Figure 1.
Cnn recruits γ-tubulin complexes via its CM1 domain and independently of Grip71 and the GCP4/5/4/6 core. (A) Fluorescence images of either interphase or mitotic Drosophila brain cells from either wild-type, spd-2 mutant, or spd-2, grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant third instar larval brains immunostained for γ-tubulin (green), mitotic DNA (blue), and Asl (centrioles, magenta). Both mutants carry the mutant spd-2 alleles to reveal the Cnn pathway of recruitment. The scale bar is 5 μm and applies to all images. (B) Graph showing average centrosomal fluorescence intensities of γ-tubulin (relative to wild type) of interphase (blue dots) and mitotic (black dots) centrosomes from different genotypes (as indicated below). Each datapoint represents the average centrosome value from one brain. N = 5 for each condition. Mean and SEM are indicated. A one-way ANOVA with a Sidak’s multiple comparisons test was used to make the comparisons indicated by P values in the graph. Note that there is only a small reduction in mitotic centrosomal γ-tubulin levels in spd-2 mutants and in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutants, showing that Cnn can still efficiently recruit γ-tubulin complexes to mitotic centrosomes when only γ-TuSCs are present. (C) Fluorescence images of either interphase or mitotic Drosophila brain cells from either wild-type or cnn∆CM1,spd-2 mutant third instar larval brains immunostained for γ-tubulin (green), mitotic DNA (blue), and Asl (centrioles, magenta). The scale bar is 5 μm and applies to all images. (D) The graph is in the same format as in B, revealing no significant increase of centrosomal γ-tubulin signal from interphase to mitosis in cnn∆CM1;spd-2 mutant cells, showing that Cnn requires its CM1 domain to recruit γ-tubulin complexes to centrosomes. Two-sided paired t tests were used to compare mean values of interphase and mitotic centrosomes within each genotype. (E) Multiprotein sequence alignment of part of the CM1 domain containing the key binding residues (indicated by red boxes) in budding yeast and humans that we mutated in Drosophila. (F and G) Western blots probed for MBP and γ-tubulin showing the results of IP experiments from wild-type embryo extracts using bacterially purified MBP-tagged N-terminal (aa1–255) Cnn fragments containing point mutations to relieve Cnn-C autoinhibition (T27E and S186D; Tovey et al., 2021) and to perturb the CM1 domain’s ability to bind γ-TuRCs (R101Q, E102A, and F115A). Source data are available for this figure: SourceData F1.

Cnn recruits γ-tubulin complexes via its CM1 domain and independently of Grip71 and the GCP4/5/4/6 core. (A) Fluorescence images of either interphase or mitotic Drosophila brain cells from either wild-type, spd-2 mutant, or spd-2, grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutant third instar larval brains immunostained for γ-tubulin (green), mitotic DNA (blue), and Asl (centrioles, magenta). Both mutants carry the mutant spd-2 alleles to reveal the Cnn pathway of recruitment. The scale bar is 5 μm and applies to all images. (B) Graph showing average centrosomal fluorescence intensities of γ-tubulin (relative to wild type) of interphase (blue dots) and mitotic (black dots) centrosomes from different genotypes (as indicated below). Each datapoint represents the average centrosome value from one brain. N = 5 for each condition. Mean and SEM are indicated. A one-way ANOVA with a Sidak’s multiple comparisons test was used to make the comparisons indicated by P values in the graph. Note that there is only a small reduction in mitotic centrosomal γ-tubulin levels in spd-2 mutants and in spd-2,grip71,grip75GCP4,grip128GCP5-RNAi,grip163GCP6 mutants, showing that Cnn can still efficiently recruit γ-tubulin complexes to mitotic centrosomes when only γ-TuSCs are present. (C) Fluorescence images of either interphase or mitotic Drosophila brain cells from either wild-type or cnn∆CM1,spd-2 mutant third instar larval brains immunostained for γ-tubulin (green), mitotic DNA (blue), and Asl (centrioles, magenta). The scale bar is 5 μm and applies to all images. (D) The graph is in the same format as in B, revealing no significant increase of centrosomal γ-tubulin signal from interphase to mitosis in cnn∆CM1;spd-2 mutant cells, showing that Cnn requires its CM1 domain to recruit γ-tubulin complexes to centrosomes. Two-sided paired t tests were used to compare mean values of interphase and mitotic centrosomes within each genotype. (E) Multiprotein sequence alignment of part of the CM1 domain containing the key binding residues (indicated by red boxes) in budding yeast and humans that we mutated in Drosophila. (F and G) Western blots probed for MBP and γ-tubulin showing the results of IP experiments from wild-type embryo extracts using bacterially purified MBP-tagged N-terminal (aa1–255) Cnn fragments containing point mutations to relieve Cnn-C autoinhibition (T27E and S186D; Tovey et al., 2021) and to perturb the CM1 domain’s ability to bind γ-TuRCs (R101Q, E102A, and F115A). Source data are available for this figure: SourceData F1.

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