PMC

S2 cells lack a γ-tubulin–containing MTOC during interphase. (A–F) S2 cells stained for MTs (red), γ-tubulin (green), and phosphohistone H3 (blue). Representative cells in early prophase (A), metaphase (B and E), telophase (C), and interphase (D and F). (E and F) γ-tubulin-GFP (green)-expressing stable line. Scale, 5 μm.

Drosophila embryos lack a γ-tubulin–containing MTOC during interphase. An embryo in the process of dorsal closure was fixed and stained for α-tubulin (A) and γ-tubulin (B). Inset shows embryo model with red box showing relative orientation of images in A and B. Three different cell populations are boxed in A, corresponding to G2-arrested amnioserosa cells (1), G1-arrested leading-edge cells (2) and epithelial cells undergoing mitosis (3). Boxed regions are displayed at higher magnification. Insets show a bipolar spindle from a mitotic domain of a younger developing embryo with γ-tubulin labeling at the poles.

Interphase microtubule nucleation is independent of centrioles and γ-tubulin. (A–F) Projections of GFP-SAS-6 (red) and EB1-mRFP (green) MT growth patterns visualized by merging short segments of a 1-min time-lapse recording into a single flattened image. Tracks of SAS-6 spots in the panels correspond to their movement over time. Acentriolar MT nucleation occurs in interphase S2 cells (A) but is associated with centrioles during mitosis (B). In embryos, MT nucleation (EB1-GFP; green) is not associated with centrioles (mCherry-SAS-6; red) in interphase amnioserosal (C) or leading edge (D) cells but is associated with spindle poles in mitotic domains (E). White dotted-line (C) traces the cell border. (F) A similar pattern of MT growth is observed after γTub23C RNAi. Scale, 5 μm (A, B, and F); 10 μm (C–E).

CLIP-190 or Msps RNAi delays MT regrowth in S2 cells but is not observed with either MAST RNAi or codepletion with γ-tubulin. (A) Distribution histograms of total integrated MT fluorescence intensity at steady state in 5000 RNAi-treated α-tubulin–immunolabeled S2 cells using quantitative HTM; (y-axis) cell number, (x-axis) arbitrary units. Approximately 5000 cells were scanned in each treatment, which produced a significant difference (p < 0.05) compared with the control RNAi using a nonparametric Kruskal-Wallis one-way analysis of variance followed by a Dunn's post-test. (B) Time points show representative day 6 RNAi-treated S2 cells stained for MTs during MT regrowth. Each row of micrographs is aligned with the dsRNA(s) that were used in A. Scale, 10 μm.

Centrioles selectively recruit γ-tubulin during mitosis but not interphase. GFP-SAS-6 (green)-expressing S2 cells in metaphase (A) or interphase (B) stained for γ-tubulin (red) and phosphohistone H3 (blue). GFP-SAS-6 localizes to a small spot inside a ring of γ-tubulin during mitosis (A, insets). 100% of mitotic γ-tubulin “spheres” contained GFP-SAS-6 (n = 100). Scale, 5 μm. Embryonic leading edge cells (C), dividing epithelial cells (D), amnioserosa cells (E), and an embryonic dividing neuroblast (E), stained for D-PLP (green), γ-tubulin (red), and actin (blue). Yellow arrowheads mark centriole positions. Cell borders are traced (blue). Scale, 10 μm. Transmission electron micrographs of S2 cells during mitosis (G) and interphase (H and I). A dense MT array radiates from a centriole pair (yellow arrowhead) during prophase (G; N, nucleus), higher magnification shown in G′. MTs (black arrowheads) do not emanate from centrioles during interphase (H and I); higher magnifications are shown in H′ and I′.

EB1 or cytoplasmic dynein (DHC) RNAi delays MT regrowth in S2 cells but is not observed with either depletion of the p150^Glued component of dynactin or codepletion with γ-tubulin. (A) Distribution histograms of total integrated MT fluorescence intensity at steady state in RNAi-treated α-tubulin–immunolabeled S2 cells using quantitative HTM; (y-axis) cell number, (x-axis) arbitrary units. Approximately 5000 cells were scanned in each treatment that produced a significant difference (p < 0.05) compared with the control RNAi using a nonparametric Kruskal-Wallis one-way analysis of variance followed by a Dunn's post-test. (B) Time points show representative day 6 RNAi-treated S2 cells stained for MTs during MT regrowth. Each row of micrographs is aligned with the dsRNA(s) that were used in A. Scale, 10 μm.

MT regrowth occurs independently of centrioles but is delayed in γ-tubulin–depleted S2 cells. (A) S2 cells were stained for MTs (green), D-PLP centrioles (red), and DNA (blue) at specific time points in a MT regrowth assay. MTs were depolymerized by cold treatment (0 min.) and brought to room temperature to allow polymerization. Cells were fixed at 5, 10, and 15 min. Centrioles (white arrowheads) are shown at higher magnifications in bottom panels (left to right; MTs, D-PLP, merge). A cell in cytokinesis is shown at 5 min with disengaged centrioles (insets). An interphase cell at 5 min contains individual MTs (yellow arrowheads) and tufts of MT foci (yellow arrow). Scale, 10 μm. (B) Mitotic (top) and interphase (bottom) S2 cells after 5 min of MT regrowth stained for MTs (green), γ-tubulin (red, insets), and DNA (blue). The white box (interphase cell) is shown at higher magnification (bottom panel) and denotes a cluster of MT foci (left to right; MTs, γ-tubulin, merge). (C) An S2 cell at 5 min of MT regrowth immunostained for MTs (green), Golgi (red), and DNA (blue). Higher magnifications of (1) individual MTs in the lamella, (2) MT foci, (3) Golgi associated with MT foci, and (4) Golgi punctae along a MT bundle. Scale, 10 μm. (D) Time points show day 7 control and γ-Tub23C/37C RNAi-treated S2 cells stained for MTs during MT regrowth. Scale, 10 μm. (E) Quantitation of MT recovery in the RNAi-treated cells displayed in D.

RNAi-mediated codepletion of γTub23C/37C does not affect steady-state MT levels in interphase S2 cells. (A) Immunofluorescence of interphase and mitotic transfected S2 cells expressing γTub37C-GFP (green) and stained for D-PLP (red) and DNA (blue). The right spindle pole in the mitotic cell is not in the focal plane. Arrowheads mark centriole positions. (B) Lysates were prepared from γ-Tub37C-GFP stable-expressing S2 cells after 7 d of control or γ-tubulin RNAi treatments and probed by Western blot using anti-γ-tubulin antibody (anti-α-tubulin was used as a loading control). (C) Bar graph shows the frequency of monopolar spindle formation after 7-d control, γ-tubulin, or MAST RNAi treatments and identified by MT staining (inset; scale, 5 μm). (D) Interphase day 7 control and γTub23C/37C RNAi-treated S2 cells stained for MTs. (E) Distribution histograms of total integrated MT fluorescence intensity in RNAi-treated α-tubulin–immunolabeled S2 cells using quantitative HTM; (y-axis) cell number, (x-axis) arbitrary units. Examples of these cells are shown in D. γ-tubulin RNAi treatments produced no significant difference (p > 0.05) as compared with control RNAi using a nonparametric Kruskal-Wallis one-way analysis of variance followed by a Dunn's post test.