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First published online November 26, 2007
doi: 10.1242/10.1242/dev.010983

Development 134, 4437-4447 (2007)
Published by The Company of Biologists 2007
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Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes

Naomi Dyer1,2,*, Elena Rebollo3,*, Paloma Domínguez3, Nadia Elkhatib4, Philippe Chavrier4, Laurent Daviet5, Cayetano González3 and Marcos González-Gaitán1,6,{dagger}

1 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
2 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.
3 Institut de Recerca Biomèdica (IRB) and Institució Catalana de Recerca i Estudis Avançats (ICREA), Parc Cientific Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain.
4 CNRS UMR144, Institut Curie, Membrane and Cytoskeleton Dynamics Group, 26 Rue d'Ulm 75005 Paris, France.
5 Hybrigenics SA, 3-5 Impasse Reille, 75014 Paris, France.
6 Département de biochimie, Sciences II, 30, Quai Ernest Ansermet CH-1211 Genève 4, Switzerland.


Figure 1
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  		Fig. 1. Drosophila ARF6 is required for cytokinesis in testes. (A) The portion of genomic region 51f encoding Drosophila ARF6 (blue), and protein coding sequences of arf6 (red). arf61, arf62 and arf63 (below) are deletions produced by imprecise excision of EP(2)2612. (B-D) Phase-contrast images of spermatids after the second meiotic division, showing white nuclei (arrows) and black mitochondrial derivatives (Nebenkerne, arrowheads). (B) WT, (C) arf61 homozygotes: two or four nuclei per cell. (D) A rescue construct expressing arf6 from the endogenous promoter restores a 1:1 ratio of nucleus to Nebenkern per cell. Scale bars: 20 µm. (E) Frequency of nuclei per cell in WT, arf61, arf61 rescued by the arf6 endogenous rescue construct and arf63 rescued by expression of ARF6HA. n, number of cells counted.

 

Figure 2
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  		Fig. 2. ARF6 is not required for central spindle or contractile ring formation. (A-E) Time-lapse images of Pav-GFP (A-C) and Sqh-GFP (D,E) during cytokinesis in control (A,D), arf6 (B,E) and chic13E mutant spermatocytes (C). Times are minutes:seconds after anaphase onset (min AA). Scale bars: 5 µm. (A) Control, Pav-GFP accumulates at the central spindle during anaphase B (arrowhead, 03:29). Central spindle microtubules labelled with Pav-GFP bundle and compact into a dense midbody (arrowheads, 09:14-59:54). (B) arf61, anaphase B Pav-GFP central spindle accumulation occurs (arrowhead, 06:54). Pav-GFP-labelled microtubules bundle (arrowhead, 15:20), a cleavage furrow initiates, but central spindle Pav-GFP signal declines (arrowhead, 24:21). After furrow regression, only a tiny amount of Pav-GFP remains at the cortex (arrowhead 50:21). (C) chic13E, anaphase B Pav-GFP central spindle accumulation occurs (arrowhead, 03:00, 06:54), but no furrow is initiated. 17:30 min AA: very little Pav-GFP remains at the central spindle. (D) Control, Sqh-GFP transfers to the cortex (arrowhead, 04:55), accumulates at the future cleavage furrow site (arrowheads, 07:05, 08:11) which then invaginates (arrowhead, 22:14). (E) arf63, Sqh-GFP transfers to the cortex (arrowhead, 04:58) concentrating at the future cleavage furrow site (arrowheads 07:01, 14:09). Sqh-GFP remains at the cortex during and after regression (arrowhead, 25:22). Genotypes: w; arf61/CyO; Pav-GFP/TM6B (A), w; arf61/arf61; Pav-GFP/TM6B (B), chic13E/chic13E; Pav-GFP/TM6B (C), y w sqhAX3; +; P (w+ sqh-gfp) (D), y w sqhAX3; arf63/arf63; P (w+ sqh-gfp) (E).

 

Figure 3
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  		Fig. 3. ARF6 is required for rapid cleavage furrow invagination. (A) Representative results for control, arf6 mutant `early regressor', `late regressor' and chic13E illustrate furrow progression, timing of Pavarotti concentration at central spindle (arrowheads) and indented cleavage furrow appearance (arrows). Diameter is measured at furrow tip or future furrow site. (B) Furrow ingression rates in control (black), arf6 late regressors (blue) chic13E (green) and arf6 early regressors (red). Furrow ingression is significantly impaired in arf6 mutants compared with control cells: *P<0.01, Student's t-test. min AA, minutes after anaphase onset.

 

Figure 4
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  		Fig. 4. ARF6 is required for rapid growth of the plasma membrane during cytokinesis. (A) Representative cells for control, arf6 early regressor and chic13E cells illustrate perimeter kinetics during anaphase and cytokinesis. (B) Rate of perimeter change in control, arf6 early and late regressors, and chic13E cells. Membrane insertion is significantly impaired in arf6 from 5 min AA onwards: *P<0.05 and #P<0.001, Student's t-test. Membrane insertion is not significantly impaired in chic13E mutants until central spindle disassembly (cf. 5-10 min AA with 10-20 min AA). Error bars represent s.e.m. (C) Timeline of the events of cytokinesis and the rates of cytokinesis furrow ingression and perimeter change (membrane growth) in control and arf6 early regressors, with respect to central spindle Pav-GFP appearance. Black and white bars represent 1-minute intervals. CS, central spindle; CR, contractile ring.

 

Figure 5
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  		Fig. 5. ARF6 endosomes at the Pav central spindle during cytokinesis. (A,B) Fixed primary spermatocytes. (A,B) ARF6HA (red) and Pav (blue) immunostaining and Rab-GFP [green; Rab4 (A) and Rab11 (B)]. ARF6HA colocalizes with GFP-Rab4 (A) and GFP-Rab11 (B) at the central spindle (arrowheads). (C) Colocalization frequency of GFP-Rab4 (n=17 cells) and GFP-Rab11 (n=13 cells) with ARF6HA at the central spindle during cytokinesis (grey) or elsewhere in the cell (blue). Error bars represent s.e.m. (D) Primary spermatocyte initiating furrow. ARF6HA (red) is already partially localized to the central spindle (arrowheads), labelled with Pav-GFP (green). DAPI labels chromosomes (blue). Scale bars: 5 µm.

 

Figure 6
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  		Fig. 6. ARF6 is not required for the localization of recycling endosomes to the central spindle. (A-D) Live primary spermatocytes in meiosis I, expressing GFP-Rab4 (A,B) or GFP-Rab11 (C,D) in control (A,C) and arf6 early (B) or late regressor cells (D). Arrowheads, GFP-Rab4 and GFP-Rab11 at the central spindle in control and arf6 mutants. Red, cell outlines determined by DIC. Scale bars: 5 µm.

 

Figure 7
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  		Fig. 7. Physical interaction of Pav and ARF6. (A) Growth on medium lacking histidine (-His) indicates a positive two-hybrid interaction of Pav residues 583-865, in the yeast two-hybrid screen with ARF6Q67L. pGAD, pLEX, empty vectors. (B) Pav protein domains. Five pav clones interacted with ARF6Q67L in yeast two-hybrid analysis (black bars) defining a minimum overlapping domain (ARF6 int, red). (C) Binding assay of ARF6 with Pav. Purified GSTPav655-865, but not GST alone (negative control) pulled down ARF6HA and ARF6Q67LHA from HeLa cell lysates.

 

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© The Company of Biologists Ltd 2007