magenta) after 30 s of warming post cooling from either cnn,grip71,grip163 or cnn,grip71,grip163,msps mutants, as indicated. N numbers are indicated, and each N represents a single cell. Only cells that had centrosomes but that had not yet formed a spindle were analyzed. Note how centrosome asters are frequently absent in cnn,grip71,grip163,msps.

Depletion of Msps strongly perturbs the ability of centrosomes to organize and nucleate microtubules in the absence of γ-tubulin complexes. (A–E) Fluorescence images of Drosophila brain cells in either prophase or prometaphase from either wild-type (A), cnn,grip71,grip163 (B), cnn,grip71,grip163,msps (C), cnn,grip71,grip163,tacc (D), or cnn,grip71,grip163,mei38-RNAi cell (E) immunostained for alpha-tubulin (microtubules, green), mitotic DNA (blue), and Asl (centrioles, magenta). Note that some cells lacking Cnn have abnormal numbers of centrosomes due to centrosome segregation problems during cell division (Conduit et al., 2010). (F) Graph showing the percentage of prophase cells in which microtubules are associated with at least one centrosome within the various genotypes, as indicated. The number of cells analyzed (n) is indicated. Datasets were compared to the cnn,grip71,grip163 dataset using one-way Chi-squared tests. Note there is a no significant reduction between wild-type and cnn,grip71,grip163 mutant cells in the proportion of cells displaying centrosome-associated microtubules, but there are significant reductions between cnn,grip71,grip163 mutant cells and cells that are also depleted for either Msps, TACC, or mei-38, indicating that Msps, TACC, and mei-38 have a role in γ-TuRC-independent microtubule nucleation. (G and H) Graphs of raw data (G) and log transformed data (H) showing the distance of centrosomes from spindle poles during prometaphase in the different genotypes, as indicated. The percentage of centrosomes that were farther than 1 μm from the spindle poles is indicated above each dataset in the graph in G. Increased distance from the spindle pole is indicative of a failure to properly organize microtubules. Kruskal-Wallis tests were used to compare the distribution of the cnn,grip71,grip163 dataset with those of the other genotypes. Each datapoint represents an individual centrosome. N numbers are: WT 62, cnn,grip71,grip163 72, cnn,grip71,grip163,msps 133, cnn,grip71,grip163,tacc 139, and cnn,grip71,grip163,mei38-RNAi 134. Note that there is a significant difference only between cnn,grip71,grip163 and cnn,grip71,grip163,msps mutant cells, indicating that Msps is particularly important for microtubule organization at centrosomes in the absence of γ-TuRCs. (I and J) Parts of a whole graph (I) and images (J) represent analyses of centrosomal aster types in cells fixed and immunostained for alpha-tubulin (microtubules, green), mitotic DNA (blue), and Asl (centrioles, magenta) after 30 s of warming post cooling from either cnn,grip71,grip163 or cnn,grip71,grip163,msps mutants, as indicated. N numbers are indicated, and each N represents a single cell. Only cells that had centrosomes but that had not yet formed a spindle were analyzed. Note how centrosome asters are frequently absent in cnn,grip71,grip163,msps.

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