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. 2023 Sep 12;222(10):e202212043. doi: 10.1083/jcb.202212043

Figure 4.

Figure 4.

Mitotic centrosomes that fail to accumulate γ-tubulin nucleate microtubules that are cold-resistant. (A–C) Fluorescence images of mitotic Drosophila brain cells from either wild-type third instar larvae, cnn∆CM1,grip71,grip163 mutant third instar larvae, or cnn∆CM1,grip71,grip163 mutant third instar larvae also expressing GFP-PACT and Jupiter-mCherry, immunostained for γ-tubulin (green), mitotic DNA (blue), and Asl (centrioles, magenta). Note that GFP and mCherry fluorescence signals are destroyed during the fixation process due to the addition of acetic acid. 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 either wild-type or cnn∆CM1,grip71,grip163 mutants. Each datapoint represents the average centrosome value from one brain. N = 4 for WT and 5 for cnn∆CM1,grip71,grip163 for both interphase and mitosis. Mean and SEM are indicated. A two-sided paired t test was used to compare mean values of interphase and mitotic centrosomes, showing that there is no accumulation of γ-tubulin at mitotic centrosomes within the cnn∆CM1,grip71,grip163 mutant genotype. (C–E) Fluorescent images (C and D) and graph (E) documenting the behavior of Jupiter-mCherry within living Drosophila control (C) or cnn∆CM1,grip71,grip163 mutant (D) third instar larval brain cells as they were cooled to 5°C for 5 min and then rapidly warmed to 20°C. Time in seconds relative to the initiation of warming (0 s) is indicated. Note that the GFP-PACT signal used to locate centrosomes is not displayed. The graph in E plots the mean and SEM centrosomal signal (after subtraction of cytosolic signal) of 12 and 11 centrosomes from 8 and 9 control and cnn∆CM1,grip71,grip163 mutant cells, respectively. The data is normalized to the average signal at centrosomes in control cells prior to cooling. Note that a relatively large fraction of the centrosomal Jupiter-mCherry signal remains at centrosomes during cooling in cnn∆CM1,grip71,grip163 mutant cells, showing that the microtubule nucleated by these centrosomes are very cold-resistant. (F and G) Graphs show the depolymerization (F) and nucleation/regrowth phases (G) phases from the graph in E. One-phase exponential decay models and “exponential plateau” models generated in GraphPad Prism are fitted. The fits were compared using an extra sum-of-squares F test. Note how the centrosomal Jupiter-mCherry signal decreases faster upon cooling, but increases slower upon warming, in cnn∆CM1,grip71,grip163 mutant cells.