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First published online 10 October 2006
doi: 10.1242/jcs.03210

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RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis

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

View larger version (68K): [in a new window]   Fig. 1. Inactivation of the centralspindlin complex prevents furrowing without affecting astral microtubule dynamics. (A) Western blot analysis of protein levels after RNAi treatment. Tubulin::GFP cells were incubated with dsRNAs directed against PAV (pav RNAi), RacGAP50C (GAP RNAi) or no dsRNA as a control. After 48 hours, the proteins were extracted and separated on a 10% SDS gel, transferred onto a PVDF membrane and probed with antibodies against PAV, RacGAP50 and -tubulin. (B) Selected images from time-lapse recordings of Drosophila S2 cells expressing a Tubulin::GFP transgene. Cells were treated for 48 hours with dsRNAs directed against the two members of the centralspindlin complex: PAV (pav RNAi) and RacGAP50C (GAP RNAi) or no dsRNA as a control. The arrowheads indicate the astral microtubules contacting the equatorial cortex. Time is in minutes:seconds relative to anaphase onset. Bar, 10 µm.

View larger version (61K): [in a new window]   Fig. 2. Centralspindlin localizes to the equatorial cortex just before furrow ingression. (A) Localization of PAV::GFP and RacGAP::GFP fusion proteins in cleaving S2 cells. Cells were fixed and stained to detect GFP (green in the merged panels), DNA (blue in the merged panels) and either RacGAP50C or PAV (red in the top and bottom merged panels, respectively). (B) Selected frames from a time-lapse series showing PAV::GFP dynamics in S2 cells. The white arrowheads indicate PAV::GFP localization at the cell equator. Time is in minutes:seconds relative to anaphase onset. Bars, 10 µm.

View larger version (113K): [in a new window]   Fig. 3. PAV::GFP dynamics in primary spermatocytes. (A) Selected frames from a time-lapse series showing PAV::GFP dynamics in Drosophila primary spermatocytes. Corresponding DIC images are at the top. The black arrows indicate furrow ingression whereas the white arrowheads mark PAV::GFP localization at the cell equator. The boxed regions indicate the area magnified in B. Time is in minutes relative to anaphase onset. (B) Magnified selected frames from the time-lapse recording of the cell shown in A. Bars, 10 µm.

View larger version (32K): [in a new window]   Fig. 4. Expression and localization of the CD8::GFP and CD8::GFP::RacGAP50C fusion proteins. (A) Schematic diagram of the transgenes used to generate the stable cell lines. (B) Western blot analysis of CD8::GFP::RacGAP50C expression. CD8::GFP::RacGAP50C and CD8::GFP cells were cultured in completed medium with (+) or without (–) 0.7 mM CuSO4 for 5 hours and then harvested for protein extraction. Protein extracts were separated on a 10% SDS gel, transferred onto a PVDF membrane and probed with a RacGAP50 antibody. The asterisk indicates a breakdown product that corresponds in size to a GFP::RacGAP50C protein. (C,D) Localization of the CD8::GFP and CD8::GFP::RacGAP50C fusion proteins in S2 cells. Expression of CD8::GFP and CD8::GFP::RacGAP50C was induced by incubating the cultures in complete medium containing 0.7 mM CuSO4 for 5-7 hours. The cells were then fixed and stained to detect GFP (green in the merged panels), DNA (blue in the merged panels) and tubulin (red in the merged panels in C). Interphase and metaphase cells are shown in C and D, respectively. Bars, 10 µm.

View larger version (63K): [in a new window]   Fig. 5. Mislocalization of RacGAP50C causes ectopic furrowing. Expression of CD8::GFP and CD8::GFP::RacGAP50C was induced by incubating the cultures in complete medium containing 0.7 mM CuSO4 for 5-7 hours. The cells were then fixed and stained to reveal GFP (green in the merged panels), DNA (blue in the merged panels) and either tubulin (A), Anillin (B), Peanut (C) or PAV (D) (red in the merged panels). The arrows indicate the ectopic furrows whereas the arrowhead indicates the equatorial furrow. Note the misshapen and bisected nuclei in A and B and the small cytoplast in C. Bars, 10 µm.

View larger version (39K): [in a new window]   Fig. 6. The membrane-tethered version of RacGAP50C can induce ectopic furrows in the absence of PAV. CD8::GFP and CD8::GFP::RacGAP50C cells were incubated with dsRNA directed against PAV for 48 hours and then the expression of transgenes induced by incubating the cultures in complete medium containing 0.7 mM CuSO4 for 5-7 hours. The cells were then fixed and stained to reveal GFP (green in the merged panels), tubulin (red in the merged panels) and DNA (blue in the merged panels). The arrows mark the ectopic furrows. Note that the size of the nuclei of the CD8::GFP::RacGAP50C cell shown at the bottom indicates that this cell is tetraploid and therefore already failed cytokinesis once. Nonetheless, multiple ectopic furrows still formed and CD8::GFP::RacGAP50C co-localized with the remnant of the central spindle. Bar, 10 µm.

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