QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 21, 2007
doi: 10.1242/10.1242/jcs.000604

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Basto, R. Articles by Raff, J. W. Search for Related Content PubMed PubMed Citation Articles by Basto, R. Articles by Raff, J. W. Social Bookmarking                         What's this?

Hsp90 is required to localise cyclin B and Msps/ch-TOG to the mitotic spindle in Drosophila and humans

Renata Basto^1,*, Fanni Gergely^1,, Viji M. Draviam^1,, Hiroyuki Ohkura², Kathryn Liley³ and Jordan W. Raff^1,*

¹ The Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology. University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
² Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, EH9 3JR, UK
³ University of Cambridge, Department of Biochemistry Building O, Downing site, Cambridge, CB2 1QW, UK

View larger version (46K): [in this window] [in a new window]   Fig. 1. Cyclin B interacts with Hsp90 and Msps. (A) A Coomassie-Blue-stained gel of the proteins that bind to MBP-CBFL after incubation with embryo extract. Lane 1, the high speed embryo extract that was loaded onto the amylose column; lane 2, the proteins that flowed through the amylose column; lane 3, the proteins that were eluted from the amylose column. The MBP-CBFL protein is highlighted with a black arrow. Hsp90 (Hsp83) and Msps are indicated with black arrowheads. (B) A western blot analysis of an immunoprecipitation experiment performed with Drosophila embryo extracts (lane 1), random rabbit IgG (lane 2), affinity-purified anti-cyclin B antibody (lane 3), affinity-purified anti-Msps antibody (lane 4) and anti-Hsp90 antibody (lane 5). The membrane was probed with anti-Msps, anti-Hsp90 and anti-cyclin B antibodies as indicated. (C) A schematic diagram of the MBP cyclin B fusion proteins used in the MBP pull-down experiment shown in D: MBP-cyclin B full length (MBP-CBFL); MBP-cyclin B N-terminal (MBP-CBNT; contains the D-box and N-terminal helix, but no cyclin boxes); MBP-cyclin B C-terminal 1 (MBP-CBC1; contains the N-terminal helix and the two cyclin boxes); MBP-cyclin B C terminal 2 (MBP-CBC2; contains only the two cyclin boxes). (D) A western blot of an MBP pull-down experiment performed with affinity-purified anti-MBP antibodies and probed with anti-Hsp90 and anti-Msps antibodies as indicated.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Cyclin B is mislocalised in Hsp90 mutants. (A-D) Immunostaining of Drosophila third instar larvae neuroblasts. Cyclin B is shown in red (left panel), -tubulin in green (middle panel), and DNA in blue. (A) In WT prophase cells (A) cyclin B accumulates on centrosomes. (B) In Hsp90 mutant prophase cells cyclin B can be detected on centrosomes (white arrows) but this localisation is weaker and more diffuse than normal. (C) In WT metaphase cells cyclin B stains the centrosomes, the spindle MTs and the spindle mid-zone. (D) In Hsp90 mutant metaphase cells cyclin B strongly stains the spindle mid-zone, but staining on the spindle and centrosomes is much weaker than normal. (E-G) Bar graphs showing the quantification of cyclin B staining on centrosomes or on MTs in WT (green bars) and Hsp90 mutant (red bars) Drosophila neuroblasts (see Materials and Methods) in prophase (E), prometaphase (F) and metaphase (G). Bar, 5 µm (A-D).

View larger version (46K): [in this window] [in a new window]   Fig. 3. Cyclin B is mislocalised in HeLa cells after geldanamycin treatment. (A-F) Immunostaining of HeLa cells either after incubation with DMSO (A,C,E) as a control or with geldanamycin (B,D,F). Cyclin B is shown in red (left panel) and centrosomes and MTs are visualised with anti-TACC3 antibodies in green (middle panel); DNA is shown in blue in the merged images (right panel). Before NEB, cyclin B can be detected at centrosomes in both control cells and in GA-treated cells (A and B, white arrows). In metaphase control cells, cyclin B decorates the mitotic spindle (C) whereas after GA incubation (D) cyclin B cannot be detected on the MTs. (E,F) Metaphase cells viewed from one end of the spindle. In control cells (E) cyclin B is seen on the centrosomes and MTs whereas after GA treatment (F) no clear centrosomal or MT staining is observed. (G) Bar graphs showing the quantification of cyclin B staining on centrosomes or on MTs in control (green bars) and GA-treated cells (red bars). Bar, 10 µm (A-F).

View larger version (44K): [in this window] [in a new window]   Fig. 4. Msps/ch-TOG is not required for the centrosomal or MT localisation of cyclin B. (A,B) Immunostaining of Drosophila neuroblasts cells showing cyclin B (red, left panel), tubulin (green, middle panel) and DNA (blue in merged panel). (A) msps mutant neuroblast cell in prophase. Although the poles are somewhat disorganised in this cell, cyclin B is associated with the centrosomes. (B) msps mutant neuroblast cell in metaphase. Cyclin B is present at the poles and is associated with the mitotic spindle. (C,D) Bar graphs showing the quantification of cyclin B staining on centrosomes or on MTs in WT (green bars) and msps mutant (red bars) neuroblasts in prophase and prometaphase + metaphase. Bar, 5 µm (A,B). (E,F) Immunostaining of control and ch-TOG-depleted HeLa cells with cyclin B (red, left panel) and ch-TOG (green, middle panel); DNA is shown in blue in the merged panel. (E) In mock-depleted HeLa cells, both cyclin B and ch-TOG can be detected on centrosomes and MTs. Note that cyclin B has already disappeared from the centrosome in this cell that has completely aligned its chromosomes at the metaphase plate (Clute and Pines, 1999). (F) In ch-TOG partially depleted cells cyclin B associates with the spindle. Bar, 10 µm (C,D).

View larger version (52K): [in this window] [in a new window]   Fig. 5. Hsp90 activity is required for Msps/ch-TOG localisation to spindle MTs. (A,B) Immunostaining of Drosophila third instar larvae neuroblasts with Msps (red, left panels), and -tubulin (green, middle panels); DNA is shown in blue in the merged panels. (A) In WT metaphase cells Msps stains the centrosomes and MTs. (B) In Hsp90 mutant cells Msps can be detected on the centrosomes but not on the spindle MTs. (C,D) Immunostaining of HeLa cells either after incubation with DMSO (C) or with geldanamycin (D). In control cells, ch-TOG (red, left panels) strongly decorates the MTs (green, middle panels) of the mitotic spindles but in GA-treated cells there is a decrease in MT staining. (F) Bar graphs showing the quantification of Msps staining in WT (green bars) and Hsp90 mutant (red bars) Drosophila neuroblasts in prometaphase and metaphase. (G) Bar graphs showing the quantification of CH-TOG staining in control (green bars) and GA-treated (red bars) HeLa cells in prometaphase and metaphase. Bars, 5 µm (A,B); 10 µm (C,D).

View larger version (63K): [in this window] [in a new window]   Fig. 6. Hsp90 is not required for the recruitment of CP190, D-TACC, CNN and -tubulin. Immunostaining of Drosophila hs90p mutant neuroblasts cells (A-C). (A,B) CP190 and D-TACC are shown in the left panel and in green in the merged images. MTs are shown in red and DNA in blue in the merged images. (C) CNN is shown separately in the left panel and in green in the right panel. -tubulin is shown in red and DNA in blue in the merged images (right panel). Bar, 5 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 7. Polo kinase is not required for the centrosomal and MT localisation of cyclin B and Msps. Immunostaining of Drosophila polo mutant neuroblasts cells. (A,B) Cyclin B and Msps are shown in the left panel, and in red in the merged images. MTs are sown in green and DNA in blue in the merged images. In polo mutants both cyclin B and Msps can localise to the mitotic apparatus just as in control cells. Compare Fig. 7A with Fig. 2C, and Fig. 7B with Fig. 5A. Bar, 5 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Cyclin B and Msps are not destabilised in the absence of Hsp90, and cyclin B can interact directly with MTs in vitro. (A) Western blot of wild-type and Hsp90 neuroblasts. The brains were dissected and either incubated at 25°C for 2 hours or heat shocked at 37°C for the same amount of time. The filters were probed with anti-Msps, anti-cyclin B and anti-Polo antibodies. CP190 was used as loading control. In Hsp90 mutants, Polo protein is not stabilised (see de Carcer et al., 2001) but levels of Msps and cyclin B are comparable to, or even higher than, levels in WT. (B) Cyclin B can associate directly with MTs in vitro. Western-blot of a MT co-pelleting assay developed with affinity purified MBP antibodies. Supernatants (S) and pellets (P) from a control sedimentation assay with no MTs (left) or with MTs (right) performed with the same amount of the following proteins (top to bottom) MBP; MBP-CBFL, MBP-CBNT, MBP-CBC2. When MTs are not present all the proteins are detected in the (S) fraction. When incubated with MTs, MBP is still detected in the S fraction but a shift in MBP-CBFL and MBP-CBNT to the P fraction is detected. MBP-CBCT1 seems to be equally distributed into S and P fractions, suggesting that the N-terminal domain of cyclin B is required for MT binding.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter