Expression of GFP-tagged MCPH1 isoforms in MCPH1-deficient 562T fibroblasts.

<p>(A) Cells were transduced with GFP-tagged coding sequence of full-length MCPH1 cDNA in a conditional, doxycycline (DOX)-dependent construct with a second regulatory construct trKRAB. Cultures were exposed to increasing DOX concentrations as indicated. Whole-cell extracts were prepared 72 h later and analyzed for the expression of MCPH1 using immunoblotting with an antibody against GFP. (B) The graph shows MCPH1 band intensity relative to the loading control p84 plotted against DOX concentrations. Data represent means ± one S.D. of three independent assays. (C) Immunoblot analysis of whole-cell extracts from non-transduced (NT) 562T cells (lane 1), 562T cells transduced with the regulatory construct only (lane 2), with GFP alone (30 kDa, lane 3) or with GFP fused to MCPH1-FL (120 kDa, lane 4), MCPH1Δe9–14 (94 kDa, lane 5), MCPH1Δe8 (78 kDa, lane 6), or MCPH1Δe1–7 (96 kDa, lane 7) with an antibody against GFP.. Nuclear matrix protein p84 served as the loading control.</p

Cell cycle-dependent regulation of MCPH1 transcripts.

<p>(A) HeLa cells were arrested in G1 phase by double thymidine block. Cultures harvested at various time points after release were analyzed using flow cytometry. (B) Plots represent numbers of cells as a function of their DNA content. A total of 90% of the cells synchronously progressed into S phase (0–4 h), entered G2 phase (4–6 h), started passing through mitosis after 7 h, and were completely in G1 phase after 12 h. (C) Levels of MCPH1-FL (diamonds, solid line), MCPH1Δe9–14 (squares, dotted line), and MCPH1Δe8 (circles, dashed line) mRNA. Data represent means ± S.E.M. of three independent experiments and are normalized to the expression levels of <i>GAPDH</i> and <i>B2M</i>.</p

The human <i>MCPH1</i> gene, its transcripts and predicted polypeptides.

<p>(A) Exon (filled boxes) and intron (open boxes) organization of the 241 906-bp encompassing <i>MCPH1</i> gene locus. Red arrows indicate the positions of the regular and of the alternative (*) polyadenylation sites (polyA). (B) The full-length (FL) and the alternative transcripts Δe9–14, Δe1–3, and Δe8: numbered boxes indicate exons, black filled areas illustrate the entire coding regions (CDS), and colored areas show untranslated regions (UTR) as indicated. (C) Predicted polypeptides representing MCPH1 isoforms: blue boxes depict the positions of BRCT domains, while green boxes represent the site of the canonical nuclear localization signal sequence (NLS). Two additional amino acids, S and M, are included into MCPH1Δe9–14 prior to premature termination (#). (D) Expression of MCPH1 transcript variants. Columns represent the levels of MCPH1 transcripts in indicated adult and fetal tissues determined using quantitative real-time PCR. Data represent means ± one S.D. of three independent experiments and are normalized to the geometric mean levels of <i>UBC</i>, <i>GAPDH</i>, <i>B2M</i>, and <i>HPRT1</i> cDNA.</p

Intracellular distribution of MCPH1 isoforms.

<p>(A) MCPH1-deficient fibroblasts expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fractionated and cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were analyzed using immunoblotting with an antibody against GFP. The nuclear matrix protein p84 and GAPDH were used as index proteins and loading controls. (B) Cells indicated in A stained with an anti-GFP antibody (green), counterstained with DAPI (blue) and analyzed using fluorescence microscopy. Arrows indicate the prophase-like nuclei. Scale bar = 10 µm. All MCPH1 isoforms exhibit unambiguous nuclear localization.</p

Colocalization of MCPH1 and γH2AX in ionizing irradiation-induced nuclear foci.

<p>(A) Non-transduced (NT) MCPH1-deficient 562T cells and 562T stably expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fixed 2 h after irradiation with 10 Gy and co-stained with antibodies against γH2AX (red) and GFP (green). Nuclei were counterstained with DAPI (blue). Rectangles frame areas, which are shown enlarged in the bottom row. MCPH1 focus formation was observed for MCPH1 isoforms containing the C-terminal BRCT tandem. (B) Quantification of cells expressing foci containing γH2AX and/or (C) MCPH1. Error bars indicate the S.D. of three different measurements, counting approximately 300 nuclei. * <i>p</i>≤0.05 vs. NT as calculated using the Student's <i>t</i>-test.</p

Nuclear localization signals (NLSs) in human MCPH1.

<p>(A) The positions of the putative NLS motifs and their amino acid sequences are highlighted in the diagram of the full-length MCPH1. (B) Subcellular distribution of GFP-tagged wild-type (wt) MCPH1 and mutants with deleted NLSs as indicated were transiently expressed in HeLa cells. Cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were immunoblotted with an antibody against GFP (left panel). GAPDH (center panel) and the nuclear matrix protein p84 (right panel) served as index proteins and loading controls. (C) Ratios of relative GFP band intensity in the cytoplasmic (Cyt) vs. nuclear (Nuc) fractions. Absolute numbers were assessed using densitometry and normalized to the loading controls. Columns designate means, and error bars represent the S.D. from three different experiments. Significant differences to wt MCPH1 are indicated by asterisks denoting <i>p</i><0.05 (Student's <i>t</i>-test). Scale bar = 10 µm.</p

Complementation of PCC in patient fibroblasts.

<p>Cells are derived from patient with homozygous truncating mutation c.427dupA (p.T143NfsX5) in <i>MCPH1</i>. Chromosome preparations from (A) non-transduced cells and (B) cells expressing GFP only, or GFP fusions with (C) full-length, (D) Δe9–14, (E) Δe8, or (F) Δe1–7 MCPH1. Arrows indicate nuclei of prophase-like cells (PLCs). (G) Mean rates of PLCs (filled columns) of slides from A-F. Open columns represent mean mitotic indices. Error bars denote the S.D. of counts of approximately 1000 cells each from three independent experiments.</p