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First published online 18 March 2008
doi: 10.1242/jcs.026716

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Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site

¹ Cancer Research UK Cell Cycle Genetics Research Group, Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK
² Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK

View larger version (24K): [in this window] [in a new window]   Fig. 1. Isolation of proteins interacting with Anillin in Drosophila cells. (A) Cells stably expressing the Ani::PtA transgene were fixed and stained to detect Protein A (red in merged panels), tubulin (green in merged panels) and DNA (blue in merged panels). (B) Colloidal Coomassie-stained gel of one the two purifications from Ani::PtA cells. The position of some of the proteins identified by mass spectrometry (MS) is indicated on the right (also see Table 1). The bait, Ani::PtA, is in red. The numbers on the left indicate the size in kD of the molecular-weight marker. Scale bar: 10 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Colocalization of Anillin and RacGAP50C during cell division. (A) Cells were treated with DMSO (control) or Lat-A for 1 hour, and were then fixed and stained to detect Anillin (red in merged panels), tubulin (green in merged panels) and DNA (blue in merged panels). The rightmost panels show a magnification of the microtubules contacting the equatorial cortex in a Lat-A-treated cell; the spindle axis and chromosomes are not contained in this field. The arrowhead marks Anillin localization at the microtubule plus ends. (B) Cells were treated with DMSO (control) or Lat-A for 1 hour, and were then fixed and stained to detect Anillin (red in merged panels), RacGAP50C (green in merged panels) and DNA (blue in merged panels). The bracket and arrowheads mark the sites of Anillin and RacGAP50C colocalization (orange/yellow in merged panels). Note the colocalization at the microtubule plus ends in Lat-A-treated cells. (C) Cells were first incubated with dsRNA directed against RacGAP50C for 48 hours and then treated with DMSO (control) or Lat-A for 1 hour. The samples were then fixed and stained to detect Anillin (red in merged panels), tubulin (green in merged panels) and DNA (blue in merged panels). Note the absence of Anillin rod-shaped structure at the microtubule plus ends (marked by the arrow) in Lat-A-treated cells. Scale bars: 10 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Anillin and RacGAP50C interact in vitro. (A) At the left is shown a schematic representation of the full-length Anillin protein and of the several fragments used for in vitro binding assays. The PH domain and myosin (My)- and actin (Ac)-binding regions are indicated. The thick grey line marks the region showing the highest homology among different species (Anillin Homology Domain, AHD). The autoradiograph of the pull-down assay along with a Coomassie-stained gel (CBB) showing the amounts of bait proteins used for the binding assay are shown at the right. The top bands in the Coomassie gel correspond to the full-length GST-tagged baits. (B) Subcellular localization of GFP-tagged Anillin fragments. A schematic description of the fragment tagged with GFP is shown at the left and their relative subcellular localization at the right. The cells were fixed and stained to detect tubulin (red in merged panels), GFP (green in merged panels) and DNA (blue in merged panels). The arrowhead marks the spindle midzone localization of Ani410-1104. (C) At the left is shown a schematic representation of the full-length RacGAP50C protein and of the several fragments used for in vitro binding assays. The GAP and C1 domains, and Pav-KLP (Pav)- and Pebble (Pbl)-binding regions are indicated. The autoradiographs of the pull-down assays are shown at the right. The amount of GST and GST-A4 proteins used is the same as in A. Scale bar: 10 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Anillin recruits Pnut and Sep2 to the cleavage furrow. (A) Cells were treated for 48 hours with dsRNAs directed against either Anillin or GFP (control), and were then fixed and stained to detect tubulin (green in merged panels), DNA (blue in merged panels) and either Pnut or Sep2 (red in merged panels). (B) Western blot analysis of protein levels after RNAi treatment. Cells were incubated with dsRNAs directed against either Anillin or GFP (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 Anillin and -tubulin. (C) Cells were treated for 96 hours with dsRNAs directed against GFP (control), pnut, Sep2 or both septins simultaneously, and then fixed and stained to detect Anillin (red in merged panels), tubulin (green in merged panels) and DNA (blue in merged panels). (D) Western blot analysis of protein levels after RNAi treatment. Cells were incubated with dsRNAs directed against GFP (control), pnut, Sep2 or both septins simultaneously. After 96 hours the proteins were extracted and separated on a 10% SDS gel, transferred onto a PVDF membrane and probed with antibodies against Pnut, Sep2 and -tubulin. Scale bar: 10 µm.

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