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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00616

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The role of PAR-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium

Hélène Doerflinger, Richard Benton^*, Joshua M. Shulman and Daniel St Johnston

The Wellcome Trust/Cancer Research UK Institute and the Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (100K): [in a new window]   Fig. 1. Loss of PAR-1 causes partially penetrant defects in follicle cell polarity. In all the figures, the follicular epithelium is shown with its apical side (which faces the oocyte) towards the top of the picture and its basal side towards the bottom. (A) Stage 6 egg chamber containing a par-1 mutant clone induced early in oogenesis, marked by the loss of nuclear GFP (in this and all subsequent figures of clones, GFP is shown in the first column, and is shown in green in the merged images in the third column). Mutant cells lose their epithelial organisation, and fail to localise DaPKC apically (centre panel: red in merged image). (B) A stage 10a egg chamber containing a smaller clone induced later in oogenesis, showing normal epithelial organisation and DaPKC localisation. Note that the nuclei are no longer in a consistent position in mutant cells. (C) Stage 9 egg chamber containing three mutant cells stained for the apical marker Neurotactin (Nrt). Most mutant cells in small clones show a wild-type apical localisation of Nrt (top right mutant cell), but some cells show reduced localisation (middle) or no localisation at all (bottom left). (D) Stage 9 egg chamber containing a small mutant clone stained for Notch, which localises apically as in wild type, even when the mutant cells form a double layered epithelium.

View larger version (62K): [in a new window]   Fig. 4. Loss of PAR-1 destabilises MTs. (A) A par-1 clone in a stage 10 egg chamber that has been treated with the MT-depolymerising drug, colcemid, and then fixed under optimised conditions and stained for -Tubulin. The par-1 mutant cells lack MTs, whereas the wild-type cells show a similar MT organisation to untreated cells. (B) Cold shock for 1 hour leads to the disappearance of the MTs in par-1 clones. Note that the cold shock also reduces the nuclear localisation of nls-GFP, and partially disperses the cortical bundles of MTs in wild-type cells. (C) Cold shock followed by 5 minutes of recovery at room temperature. The MTs have started to re-grow in mutant cells, but are still less dense than in the wild-type cells. (D) Cold shock followed by 10 minutes of recovery at room temperature. The mutant cells now contain more MTs than wild-type cells, as in untreated ovaries. GFP, green; -Tubulin, red.

View larger version (113K): [in a new window]   Fig. 2. ß-spectrin and F-actin are enriched laterally in par-1 clones. (A) ß-Spectrin localisation in a stage 9 egg chamber containing two small par-1 clones. ß-Spectrin still localises to the lateral cortex of mutant cells, but is present in higher amounts than in wild-type cells. (B,C) Small clones in stage 10 egg chambers in which F-actin has been labeled with rhodamine-phalloidin. Mutant cells show an increase in F-actin along the lateral cortex. (B) Sagittal view. (C) Horizontal view at the level of the nuclei. GFP, green; ß-Spectrin or Actin, red.

View larger version (56K): [in a new window]   Fig. 3. MT density is increased in all par-1 mutant clones. (A) A stage 10 egg chamber containing two small par-1 mutant clones, fixed under optimised conditions for preserving MTs (8% PFA) and stained for -Tubulin. The mutant cells contain more MTs than the adjacent wild-type cells. (B) Expression of a GFP-PAR-1 fusion protein rescues this phenotype. The mutant cells that express the transgene can be identified by the presence of lateral GFP-PAR-1 signal and by the loss of nuclear GFP, and show a MT network that is similar to that in the neighbouring wild-type cells. (C) Under standard fixation conditions (4% PFA), par-1 clones appear to lack MTs, suggesting that MTs are less stable than in wild type. (D) Quantification of the intensity of the GFP and the -Tubulin staining in wild-type and mutant follicle cells. Fluorescent signal was measured along the broken white line using the Laser Pix4 software (BioRad). The par-1 mutant cells, which are marked by the decrease in the GFP fluorescence (green line), show twice as much microtubule staining (red line) as the wild-type cells. GFP, green; -Tubulin, red.

View larger version (69K): [in a new window]   Fig. 5. The loss of PAR-1 alters the distribution of MT plus-ends. (A) A par-1 clone in a stage 10 egg chamber in which the MT minus end marker, Nod:ß-Gal, is expressed in the follicle cells. Nod:ß-Gal localises apically in both mutant and wild-type cells. (B): A par-1 clone in a stage 10 egg chamber in which the MT plus-end marker, Kin:ß-Gal, is expressed in the follicle cells. Kin:ß-Gal localises basally in wild-type cells, but accumulates in the centre of mutant cells. GFP, green; ß-Gal, red. (C) -Tubulin stainings in wild-type follicle cells (WT) show MTs extending from the apical to the basal cortex, with a lower density along the basal membrane. par-1 mutant follicles cells (par-1-) show some MTs along their basal membrane.

View larger version (51K): [in a new window]   Fig. 6. Identification and characterisation of Drosophila Tau as a candidate PAR-1 substrate. (A) Organisation of the tau locus, showing the intron/exon structure of tau (UTRs in white) and the location of the S10 gene and the EP element insertions within the first intron. The position of the deficiency uncovering tau, Df(3R)MR22(tau) is shown below. (B) Domain structure of human and Drosophila Tau, illustrating the percent identity (similarity) between the N-terminal projection domains and the MTBD repeats (in grey); an alignment of these repeats is shown below. The putative PAR-1 target serine within the KXGS motif is conserved in four of the five Drosophila repeats (arrowhead). (C) Western blot of 12-18 hour and tau mutant embryos probed with an antibody raised against the MT-binding domain of Drosophila Tau. (D) MT spin-down assay, revealing cosedimentation of Tau with Taxol-induced polymerised Tubulin in the pellet (P) fraction. In the absence of Taxol, both remain in the supernatant (S). (E) MT localisation of Tau:GFP in a living Drosophila ovary. (F) PAR-1 kinase assay with GFP:PAR-1 immunoprecipitated from ovarian extracts and MBP:Tau MTBD substrates, containing (KXGS) or lacking (KXGA) the four putative PAR-1 target sites. (G) Stage 10 egg chamber containing two large mutant clones for Df(3R)MR22(tau) stained for DaPKC (blue) and -tubulin (red). DaPKC and -tubulin localise normally in tau mutant clones.