Fig 1.
Drosophila cells without centrioles can form acentriolar microtubule organizing centers (aMTOCs).
(A) Drosophila larval brain cells were stained with antibodies against Ana1 (green) to mark the centrioles, Cnn (red) to mark the PCM and α-tubulin (white) to mark the spindle. DNA is in blue. Sas-4 mutants do not have centrioles as indicated by the lack of Ana1 on the spindle poles, but often have some Cnn detectable at either one (middle panel) or both (lower panel) of their mitotic spindle poles (arrows). WT and Sas-4 cells were recorded using identical microscope settings, visualising the much fainter staining of Cnn on spindle poles in Sas-4 mutant cells. Scale bars represent 5μm. (B) Quantification of Cnn recruitment to mitotic spindle poles in WT and Sas-4 mutant cells (as judged by Cnn staining on at least one spindle pole) shows that Cnn staining on spindle poles can be detected in 100% ± 0 of WT cells and 55.93% ± 3.16 of Sas-4 cells. A minimum of 120 cells from 4 brains were quantified per genotype. Data are expressed as mean±SEM with average per brain representing one data point. (C, D) Quantification of Cnn intensity (C) and the size of the Cnn foci (D) in WT or Sas-4 mutant Drosophila brain cells stained with anti-Ana1 as centriole marker, anti-α-tubulin to visualise spindles and anti-Cnn. Cnn foci on 20 different spindle poles were analysed for each genotype. Data are expressed as mean±SEM with Cnn staining on each spindle pole representing one data point. P-values were calculated using a Mann-Whitney test.
Fig 2.
The formation of aMTOCs in living third instar larval brain cells.
(A) Panels show images from time-lapse movie illustrating spindle formation in Sas-4 mutant larval brain cells expressing GFP-Cnn (red) and Jupiter-mCherry (green). Time (secs) is indicated relative to nuclear envelope breakdown (NEBD). GFP-Cnn foci form in the cytoplasm and nucleate microtubules a few minutes prior to NEBD (arrowheads). These subsequently cluster at the spindle poles by metaphase (arrows). aMTOCs were present at metaphase in 54% of cells filmed (n = 55). aMTOCs are present in prometaphase and metaphase images from Sas-4 mutant brain cells expressing (B) Spd-2-GFP (red) and Jupiter-mCherry (green) (46%, n = 13), (C) Asl-GFP (red) and Jupiter-mCherry (green) (46%, n = 24), (D) γ-Tubulin-GFP (red) and Jupiter-mCherry (green) (54%, n = 59). All scale bars represent 5μm.
Fig 3.
The PCM recruitment factor Asl is essential for aMTOC formation.
(A) Larval brain cells of acentriolar Drosophila mutants were stained with antibodies against α-tubulin (green) and Cnn (red). DNA is in blue. aMTOCs are indicated by the arrows; note the absence of aMTOCs in asl mutants. (B) Quantification of aMTOC formation in mitotic brain cells (as judged by Cnn staining on at least one spindle pole) in acentriolar mutants shows that Cnn staining on spindle poles can be detected in 100% ± 0 of WT cells, 55.93% ± 3.16 of Sas-4 cells, 60.46% ± 2.42 of ana2 cells, 79.22% ± 3.87 of Sas-6 cells and 0.68% ± 0.44 of asl cells. (A minimum of 120 cells from 4 brains were quantified per genotype). Data are expressed as mean±SEM with average per brain representing one data point. P-values were calculated using a Mann-Whitney test. (C) Spindle formation was followed in live asl mutant brain cells. No aMTOC formation can be observed in asl mutants throughout mitosis in stocks expressing Jupiter-mCherry (green) and either GFP-cnn (red) (n = 21), Spd-2-GFP (red) (n = 12) or γ-Tubulin-GFP (red) (n = 17). All scale bars represent 5μm.
Fig 4.
The PCM scaffolding proteins Spd-2 and Cnn are required for efficient aMTOC formation.
(A) Sas-4 mutant cells and Spd-2 Sas-4 and cnn; Sas-4 double mutant cells were stained with antibodies against Cnn (red) and γ-tubulin (green). DNA is in blue. Scale bar represents 5μm. The aMTOCs present in Sas-4 and Spd-2 Sas-4 mutant cells are indicated by arrows. (B) Quantification of aMTOC formation in mitotic brain cells (as judged by γ-tubulin staining on at least one spindle pole) shows that γ-tubulin staining on spindle poles can be detected in 50.69% ± 0.83 of Sas-4 mutant cells, 27% ± 4.33 of Spd-2 Sas-4 double mutant cells and 1.23% ± 0.67 of cnn; Sas-4 double mutant cells (a minimum of 80 cells from 3 brains were quantified per genotype). (C) Quantification of Cnn recruitment to mitotic spindle poles shows that Cnn staining on spindle poles can be detected in 50.69% ± 0.83 of Sas-4 mutant cells and 27% ± 4.33 of Spd-2 Sas-4 double mutant cells. (A minimum of 137 cells from 4 brains were quantified per genotype). All data are expressed as mean±SEM with average per brain representing one data point. P-values were calculated using a Mann-Whitney test.
Fig 5.
aMTOCs do not detectably contribute to spindle assembly in the absence of centrosomes.
(A) WT, asl and Sas-4 neuroblasts expressing GFP-PACT to mark centrioles and Jupiter-mCherry to visualise spindle formation were filmed to compare spindle assembly dynamics (asl is shown here as an example, time (secs) is relative to NEBD). Note how the GFP-PACT signal present in the nucleus at the first timepoint (-240s) is lost at NEBD. Scale bar represent 5μm. (B) Time between NEBD and anaphase onset was measured for WT (n = 27; 494s ± 35s), Sas-4 (n = 50; 665s ± 25.6s) and asl (n = 18; 637s ± 36s) cells. Spindle assembly takes significantly longer in the mutant cells, but does not vary significantly between Sas-4 and asl mutants indicating that aMTOCs do not increase the efficiency of spindle assembly. Data are expressed as mean±SEM. P-values were calculated using a Mann-Whitney test.
Fig 6.
aMTOCs aid formation of monopolar spindles in acentriolar cells that also lack misato.
(A) Larval brain cells of WT, mst; Sas-4 and mst; asl double mutants were stained with antibodies against α-tubulin (red) and Cnn (green), DNA is in blue. mst; Sas-4 cells have aMTOCs (arrow) on the poles of monopolar spindles. Scale bar represents 5μm. (B) Quantification of monopolar spindles in mst; Sas-4 and mst; asl cells (a minimum of 263 cells in 8 brains were quantified per genotype). In mst; Sas-4 mutants 59.32% ± 3.81 of cells form monopolar spindles while in mst; asl only 25.15% ± 3.34 of cells form monopolar spindles (the remaining cells do not exhibit any monopolar or bipolar spindles). (C) Quantification of regrowth of monopolar spindles after cold-induced depolymerisation at different time points after release from cold treatment. In mst; Sas-4 brains 0% ± 0 of cells formed monopolar spindles at 0 minutes, 8% ± 4.25 at 2 minutes, 12.5% ± 7.89 at 5 minutes, 24.24% ± 6.1 at 10 minutes, 38.27% ± 3.24 at 20 minutes and 47.96% ± 3.09 at 30 minutes. In mst; asl brains 4.7% ± 2.62 of cells formed monopolar spindles at 0 minutes, 3.03% ± 1.55 at 2 minutes, 8.93% ± 2.9 at 5 minutes, 2% ± 3.93 at 10 minutes, 0% ± 0 at 20 minutes and 3.74% ± 2.74 at 30 minutes (a minimum of 43 cells from 4 brains were quantified for each timepoint). All data are expressed as mean±SEM with average per brain representing one data point. P-values were calculated using a Mann-Whitney test.
Fig 7.
aMTOCs are essential for acentriolar spindle pole focusing by dynein.
(A) Larval brain cells of Sas-4 ncd and asl ncd were stained with antibodies against Cnn (green) and α-tubulin (red). DNA is in blue. The different spindle phenotypes observed are illustrated; aMTOCs are present in Sas-4 ncd mutant cells (arrow). (B) Quantification of bipolar spindle formation ability in mitotic cells of different mutants (a minimum of 70 cells from 5 brains were quantified per genotype). 82.74% ± 2.94 of WT cells, 55.24% ± 8.11 of Sas-4 cells, 47.08% ± 4.5 of asl cells, 83.65% ± 5.58 of ncd cells, 25.5% ± 6.47 of Sas-4 ncd cells, 4% ± 2.36 of asl ncd cells, 0% ± 0 of cnn; Sas-4 ncd cells, 0.73% ± 0.55 of Sas-4 ncd dhc cells, 71.95% ± 2.39 of dhc cells, 51.71% ± 5.67 of Sas-4 dhc cells and 2.72% ± 1.8 of ncd dhc cells formed bipolar spindles. All data are expressed as mean±SEM with average per brain representing one data point. P-values were calculated using a Mann-Whitney test. aMTOCs cooperating with dynein ameliorate the spindle formation defects observed in the absence of centrioles and Ncd (for a more detailed explanation see main text). Note that Sas-4 and asl have fewer mitotic cells with formed spindles as spindle formation takes longer in the absence of centrioles (Fig 4). (C) Sas-4 ncd dhc triple mutant cells stained with antibodies against Cnn (green) and α-tubulin (red); these cells have a few misformed spindles with splayed spindle poles. (D) WT and Sas-4 mutant cells stained with antibodies against Cnn (green) and Dynein light intermediate chain (red); Dynein localises to centrosomal MTOCs and aMTOCs (arrows). (E) Prometaphase and metaphase images from Sas-4 mutant brain cells expressing Dlic-GFP (red, localising to aMTOCs as marked by the arrows) and Jupiter-mCherry (green) (The punctate signals in the middle region of the cell in prometaphase are likely to be localisation of Dlic-GFP to kinetochores). All scale bars represent 5μm.
Fig 8.
Acentriolar spindle pole focusing by dynein can ameliorate proliferation defects observed in the absence of centrioles and Ncd.
(A) Panels show third instar larval brains from WT and different mutant strains. Note how asl ncd and Sas-4 ncd dhc brain lobes are much smaller than WT or Sas-4 ncd brains and lack fully developed imaginal discs. (B) Quantification of third instar larval brain size in WT, Sas-4 ncd, asl ncd and Sas-4 ncd dhc 3rd instar wandering larvae. Brain size was assessed by measuring brain lobe circumference and calculating brain lobe volume under the assumption that the lobes were spherical. Each data point represents the averaged brain lobe volume of each two lobes of a brain (a minimum of 8 brains were analysed per genotype). A Mann-Whitney test shows that brains that lack aMTOCs and Ncd (asl ncd) or have aMTOCs but lack dynein and Ncd (Sas-4 ncd dhc) brains are both significantly smaller than Sas-4 ncd brains (that lack Ncd but have aMTOCs and dynein) suggesting that acentriolar spindle focusing by aMTOCs and dynein can ameliorate the proliferation defects observed in the absence of Ncd and centrioles.
Fig 9.
A model for acentriolar spindle pole focusing by the molecular motors dynein and Ncd.
(A) In Drosophila cells with centrosomes K fibers (light blue) are thought to be predominantly crosslinked by the minus end-directed motor Ncd (red), while the minus-end directed motors dynein (purple) transports the K fibers along centrosomal MTs (light green) to the centrosome [9]. (B) In cells lacking centrosomes, Ncd can still focus acentriolar spindle poles by crosslinking the K fibers (as, for example, in an asl mutant cell). (C) Loss of Ncd in acentriolar cells which also lack aMTOCs leads to severe impairment of spindle focusing as dynein cannot crosslink MT minus ends (as, for example, in asl ncd mutant cells). (D) In acentriolar cells with aMTOCs (as, for example, in Sas-4 ncd mutant cells), dynein can transport K fibers towards aMTOCs and so allow some spindle pole focusing even in the absence of Ncd.