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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.045377

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Targeting of p0071 to the midbody depends on KIF3

Institute for Pathophysiology, Division of Pathobiochemistry, Martin-Luther-University Halle, Hollystrasse 1, 06114 Halle, Germany

View larger version (42K): [in this window] [in a new window]   Fig. 1. P0071 and Ect2 are transported independently to the midbody. HeLa cells were synchronized and stained for p0071, Ect2 and -tubulin as indicated. (A) During anaphase and at the beginning of furrowing, Ect2 localized at the microtubule midzone. At this stage, p0071 was essentially absent from the midzone. (B,C) Along with progression through telophase and cytokinesis, p0071 became strongly enriched at the midbody and revealed a pronounced colocalization with Ect2. Scale bars: 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 2. Knockdown of Ect2 and MKLP1 affects p0071 midbody association. (A) HeLa cells were transfected with siRNAs as indicated and protein extracts analyzed by western blotting for -tubulin, Ect2 and MKLP1 at 72 hours after transfection. Quantification revealed knockdown efficiencies of 36% ± 5% for Ect2 and 48% ± 7% for MKLP1. (B) In cells transfected with control siRNA, Ect2 and p0071 strongly accumulated at the midbody during cytokinesis. (C,D) After transfection of Ect2 siRNA (C) or MKLP1 siRNA (D), those cells showing furrow ingression revealed reduced p0071 accumulation at the midbody. Scale bars: 10 µm; enlargements, 5 µm. (E) Furrow ingression was analyzed in synchronized cells at 90 minutes post nocodazole release. Due to the nocodazole treatment, approx. 35% of control siRNA-transfected cells revealed disturbed or delayed furrowing. Most of the Ect2 and MKLP1 siRNA-transfected cells did not furrow regularly (87% and 94%, respectively). Mean values + s.d. of three independent experiments counting >100 cells each are shown. * indicates P0.01.

View larger version (76K): [in this window] [in a new window]   Fig. 3. KIF3b colocalizes with p0071. (A) MCF-7 cells were co-stained for KIF3b and p0071 or KIF3b and -tubulin, respectively. In the upper panel a monoclonal KIF3b antibody (clone 35, BD Transduction Laboratories) was used and in the lower panel KIF3b was stained using a polyclonal antibody (H60, Santa Cruz). In interphase, KIF3b (green) is localized at the centrosome and colocalizes with p0071 (red, upper panel) and -tubulin (red, lower panel). Both KIF3b antibodies (green) stain primarily the centrosomal region. (B) HeLa cells were synchronized and analyzed during cytokinesis. For staining of KIF3b the polyclonal antibody was used. P0071 (green) accumulated at the contractile ring whereas KIF3b (red) localized at the microtubule midzone, which is closely associated with the contractile ring. Scale bars: 10 µm, enlargements 5 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 4. KIF3b interacts with p0071 in vitro and in vivo. (A) The interaction between p0071 and KIF3b was examined in the yeast 2-hybrid system. YRG2 yeast cells were co-transformed with the p0071 repeat domain or truncated variants of the p0071 repeat domain and wild-type KIF3b, KIF3b N-terminal (NT) and KIF3b C-terminal (CT) domains as indicated and plated on selection plates without tryptophane and leucine (–WL) and reporter plates without trytophane, leucine and histidine (–WLH). Colonies grown on selection plates (–WL) were probed for β-galactosidase activity (lacZ). (B,C) Overexpressed and endogenous KIF3b interact with the p0071 repeat domain in a GST pulldown experiment. The glutathione-agarose immobilized recombinant GST-p0071 repeat domain precipitated KIF3b from wild-type KIF3b-HA overexpressing (B) and nontransfected HEK293 cell lysates (C). Binding of KIF3b was determined by western blotting using an HA-specific (B) or a KIF3b-specific (C) antibody (PD: pull down). (D) A FLAG-IP approach revealed an association between p0071 and KIF3b in vivo. After overexpression of FLAG-tagged wild-type KIF3b and/or HA-tagged wild-type p0071 in HEK293 cells, KIF3b was precipitated from total lysates using anti-FLAG-agarose beads. Eluates were analyzed for p0071. (E) A Co-immunoprecipitate of in vitro translated Myc-tagged wild-type KIF3b and HA-tagged wild-type p0071 revealed a direct interaction between both proteins. Myc-tagged wild-type KIF3b and HA-tagged wild-type p0071 were in vitro transcribed and translated. Myc-tagged wild-type KIF3b was precipitated using monoclonal anti-Myc antibody coupled to protein A agarose. Eluates were analyzed for p0071 binding using a polyclonal HA-antibody.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Localization of p0071 at the midbody critically depends on KIF3b. (A) Knockdown efficiencies of KIF3b and p0071 siRNAs in HeLa cells were analyzed by western blotting at 72 hours post transfection. -tubulin was detected as a loading control. (B,C) siRNA-mediated knockdown of KIF3b (C) strongly decreased the accumulation of p0071 at the midbody compared with an unspecific siRNA (B). After transfection and synchronization, cells were fixed and stained for p0071 (green) and -tubulin (blue). Scale bars: 10 µm. (D) Quantification of p0071 localization at the midbody. Fluorescence intensities were determined in control siRNA- and KIF3b siRNA-transfected cells. For quantification p0071 fluorescence intensity at the midbody (white circle; 2.65 µm2) was divided by p0071 fluorescence intensity of the whole cell (encircled by the yellow line). The graph displays mean fluorescence intensities + s.d. for 20 cells each. (E) Mitotic microtubules were prepared at 90 minutes after nocodazole release (cytokinesis stage in control cells) and preparations from p0071 siRNA-, KIF3b siRNA- or p0071+KIF3b siRNA-transfected cells were probed for the presence of p0071. -tubulin was used as a loading control in the lysates as well as in the microtubule-enriched fractions. (F) Quantification of p0071 detected in mitotic microtubule preparations compared with total p0071. Amounts of total p0071 and amounts of p0071 detected in the microtubule-enriched preparations were normalized to the -tubulin content in the corresponding fractions. The graph displays the relative p0071 amounts + s.d. for three independent experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 6. RhoA and Ect2 localization are essentially unaltered after the KIF3b knockdown. (A) KIF3b siRNA-transfected cells were stained for p0071 (green), Ect2 (red) and -tubulin (blue). Whereas Ect2 recruitment to the midbody is not affected by the KIF3b knockdown, p0071 midbody localization is strongly reduced. Scale bars: 10 µm; enlargements, 5 µm. (B,C) Quantification of Ect2- (B) and RhoA- (C) fluorescence at the midbody. Fluorescence intensities were determined after control siRNA and KIF3b siRNA transfections. For quantification the respective fluorescence intensity at the midbody (white circle; 2.65 µm2) was divided by the fluorescence intensity of the whole cell (encircled by the yellow line). The graph displays mean fluorescence intensities + s.d. for 20 cells each.

View larger version (62K): [in this window] [in a new window]   Fig. 7. A KIF3b-binding-deficient mutant of p0071, p0071rep1, does not localize at the midbody. MCF-7 cells were transfected with wild-type p0071-DsRed (A) or p0071rep1-DsRed (B) and stained for -tubulin and DNA. (A) Wild-type p0071 associated with the plasma membrane in interphase cells (upper panel). During cell division wild-type p0071 localized at mitotic spindles (lower panel). (B) p0071rep1 exhibited plasma membrane association in interphase cells (upper panel) but was mislocalized during cytokinesis (lower panels). Scale bars: 10 µm.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Knockdown of p0071 and KIF3b induces multinucleation. (A) HEK293 cells were transfected with unspecific control siRNA, p0071 or KIF3b-specific siRNAs, respectively, p0071+KIF3b siRNAs and GFP-tagged Histone2b to identify transfected cells. Subsequently, cells were synchronized by a double thymidine block, released for 12 hours, then fixed and stained with Alexa-Fluor-594-conjugated phalloidin. At this point, many cells were bi- or multinucleated in response to the knockdown of p0071 or KIF3b. Scale bars: 10 µm. (B) The number of multinucleated cells was quantified. Mean values + s.d. of three independent experiments counting >100 cells each are shown. *** indicates P0.005. (C) HeLa cells were synchronized by successive thymidine and nocodazole treatment. At 30 minutes after nocodazole release cells were imaged for progression through cytokinesis. Cells transfected with control siRNA furrowed normally at 60 minutes after nocodazole release (1800 seconds, upper panel). P0071 knockdown cells exhibited strongly delayed and disturbed furrowing, resulting in failure of cytokinesis (lower panel). Scale bars: 10 µm. (D) HeLa cells were transfected with siRNAs as indicated, then synchronized, fixed at 90 minutes after nocodazole release and counted for normal and disturbed furrowing. Mean values + s.d. of three independent experiments counting >100 cells each are shown. * indicates P0.01.

View larger version (56K): [in this window] [in a new window]   Fig. 9. The phenotype of the KIF3b knockdown strongly resembles the phenotype of the p0071 knockdown. (A) HeLa cells were transfected with control siRNA, p0071-specific siRNA and KIF3b-specific siRNA as indicated, then synchronized and stained for actin (green) and phospho-MLC (p-MLC, red). Knockdown of p0071 and KIF3b considerably reduced the accumulation of actin and phosphorylated myosin light chain at the region of furrow ingression. Scale bars: 10 µm; enlargements 5 µm. (B) The level of p-MLC was reduced to a similar level in p0071 and KIF3b siRNA-transfected cells, respectively (40%). The graph displays the relative p-MLC amounts normalized to -tubulin + s.d. for three independent experiments. * indicates P0.01. (C) KIF3b and p0071 knockdown interfered with RhoA activation in telophase and cytokinesis. Cells were synchronized and probed for active RhoA at 90 minutes after nocodazole release. Active RhoA was precipitated by the GST-ROCK-Rho-binding-domain coupled to glutathione-agarose. The graph displays the relative amounts of active RhoA normalized to total RhoA +s s.d. for three independent experiments. * indicates P0.01.

View larger version (35K): [in this window] [in a new window]   Fig. 10. A p0071–MKLP1-motor-domain fusion protein rescues the KIF3b knockdown induced decrease in phospho-MLC. (A,B) HEK293 cells were transfected with KIF3b siRNA and p0071-DsRed expression vectors as indicated. (A) Phospho-MLC (p-MLC) was detected in the cell lysates. -tubulin was determined as a loading control. KIF3b knockdown and p0071 expression were verified using KIF3b and p0071 antibodies, respectively. Only the p0071–MKLP1-fusion protein was able to rescue the decrease in p-MLC observed after KIF3b knockdown. The graph displays the relative p-MLC amounts normalized to -tubulin + s.d. for three independent experiments. * indicates P0.01. (B) Cells were treated as described in (A) and the number of multinucleated cells was quantified. Mean values + s.d. of three independent experiments counting >100 cells each are shown.

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