Fig 1.
Rescue of funnel cakes and onion rings mutant cells by Sec8 and Exo84.
(A) Phase contrast microscopy of wild type, funz1010/Df(3R)Exel6145 mutant (B) and onrz4840/Df(3R)Espl3 mutant (C) male germline cells. In fun and onr mutant cells, cell division fails and multiple nuclei (white spherical objects) are observed in association with enlarged nebenkern (black spherical objects). In wild-type cells, single nuclei are found in association with nebenkern of approximately equal size. A single copy of a transgene containing either genomic Sec8 (D) or genomic Exo84 (E) rescues cytokinesis defects in funz1010/Df(3R)Exel6145 (D) and onrz4840/Df(3R)Espl3 (E) mutant cells. Scale bar, 10 μm.
Fig 2.
Localization of exocyst complex proteins in dividing spermatocytes.
(A) Localization of Sec8 protein in wild-type primary spermatocytes. Interphase and dividing spermatocytes were stained for Tubulin (green), Sec8 (red) and DNA (blue). During interphase, Sec8 was mostly diffuse in the cytoplasm and enriched at the plasma membrane (arrowheads). In dividing spermatocytes, Sec8 appeared enriched in a broad cortical band that encircled the midzone (arrows) and was excluded from the poles. (B) Localization of Sec5 protein in wild-type dividing spermatocytes. Primary spermatocytes were stained for Tubulin (green), Sec5 (red) and DNA (blue). Note the enrichment of Sec5 in puncta at the astral microtubules (arrowhead) and at the cleavage furrow (arrows). Scale bar, 10 μm.
Fig 3.
Defective cytokinetic ring ingression in fun and onr mutant cells.
(A) Selected still frames from supplemental S1, S2 and S3 Movies. Dividing spermatocytes expressing the regulatory light chain of non-muscle myosin II, Sqh-GFP, were imaged starting from the beginning of anaphase. Numbers at the bottom of each frame indicate minutes from the beginning of imaging. Note that the Sqh-GFP ring undergoes minimal constriction (fun) or fails to constrict (onr) in mutant cells. Scale bar, 10μm. (B) Dynamics of cleavage furrows in fun and onr mutants. Furrow diameters (relative to the diameter at t = 0) in dividing spermatocytes from wild type, funz1010/Df(3R)Exel6145 (fun) and onrz4840/Df(3R)Espl1 (onr) males expressing Sqh-GFP and undergoing ana-telophase were plotted over time. (C) Furrow diameters (relative to the diameter at time = 0) were plotted at 5-minute intervals. Furrow diameters were measured in movies from dividing spermatocytes expressing Sqh-GFP and undergoing ana-telophases (n = 9 wild type, n = 8 fun and n = 8 onr). Error bars indicate standard deviations. *p = 0.0035, **p = 0.0008;***p = 0.0001, significantly different from control in the Student t test.
Fig 4.
Failure in anaphase elongation, cleavage furrow progression, and surface area addition in onr and fun mutant cells.
(A-C) Still frames from time-lapse confocal microscopy of wild-type (A), onrz4840mutant (B), and funz1010 mutant (C) male germline cells expressing PLCδd-PH-GFP and β-Tub-GFP (imaged simultaneously in single channel). Cells are shown just prior to elongation (A, B, C), immediately before ingression (A’, B’, C’), during ingression (A”, B”, C”), and after successfully completing or failing to complete cytokinesis (A”‘, B”‘, C”‘). (D-F) Representative segmented and voxelized cells of wild-type (D), onrz4840 mutant (E), and funz1010 mutant (F) cells. (G-J) Quantitative computational analysis of surface area (G), volume (H), aspect ratio (I), and convex hull volume ratio (a measurement of furrow ingression, J) in wild-type (blue), onrz4840 mutant (red), and funz1010 mutant (green) cells. Left, lines are average values of wild-type (n = 8), onr (n = 11), and fun (n = 10) segmented cells. Data from individual cells were aligned such that t = 0 is the start of anaphase elongation, while arrowheads mark the initiation of cytokinesis in wild-type cells (see Materials and Methods). Right, quantitation of percent change from t = 0 to t = 25 min. Increases in surface area, aspect ratio, and CHVR observed in wild-type cells are disrupted in onrz4840 and funz1010 mutant cells, while no significant difference is observed in volume. Prior to the start of anaphase elongation, cell volume and surface area were nearly identical in wild type, onr mutant, and fun mutant cells (p-values ranging from 0.0838 to 0.5969). All movies start during early anaphase and end after successful (wild-type) or failed (fun and onr) cytokinesis. Shaded region indicates standard error (G-J); *p<0.0001, significantly different from control in the two-sample Student t-test; n.s. = not significant, p>0.23. Scale bar, 10 μm.
Fig 5.
Defects in Golgi structure in fun and onr mutant cells.
(A) G2 primary spermatocytes from wild-type, onrz4840/Df(3R)Espl3 and funz1010/Df(3R)Exel6145 mutant males, stained for the Golgin Lva (red) and DNA (blue). Enlargements of Golgi stacks are shown on the right of each panel. Scale bar, 10 μm. (B) Average number of Golgi bodies per cell (± SEM) visualized in G2 spermatocytes from wild type (n = 50), onrz4840/Df(3R)Espl3 (onr, n = 48), or funz1010/Df(3R)Exel614 (fun, N = 48) after staining for Lva. Numbers of Golgi per cell in fun and onr mutants are significantly different from wild type in the Student t test:*p<0.0001, **p<0.0001. (C) Average area (± SEM) of Golgi bodies, quantified by ImageJ (expressed in arbitrary units), in G2 primary spermatocytes stained for Lva, Golgi sizes are significantly different in funz1010/Df(3R)Exel614 (fun) and onrz4840/Df(3R)Espl3 (onr) compared to wild type using the Student t test, *p<0.0001, **p<0.0001.
Fig 6.
Defects in morphology and ultrastructure of parafusorial membranes and Golgi bodies in fun and onr mutant cells.
Transmission electron micrographs showing parafusorial membranes (A-F), astral membranes (G-I), and Golgi bodies (J-L) in fun and onr mutant spermatocytes. Parafusorial and astral membranes (arrows) are enlarged, fragmented and vacuolated in funz1010/Df(3R)Exel6145 (B, E, H) and onrz4840/Df(3R)Espl3 (C, F, I) dividing spermatocytes. (D, E, F) panels are magnified images of areas surrounded by white squares in (A, B, C). (H, I) panels are magnified images of areas surrounded by black squares in (B, C). Golgi bodies (asterisks) show vacuolated regions in fun (K) and onr (L) mutant spermatocytes. Golgi bodies surrounded by white squares in (J-L) are magnified in insets. Scale bars are 2 μm (A-C, J, K) or 500 nm (D-I, L).
Fig 7.
onr and fun mutations disrupt localization of Rab11 protein in dividing spermatocytes.
(A) Telophase spermatocytes from wild type, funz1010/Df(3R)Exel6145 (fun) and onrz4840/Df(3R)Espl3 (onr) stained for Rab11 (green) and DNA (blue). (B) Telophase spermatocytes from wild type, funz1010/Df(3R)Exel6145 (fun) and onrz4840/Df(3R)Espl3 (onr) stained for Rab11 (green), Anillin (red) and DNA (blue). Scale bar, 10 μm.
Fig 8.
The PITP Giotto fails to concentrate at the midzone of dividing spermatocytes from fun and onr males.
Spermatocytes were stained with anti-Tubulin (green), anti-Gio (red) and DAPI (blue). Arrows indicate the cleavage site. Wild type, funz1010/Df(3R)Exel6145, and onrz4840/Df(3R)Espl3 were stained for Tubulin (green), Gio (red) and DNA (Blue). Double arrowheads point to astral membranes, arrowheads indicate parafusorial membranes. Scale bar, 10 μm.
Fig 9.
onr and fun mutations interact with mutations in Rab11.
(A) Frequencies of early spermatids containing 2, 4 or more than 4 nuclei per nebenkern in testes from either Rab1193Bi/Rab1193Bi (Rab11) or fun z1010 Rab1193Bi/+ Rab1193Bi (fun Rab11/Rab11) mutant males. (B) Frequencies of early spermatids containing multiple nuclei (2, 4 or more than 4 nuclei) per nebenkern in testes from either Rab1193Bi/Rab11E(To)3 (Rab11), funz1010/fun z1010 (fun), or funz1010 Rab1193Bi fun z1010Rab11E(To)3 (fun Rab11) mutant males. (C) Co-IP of HA-Sec8 with GFP-Exo84. Protein extracts from testes expressing either HA-Sec8 and GFP-Exo84 or HA-Sec8 alone were immunoprecipitated with anti-GFP (i.e., GFP-trap beads) and immunoblotted for either GFP, HA or Rab11. (D) Co-IP of Sec5 with YFP-Rab11. Protein extracts from testes expressing either wild-type YFP-Rab11 (wt), YFP-Rab11Q70L (Q70L) or YFP-Rab11S25N (S25N) were immunoprecipitated for YFP (using GFP-trap beads) and blotted for either YFP or Sec5.