PMID:23520495

Citation	Miyauchi, C, Kitazawa, D, Ando, I, Hayashi, D and Inoue, YH (2013) Orbit/CLASP is required for germline cyst formation through its developmental control of fusomes and ring canals in Drosophila males. PLoS ONE 8:e58220
Abstract	Orbit, a Drosophila ortholog of microtubule plus-end enriched protein CLASP, plays an important role in many developmental processes involved in microtubule dynamics. Previous studies have shown that Orbit is required for asymmetric stem cell division and cystocyte divisions in germline cysts and for the development of microtubule networks that interconnect oocyte and nurse cells during oogenesis. Here, we examined the cellular localization of Orbit and its role in cyst formation during spermatogenesis. In male germline stem cells, distinct localization of Orbit was first observed on the spectrosome, which is a spherical precursor of the germline-specific cytoskeleton known as the fusome. In dividing stem cells and spermatogonia, Orbit was localized around centrosomes and on kinetochores and spindle microtubules. After cytokinesis, Orbit remained localized on ring canals, which are cytoplasmic bridges between the cells. Thereafter, it was found along fusomes, extending through the ring canal toward all spermatogonia in a cyst. Fusome localization of Orbit was not affected by microtubule depolymerization. Instead, our fluorescence resonance energy transfer experiments suggested that Orbit is closely associated with F-actin, which is abundantly found in fusomes. Surprisingly, F-actin depolymerization influenced neither fusome organization nor Orbit localization on the germline-specific cytoskeleton. We revealed that two conserved regions of Orbit are required for fusome localization. Using orbit hypomorphic mutants, we showed that the protein is required for ring canal formation and for fusome elongation mediated by the interaction of newly generated fusome plugs with the pre-existing fusome. The orbit mutation also disrupted ring canal clustering, which is essential for folding of the spermatogonia after cytokinesis. Orbit accumulates around centrosomes at the onset of spermatogonial mitosis and is required for the capture of one of the duplicated centrosomes onto the fusome. Moreover, Orbit is involved in the proper orientation of spindles towards fusomes during synchronous mitosis of spermatogonial cysts.
Links	PubMed PMC3592921 Online version:10.1371/journal.pone.0058220
Keywords	Actins/genetics; Actins/metabolism; Animals; Centrosome/metabolism; Drosophila Proteins/genetics; Drosophila Proteins/metabolism; Drosophila melanogaster; Male; Microtubule-Associated Proteins/genetics; Microtubule-Associated Proteins/metabolism; Mitosis/physiology; Mutation; Spermatogonia/cytology; Spermatogonia/metabolism; Spindle Apparatus/genetics; Spindle Apparatus/metabolism; Stem Cells/cytology; Stem Cells/metabolism
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
DROME:CLASP		GO:0045170: spectrosome	ECO:0000314:		C		Organism: Drosophila (fruit fly) Protein: ORBIT (majorly known as CLASP; mentioned as both in paper) Figure 6. A) Orbit is localized on the spectrosome.	complete CACAO 12344
DROME:CLASP		GO:0000776: kinetochore	ECO:0000314:		C		Organism: Drosophila (fruit fly) Protein: Orbit (majorly known as Clasp, mentioned as both in paper.) Figure 6. I) Orbit is localized on kinetochores.	complete CACAO 12345
DROME:CLASP		GO:0045169: fusome	ECO:0000314:		C		Organism: Drosophila melanogaster Protein: CLIP-associating protein Figure 2A&B) (A) An early spermatocyte cyst incubated without colchicine. Note the cytoplasmic microtubule structures and distinct Orbit localization on growing fusomes. (B) An early spermatocyte cyst treated with colchicine; a branched fusome structure is intact. we concluded that Orbit localization on the fusome is independent of microtubules and that microtubules are not essential for the maintenance of the fusome structure.	complete CACAO 12648
DROME:CLASP		GO:0005737: cytoplasm	ECO:0000314:		C		Organism: Drosophila melanogaster Protein: CLIP-associating protein Figure 1B) Orbit was also concentrated in the cytoplasm of hub cells,	complete CACAO 12650
DROME:CLASP		GO:0072686: mitotic spindle	ECO:0000314:		C		Organism: Drosophila melanogaster Protein: CLIP-associating protein Figure 1A) The protein was further detected on mitotic spindles and kinetochores	complete CACAO 12652
DROME:CLASP		GO:0031965: nuclear membrane	ECO:0000314:		C		Organism: Drosophila melanogaster Protein: CLIP-associating protein Fig. S2A: Orbit was localized around the nuclear membranes during the prophase	complete CACAO 12655
DROME:CLASP		GO:0070732: spindle envelope	ECO:0000314:		C		Organism: Drosophila melanogaster Protein: CLIP-associating protein Fig. S2B: Orbit was localized around the nuclear membranes during the prophase and associated with spindle envelopes and kinetochores from the prometaphase to the metaphase	complete CACAO 12656
DROME:CLASP		GO:0070938: contractile ring	ECO:0000314:		C		Organism: Drosophila melanogaster Protein: CLIP-associating protein Fig. S2D: By the telophase, Orbit had accumulated on the prospective cleavage furrow region and contractile rings; the protein remained on the contractile rings during cytokinesis .	complete CACAO 12657
DROME:CLASP		GO:0005876: spindle microtubule	ECO:0000314:		C		Organism: Drosophila Melanogaster Protein: CLIP-associating protein Fig. 2C, D: We confirmed the complete degradation of spindle microtubules and astral microtubules after colchicine treatment.	complete CACAO 12659
DROME:CLASP		GO:0000235: astral microtubule	ECO:0000314:		C		Organism: Drosophila Melanogaster Protein: CLIP-associating protein Fig. 2C, D: We confirmed the complete degradation of spindle microtubules and astral microtubules after colchicine treatment.	complete CACAO 12660

References

See Help:References for how to manage references in GONUTS.