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doi: 10.1242/10.1242/dev.00315

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Orbit/Mast, the CLASP orthologue of Drosophila, is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis

¹ Cancer Research UK, Cell Cycle Genetics Research Group, Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EN, UK
² Drosophila Genetic Resource Center, Kyoto Institute of Technology, Sagaippongi-cho, Ukyou-ku, Kyoto, 616-8354, Japan

View larger version (86K): [in a new window]   Fig. 5. Defective spindles fail to associate with fusome in orbit6 mutants. (A-C) Germaria stained with anti-Orbit/Mast (red), anti--tubulin (green) antibodies and TOTO3 to reveal DNA (blue). (A) At metaphase, Orbit/Mast concentrates at spindle poles and localises along spindle MTs in wild-type stem cells. (B) Short bipolar (arrowheads) and monopolar (arrow) spindles seen at the tip of an orbit6 germarium. (C) Multiple short spindles at the tip of an orbit6 germarium. Arrowhead indicates a short bipolar spindle. (D-G) Germaria stained with anti-Orbit/Mast (red), anti--tubulin (green) and anti--spectrin (blue). (D) Wild-type stem cell at metaphase, showing the anterior spindle pole attached to the fusome. (E) Wild-type eight-cell germline cyst at metaphase with the spindles being attached to fusome through one of their poles. (F) Monopolar (arrow) and bipolar spindles that fail to associate with the fusome in an orbit6 germarium. (G) orbit6 germarium showing a monopolar spindle (arrow) close to the very apical region of the germarium, and a poorly organised germline cyst (arrowhead) situated more posteriorly. Both contain fusome pieces of different sizes. Scale bars: 5 µm A-E; 10 µm in F.

View larger version (65K): [in a new window]   Fig. 1. Subcellular localisation of Orbit/Mast during germline cyst divisions. Wild-type germaria were stained with anti-Orbit/Mast (red), anti-HtsF (green) antibodies and TOTO3 to reveal DNA (not shown). (A) Orbit/Mast locates on the spindle remnant (sr) during stem cell cytokinesis. The HtsF associates with the nascent fusome plug (fp), which is situated at the mid-zone of the spindle remnant. The pre-existing fusome (p-f) is spherical and resides at the anterior tip of the stem cell. (B) The stem cell (SC) and cystoblast (CB) are interconnected through an Orbit/Mast containing ring canal (rc) that divides the fusome into two unequal pieces. (C) Metaphase cyst, showing the Orbit/Mast on the spindles and pre-existing fusome (arrowhead). Only two out of four spindles in this cyst are shown. (D) Cyst featuring Orbit/Mast on spindle remnants with fusome plugs (arrow). The pre-existing fusome (arrowhead) is situated in the middle of the cyst. (E,F) Orbit/Mast accumulates in the ring canals surrounding the newly formed fusome plugs. Along the pre-existing fusome, Orbit/Mast shows a more diffuse distribution in addition to its presence in ring like structures. Some fusome plugs have already fused with the pre-existing fusome, while others are still 2-3 µm distant. Scale bar: 5 µm. (G) The dynamic changes in the subcellular localisation of Orbit/Mast and HtsF during a division cycle of a germline cyst. Two-cell cyst at metaphase: the asymmetric nature of this division is determined by the fusome-spindle interaction, as only one of the poles of each spindle is fusome anchored. Orbit/Mast accumulates at the spindle poles and decorates the spindle MTs. It is also present in the fusome and the fusome associated ring canal. Note that one of the cells contains a bigger part of the fusome. Two-cell cyst at telophase: central spindle and contractile ring assembly marks the onset of cytokinesis. Cleavage furrow arrest takes place upon contact with spindle remnants in a four-cell cyst. Orbit/Mast associates with central region of spindle remnants. Concomitantly, the fusome plug formation is initiated as inferred from the accumulation of HtsF between the spindle remnant and contractile ring. Ring canal migration: Orbit/Mast accumulates in the ring canals and to a less extent associates with the fusome plugs. The spindle remnants reorient towards the fusome and the fusome plugs together with their ring canals move towards the pre-existing fusome, changing the cyst geometry from a linear into a rosette configuration. Eventually, the pre-existing fusome fuses with fusome plugs and the spindle remnants disassemble. Once again the bigger fusome part will be retained by the cell that has inherited it during the previous division cycle.

View larger version (62K): [in a new window]   Fig. 2. Diminished levels of Orbit/Mast lead to egg chambers with a reduced number of germline cells. (A) Western blot indicating the reduced levels of Orbit/Mast in total ovarian protein extracts of the indicated genotypes. (B) Western blot showing levels of Hts (Hu-li tai shao) ADD-95 and ADD-87 isoforms in ovarian total protein extracts. The ADD-95 isoform is present at similar levels in all four extracts, while the ADD-87 isoform is absent from orbit5/orbit5 and orbit6/orbit6 ovaries. Equal amount of samples were loaded on both Western blots and confirmed by staining with an anti-tubulin antibody. (C) Western blot indicating the age-dependent reduction of the expression level of Orbit/Mast in total ovarian protein extracts of the orbit6/orbit6 ovaries as normalised to wild type. (D) Wild-type (wt) and orbit6 (orbit) egg chambers were stained with anti-lamin (green) antibody and TOTO3 for DNA (blue). The orbit6 egg chambers contain fewer than 16 germline cells. They show no oocyte differentiation and all cells adopt nurse cell fate. Scale bar: 50 µm.

View larger version (40K): [in a new window]   Fig. 3. Disruption of the ovariole niche in germaria of orbit6 mutants. Germaria are stained with anti--spectrin (red), anti--tubulin (green) antibodies and TOTO3 (blue) to reveal DNA. (A) The apical region of a wild-type germarium with two stem cells (arrowhead) that each contains a spherical fusome (inferred by -spectrin staining). Germline cysts have branched fusomes (arrow) linked to microtubule bundles. (B) The germarium of a 1-day-old orbit6 female showing normal fusome morphology in a stem cell (arrowhead) and a germline cyst (arrow). In other cysts the fusome failed to branch (star). The terminal filament cells are not visible on the picture since they are out-of-focus. (C) The germarium of a 2-day-old orbit6 female that has no stem cells. Only one germline cell-cyst is present in the germarium containing a spherical fusome (star) and some fusome fragments. Microtubule bundles are cross-linking these fusome elements and the stack of terminal filament cells looks normal (arrow). (D) The germarium of a 3-day-old orbit6 female that has no stem cells. A single germline cell-cyst is present that contains a single nurse cell-like nucleus, an elongated fusome and some fusome fragments connected through microtubule bundles. A stack of terminal filament cells is not present. Scale bar: 10 µm.

View larger version (86K): [in a new window]   Fig. 4. Anillin and Pav-KLP fail to be recruited to ring canals in orbit6 mutants. (A-E) Germaria stained with anti-Anillin (red) and anti-HtsF (green) antibodies. (A) Wild-type cyst showing Anillin recruited to the newly formed contractile rings (arrow) before the initiation of fusome plug formation. (B) Wild-type cyst with Anillin present in the constricting and migrating ring canals. (C) Wild-type 16-cell cyst that has completed the four rounds of divisions. Anillin is present in the ring canals situated along the fusome. Only eight ring canals and the corresponding fusome part are visible; the others are out of focus. (D,E) orbit6 germaria that contain one and two germline cysts respectively, each with several fusome pieces. No Anillin-containing ring canal-like structures are visible. However, as with wild-type, the interphase nuclei of mutant cysts contain Anillin. Scale bar: 5 µm. (F-H) Germaria were stained with anti-Pav-KLP (green) and anti-HtsF (red) antibodies. (F) Wild-type stem cell and cystoblast interconnected through a Pav-KLP-containing ring canal at the time of fusome partition. The nuclei of these cells contain elevated levels of Pav-KLP, suggesting that these cells are in interphase. (G) Pav-KLP recruited to the ring canals surrounding the pre-existing fusome in wild-type germline cysts. (H) orbit6 germline cyst with an abnormal fusome and Pav-KLP accumulated in a single ring canal-like structure (broad arrow). Fusome pieces seen at the posterior of this cyst are of different sizes and show little or no Pav-KLP staining. Scale bars: 5 µm in F,G; 10 µm in H.

View larger version (96K): [in a new window]   Fig. 6. Ring canal organisation is affected in egg chambers of orbit6 mutants. (A-D) Germaria were stained with rhodamine-phalloidine (red), anti-Filamin (green) antibody and TOTO3 to reveal DNA (blue). (A) Wild-type egg chamber in which Filamin and F-actin co-localise in the ring canals. Scale bar: 50 µm. (B) Egg chamber of 1-day old orbit6 female containing seven nurse cell nuclei and six ring canals. Filamin and F-actin accumulate not only in the rims but also in the lumen of the ring canals. Note the weak F-actin staining in the cortex of the nurse cells. Scale bar: 10 µm. (C) Filamin and F-actin are not restricted to the rims of ring canals in orbit6 egg chambers. Scale bar: 5 µm. (D) Severely affected egg chambers from 3-day old orbit6 female with a single large nurse cell nucleus; ring canals fail to form although filamentous structures containing Filamin and F-actin are visible. Scale bar: 10 µm. (E-G) Germaria were stained with anti-Orbit (red), anti-HtsRC (green) antibodies and TOTO3 to reveal DNA (blue). (E) Wild-type egg chamber showing the HtsRC and Orbit/Mast (inset) proteins in the ring canals. Scale bar: 25 µm. (F) Egg chamber from 1-day old orbit6 female in which HtsRC accumulates in the rims and obstructs the lumen of the ring canals. Orbit/Mast staining is weak in the cortex of the nurse cells. Scale bar: 10 µm. (G) Abnormal egg chamber of 3-day old orbit6 female showing irregular structures containing HtsRC protein, some of which resemble ring canals that have completely occluded lumens. Scale bar: 10 µm.

View larger version (74K): [in a new window]   Fig. 7. The polarised MT network fails to develop correctly in egg chambers of orbit6 mutants. (A-C) Egg chambers were stained to reveal Orbit/Mast (red), -tubulin (green) and DNA (blue). (A) Wild-type egg chambers at stages 1-, 5 and 7 showing the Orbit/Mast protein accumulates at the MTOC of the oocyte (arrowhead) and is present in the oocyte nucleus from stage 7 onwards (arrow). The punctate pattern of Orbit/Mast seems to show some correlation with the microtubule network (inset) of the egg chambers. (B) Egg chambers of 1- and 3- day old orbit6 females showing the age-dependent severity of the mutant phenotype. In egg chambers of younger females, the residual Orbit/Mast protein decorates a diminished number of microtubule bundles, which seem to contact local accumulations of Orbit/Mast (arrow). There is also reduced staining of a putative MTOC (arrowhead). (C) Colchicine-treated, wild-type stage 3 egg chamber showing no MTOC-specific localisation of Orbit/Mast. The microtubule network is destroyed and the Orbit/Mast staining is diffuse in the cytoplasm of cells. Scale bar: 10µm. (D) Wild-type egg chambers stained to reveal Orbit/Mast (red), lamin (green) and DNA (blue). Note the MTOC and nuclear localisation of Orbit and Mast, respectively. Scale bar: 20 µm.

View larger version (77K): [in a new window]   Fig. 8. Localisation of CLIP-190 is disrupted in orbit6 mutants. (A-F) Germaria and egg chambers stained to reveal CLIP-190 (red), -Tubulin (green) and DNA (blue). The monochrome panels correspond to the staining pattern of CLIP-190. (A) Wild-type four-cell cyst at metaphase with CLIP-190 localising on the spindle and fusome. (B) Wild-type 16-cell stretching (arrowhead) and lens-shaped (arrow) cysts showing specific accumulation of D-CLIP-190. (C) Wild-type stage 6-7 egg chamber; CLIP-190 displays a punctate localisation along MT bundles (inset) and accumulates in the apical cortical region of the follicle cells. (D) Mitotic cyst in orbit6 germarium with no CLIP-190 on the spindles and a fusome-like body containing CLIP-190 (arrow) that contacts a spindle pole. (E) Egg chamber of a young orbit6 female with irregular microtubule bundles that show no accumulation of CLIP-190. (F) Egg chamber of an older orbit6 female with a few irregular microtubule bundles. Scale bars: 10 µm. (G) Western blots of total ovarian extracts immunoprecipitated with anti-CLIP-190 or anti-Orbit/Mast specific antibodies, subjected to western blotting and probed with the indicated antibodies.