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First published online 25 April 2006
doi: 10.1242/jcs.02922

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Motor protein KIFC5A interacts with Nubp1 and Nubp2, and is implicated in the regulation of centrosome duplication

Andri Christodoulou¹, Carsten W. Lederer¹, Thomas Surrey², Isabelle Vernos^2,* and Niovi Santama^1,

¹ Department of Biological Sciences, University of Cyprus and Cyprus Institute of Neurology and Genetics, PO Box 20537, 1678 Nicosia, Cyprus
² Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (29K): [in a new window]   Fig. 1. (A) Deduced protein sequence of mouse KIFC5A, isolated from the hippocampus of embryonic stage E13. The motor domain is located at the C-terminal. Underlined sequences are conserved motor domain `signature motifs', including the ATP-binding site (GX4GKT). The sequence to which an anti-peptide antiserum was produced is boxed. Shading shows the sequence of recombinant KIFC5A-s, used for motility and protein interaction assays. (B) Predicted probability of coiled-coil formation and protein domains in KIFC5A. Prediction of coils was based on the algorithm of Lupas et al. (Lupas et al., 1991) and domain analysis was performed using the Predict Protein Server (Rost and Liu, 2003). A sketch of KIFC5A overall organisation is shown at the bottom of the panel. (C) Detection of KIFC5A in NIH 3T3 cells. Western blot analysis of a total protein extract from NIH 3T3 fibroblasts, using the affinity-purified anti-peptide antibody to KIFC5A, reveals a band consistent with the predicted Mr of 69.253x103.

View larger version (48K): [in a new window]   Fig. 2. Expression of KIFC5A in the brain is subject to developmental and cell-type-specific regulation. (A) Comparative analysis of levels of KIFC5A cDNA (oligo set 3), synthesised from identical amounts of RNA isolated from the hippocampus of E13 and E18 mice, juvenile (2.5 weeks) and adult (3 months) animals, as well as from pure, proliferating, glia astrocyte cultures (newborn animals) and NIH 3T3 fibroblasts. Equivalent reactions for GAPDH (oligo set 4) were used as internal standards (bottom panel). (B) Tissue-specific pattern of expression of KIFC5A. Equivalent RT-PCR reactions from various mouse tissues (Str., striated). A mock reverse transcription reaction with E13 RNA was the negative (neg.) control. Equivalent reactions for L19 (oligo set 5) were used as internal standards (bottom panel).

View larger version (41K): [in a new window]   Fig. 3. (A) In vitro motility assay of fluorescently labelled MTs in the presence of the affinity-purified recombinant truncated protein KIFC5A-s. Movement of MTs on coverslips coated with KIFC5A-s was studied. The arrow points to a gliding MT. MTs moved at a maximum speed of 1.26 µm minute-1, n=40 (95% confidence). (B) Movement of polarity-marked MTs on recombinant KIFC5A-s. The minus-end of MTs is more brightly labelled. The white bar is at a stable position in all panels. The MT shown glided towards its plus-end, indicating minus-end-directed motility of KIFC5A. Video images were taken at the indicated time intervals (s, seconds). Bar, 10 µm.

View larger version (86K): [in a new window]   Fig. 4. Localisation of KIFC5A during the phases of the cell cycle in NIH 3T3 fibroblasts. (A) NIH 3T3 cells were processed for immunofluorescence with an antibody against -tubulin (red) and an antibody against KIFC5A (green). DNA was visualised with Hoechst (blue). (See also Fig. S4, supplementary material.) Bars, 5 µm. (B) NIH 3T3 cells, transiently transfected with YFP-KIFC5A. Only overlays are shown, with -tubulin staining in red, YFP-KIFC5A in green and DNA in blue. In the left-most panel, visualising a field of cells in interphase, only one cell is transfected. YFP-KIFC5A localisation matches closely that of the endogenous KIFC5A (panels in A). Bars, 10 µm.

View larger version (86K): [in a new window]   Fig. 5. Overexpression of KIFC5A inhibits bipolar spindle formation and causes stalling of mitotic progression in prometaphase. (A) Two typical examples of NIH 3T3 cells overexpressing YFP-KIFC5A (top panel) or myc-KIFC5A (bottom panel). Cells were labelled for -tubulin (red) or myc (green). DNA was visualised with Hoechst (blue), YFP-KIFC5A is green. Bars, 5 µm. (B) Another example of the `stalled phenotype' in prometaphase with the use of anti-pericentrin (top panel). In the overlay, YFP-KIFC5A is green, -tubulin staining is red and pericentrin is blue. A normal cell in prometaphase is shown for comparison (bottom panel). Bars, 5 µm.

View larger version (64K): [in a new window]   Fig. 6. RNAi-mediated silencing of KIFC5A causes centrosome amplification and aberrant multi-polar spindles in NIH 3T3 cells. (A1) Efficiency of silencing by quantitative real-time PCR. This shows an average knockdown of KIFC5A mRNA to 27.0±10.5% (s.d.) of control-silenced levels (silencing with medium GC content control silencing oligo; see Materials and Methods), whereas mRNA of the (unrelated) mitotic motor protein Eg5 shows 109.9±24.7% of control on average. mRNA levels of the PBGD housekeeping gene were used for sample normalisation. (A2) Efficiency of silencing by western blot. This shows a visible reduction of KIFC5A protein levels in silenced cells (lane 2; approx. 35%), compared with control-silenced cells (lane 1). Dynein detection (upper band) served as an internal loading control. (A3) Verification of silencing by triple immunofluorescence. Cells were immunolabelled for -tubulin, KIFC5A and counterstained for DNA (red, green and blue in the overlay, respectively). Whereas the lower panels (control-silenced cell) show normal KIFC5A labelling of the spindle in prometaphase, labelling is hardly detectable in the KIFC5A-silenced cell (upper panels) at the same exposure (300 milliseconds). Bars, 5 µm. (B1) Quantification of the silencing effect on centrosome numbers in mitotic cells. KIFC5A-silenced mitotic cells contain a higher number of centrosomes per cell, 120 hours after initial siRNA treatment, compared with the control. Shown are the means from three independent experiments (see also Table 2). Bars represent s.d. values. (B2) Immunofluorescence of silenced cells, displaying an increase in the number of centrosomes and spindle poles. The top panel shows an example of a KIFC5A-silenced cell, immunostained for -tubulin (green), -tubulin (red) and counterstained for DNA (blue). In the bottom panel, anti-centrin, instead of -tubulin, was used. The exact match between -tubulin (green) and centrin (red) labelling can be observed. The extracted detail in insets (bottom) illustrates that supernumerary centrosomes consist of duplicated centrioles (arrows). Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 7. (A) Diagram of the KIFC5A-s cDNA used as bait in the yeast two-hybrid system. (B) Protein sequence alignment between Nubp1 and Nubp2. Identical residues are boxed and conservative substitutions are highlighted in grey. The conserved ATP/GTP-binding motif (P loop) is marked in blue, the successive MRP motifs and ß are marked in green, and the sequence to which an anti-peptide serum to Nubp2 was produced is marked in orange. (C) In vitro co-selection experiments confirming interactions between KIFC5A&Nubp2, KIFC5A&Nubp1 and Nubp1&Nubp2. In the top panels, bacterially expressed tagged His6-KIFC5A-s, immobilised on Ni2+-NTA agarose beads, was incubated with a lysate from NIH 3T3 fibroblasts expressing GFP-Nubp2 or GFP-Nubp1 (lanes 1), or GFP only (negative control, lanes 2), or GFP-Nubp2 in the absence of pre-bound His6-KIFC5A-s (additional negative control, lanes 3). Bound proteins were probed by immunoblotting with anti-KIFC5A (left panel) or anti-GFP (right panel). A positive signal (detection of GFP-Nubp2 or GFP-Nubp1) is obtained only in the presence of pre-bound His6-KIFC5A-s (no signal in the absence of bound KIFC5A indicates that Nubp2 or Nubp1 does not bind the beads non-specifically). The co-selection of GFP-Nubp2 on the beads is not caused by interaction of KIFC5A with the FP domain (absence of signal in lane 2, right panel). In the bottom panels, the set-up of the experiment and negative controls is similar. GST-Nubp2 was immobilised on glutathione-Sepharose beads and was tested against GFP-Nubp1 (lanes 1), GFP only (negative control, lanes 2) or GFP-Nubp1 in the absence of GST-Nubp2 (additional negative control, lanes 3). Again, the positive signal (detection of Nubp1) was specific only in the presence of GST-Nubp2 (lanes 1). (D) Detection of native Nubp2 in NIH 3T3 cells. Western blot analysis of a total protein extract from NIH 3T3 fibroblasts, using the affinity-purified anti-peptide antibody to Nubp2, revealed a unique band consistent with the predicted Mr of Nubp2. (E) Localisation of Nubp2 in NIH 3T3 cells. Immunofluorescence for Nubp2 (green), centrin (red) and -tubulin (red). DNA was visualised with Hoechst (blue). (E1) Cells in interphase; (E2) cell in prophase; (E3) an aberrant spindle with four asters in prometaphase; (E4) double labelling with anti-centrin and anti-Nubp2 in a cell in prometaphase; (E5) Nubp2 enrichment in centrosomes (arrows) of the fully formed metaphase spindle, shown by double labelling with -tubulin. Bars, 10 µm (E2-E4) and 5 µm (E1). (F) Silencing phenotype after double Nubp1&Nubp2 RNAi, as revealed by triple fluorescence. Cells were immunolabelled for -tubulin, centrin and counterstained for DNA (red, green and blue in the overlay, respectively). Bar, 10 µm.