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Domains of the Pavarotti kinesin-like protein that direct its subcellular distribution: effects of mislocalisation on the tubulin and actin cytoskeleton during Drosophila oogenesis

Cancer Research Campaign Cell Cycle Genetics Group, University of Cambridge, Department of Genetics, Downing Street, Cambridge CB2 3EH, UK

View larger version (43K): [in a new window]   Fig. 2. Wild-type and mutant forms of Pav-KLP introduced into the Drosophila germ line under UAS control. (A) Different GFP-tagged Pavarotti variants were subcloned into the Drosophila transformation vector pUASp for expression in the female germ line. Regions subjected to site-directed mutagenesis are indicated in yellow boxes. Linking amino acids between the GFP tag and domains of Pavarotti are represented as a blue bar. The first ATG to be translated is given in green. The proteins that derive from these constructs and are used in the text and figures are given below each drawing. (B) Western blots to indicate levels of expression of the indicated constructs in ovaries when driven by GAL4-VP16 under control of the maternal 4-tubulin promoter as described in the text.

View larger version (134K): [in a new window]   Fig. 1. Comparison of the immunolocalisation of Pav-KLP (A,C) with the localisation of GFP-tagged Pav-KLP expressed from the poly ubiquitin promoter (B,D). (A,B) In early cysts both Pav-KLP and GFP-Pav-KLP accumulate in the ring canals that connect either the germ cells (arrow in A, large arrow in B) or the follicular cells (arrowheads in A and B) with each other. GFP-Pav-KLP is also present in germ cell nuclei (small arrow in B). (C,D) At a later stage, both Pav-KLP and GFP-Pav-KLP concentrate in the oocyte nucleus (arrowhead in C and large arrowhead in D) and persist in the ring canals (large arrow in C, not visible in D). Nurse cell nuclei accumulate much higher concentrations of GFP-Pav-KLP (arrow in D) than Pav-KLP (small arrow in C). GFP-Pav-KLP is also found along actin filament bundles from stage 10B through 11 (small arrowheads in D). Bars, 20 µm (A, B, C) and 50 µm (D).

View larger version (52K): [in a new window]   Fig. 3. Defects in oogenesis resulting from overexpression of wild-type Pav-KLP. Live (A,B,D), Hoechst-stained (C) or immunostained (E-H) egg chambers expressing GFP-Pav-KLP (green) are shown. DNA is shown in red (E-H) and Staufen in blue (G-H). (A) Stage 5 oocyte with GFP-Pav-KLP in speckles within nurse cell nuclei (arrow); GFP-Pav-KLP is also present in ring canals (arrowhead). The oocyte nucleus appears to contain a filamentous network (dashed box and arrowhead in inset). (B) An egg chamber in which the oocyte nucleus (small arrowhead) is being pushed towards the posterior pole by an invading nurse cell (small arrow), which has been able to pass the nurse cell/oocyte border (large arrow). The large arrowhead points at one of the four ring canals that are normally located at the nurse cell/oocyte border. (C) Egg chamber in which the oocyte at the posterior half has been invaded by three nurse cell nuclei (arrowheads). (D) Dumpless egg chambers with nurse cells that have retained most of their cytoplasm (arrowhead). Although dorsal appendage follicle cells have migrated more or less normally, the appendages (revealed by auto-fluorescence) are broad and irregular in shape (arrow). (E,F) Egg chambers isolated from females fed for 24 hours on unsupplemented yeast paste (E) or yeast paste containing 1 mg/ml colchicine (F). Egg chambers isolated from untreated females contain nurse cells that have a clearly visible nuclear GFP-Pav-KLP network (arrow in E). A similar network is also associated with the oocyte nucleus (arrowhead in E). In colchicine-treated egg chambers GFP-Pav-KLP is still present in nurse cell nuclei, but it does not form filaments any more (arrow in F). The network associated with the oocyte nucleus has also collapsed (arrowhead in F). (G) Overexpression of GFP-Pav-KLP can lead to egg chambers, in which the oocyte (arrow) fails to localise to the posterior pole (asterisk). (H) An egg chamber in which one nurse cell (arrow) has passed the nurse cell/oocyte border without affecting Staufen localisation at the posterior pole of the oocyte (arrowhead). Bars, 20 µm (A), 50 µm (C,E-H), 100 µm (B) and 200 µm (D).

View larger version (30K): [in a new window]   Fig. 4. Localisation of the GFP-tagged stalk domain of Pav-KLP to ring canals. An egg chamber expressing GFP-PavSTALK (green) and stained for Hu-li tai shao (red) and DNA (blue). GFP-PavSTALK appears to be localised at the outer side or ring canals, whereas Hu-li tai shao is more concentrated at the inner side. Regions where the two proteins overlap appear yellow. Bar, 10 µm.

View larger version (74K): [in a new window]   Fig. 5. Stabilisation of cytoplasmic microtubules and disorganisation of nurse cell positioning following overexpression of GFP-tagged PavDEAD or PavNLS(4-7)*. Live (A,B) or fixed (C-F) egg chambers expressing either GFP-PavNLS5* (A), GFP-PavNLS(4-7)* (B,C) or GFP-PavDEAD (D,F) (green). DNA was stained with TOTO-3 (blue, C,D) or Hoechst (E-F). (A) High concentrations of GFP-PavNLS5* are present in the germ cell nuclei (arrow) and in ring canals (arrowhead). (B) GFP-PavNLS(4-7)* is excluded from the nurse cell nuclei (arrow) and accumulates in the nurse cell cytoplasm (arrowhead). (C,D) Egg chambers in which nurse cell nuclei (arrowheads) are clustered and surrounded by a dense filamentous network (arrows) associated with GFP-PavNLS(4-7)* (C) or GFP-PavDEAD (D). (E) A wild-type egg chamber in which the follicular cells have started to migrate to the posterior pole (arrow). The 15 polyploid nurse cell nuclei are all localised outside of the oocyte at the anterior half of the egg chamber (arrowhead). (F) An egg chamber containing mispositioned nurse cells (arrowheads) following GFP-PavDEAD expression. Bars, 20 µm (A,B,D,F) and 50 µm (C,E).

View larger version (116K): [in a new window]   Fig. 7. Overexpression of GFP-PavDEAD results in failure of oocyte growth, loss of interfollicular stalks, fusion of egg chambers and formation of tubulin-actin aggregates. Wild-type (C,H) and mutant egg chambers expressing either GFP-PavDEAD (B,D,I) or GFP-PavNLS(4-7)* (A,E-G) (green). DNA is in blue and Staufen (B), actin (C-G) and the Hu-li tai shao isoform ADD-60 (H-I) in red. (A) A linear projection of a series of confocal images of a germarium isolated from females expressing GFP-PavNLS(4-7)*. The earliest visible GFP fluorescence appears in ring canals of cysts located in region 2a. The cyst located in region 2b contains 16 cells, whereas the stage 1 cyst in region 3 is composed of 32 cells. (B) A young egg chamber with its oocyte localised at the anterior pole (arrowhead). Asterisk, posterior pole. (C) Nurse cells of wild-type egg chamber accumulate actin at their cortex, facilitating the identification of their cell borders (arrowhead). Oocytes (OC) always have four associated ring canals and appear to have a higher concentration of actin at their cortex. They are in direct contact with the follicle cell (FC) layer (arrow). (D) Young mutant egg chambers are not separated by inter-follicular stalks (small arrow). Nurse cells still accumulate actin at their cell borders (arrowhead). The large arrow indicates that the oocyte (OC) is not in contact with the follicular cell layer. (E) A single follicle cell (FC) layer surrounds a 32-cell cyst with two oocytes (OC). All the nurse cells (NC) are of equal size. (F) A single follicle cell layer (FC) surrounds a 32-cell mutant egg chamber with two distinct cysts of 16 cells (1 and 2). Cyst 1 contains some collapsed ring canals (RC) and nurse cells that are not separated by an actin cytoskeleton (arrowhead). (G) An egg chamber in which tubulin-actin aggregates (arrow) are embedded in a cytoplasmic pool of microtubules. Nurse cell nuclei appear as black holes. (H) A wild-type egg chamber in which Hu-li tai shao is present in the ring canals (arrowhead). (I) Hu-li tai shao accumulates in the ring canals (arrowhead) and nurse cell membranes (arrow) of mutant egg chambers. Bars, 10 µm (A,E), 20 µm (C,D,F), 50 µm (G-I) and 100 µm (B).

View larger version (90K): [in a new window]   Fig. 6. Overexpression of GFP-PavDEAD stabilises microtubules that have associated Orbit but not Msps protein. Fixed egg chambers expressing GFP-PavDEAD (green). DNA is in blue, and tubulin (A,D), Orbit (B,E) and Mini spindle protein (C,F) in red. (A-F) Extensive regions of colocalisation of tubulin (A) and GFP-PavDEAD are apparent and appear yellow in the merged image (D). Orbit (B,E) but not Mini spindle protein (C,F) is found along the microtubules (arrows in B,C,E,F). (G-I) Egg chambers isolated from females fed for 24 hours on unsupplemented yeast paste (G) or yeast paste containing 50 µg/ml (H) or 1 mg/ml (I) colchicine. (G) Overexpression of GFP-PavDEAD leads to a cytoplasmic network within the nurse cells (arrow) but not the oocyte (arrowhead). (H) Feeding females 50 µg/ml colchicine for 24 hours results in a partial depolymerisation of the network in the cytoplasm but not at the cortex (arrow). (I) Increasing the colchicine concentration to 1 mg/ml depolymerises the network almost completely, leaving just a thin ring of filaments around the nurse cell nuclei (arrow). Arrowheads in H and I point at the oocyte. Bars, 10 µm (E,F) and 25 µm (D,G-I).