Figure 1.
Splice Variants of the CDC14B Parental Gene and Subcellular Localization of Encoded Proteins
(A) Exon and protein structures of CDC14B splice variants. Color code of coding exons (exons not drawn to scale): exons shared by all variants are colored in dark blue; exons colored in yellow, light blue, green, and red are specific to transcripts CDC14B1, CDC14B2, CDC14B3, and CDC14Bpar, respectively, as indicated by the schematic representation of splicing patterns. The basic protein structure and sequence encoded by the different splice variants is indicated (only the protein sequence that differs between the splice variants is color-coded to indicate the relationship with the alternative splicing patterns of the coding exons). The retroduplication event of the CDC14Bpar transcript that gave rise to the CDC14B retrogene (CDC14Bretro) is illustrated.
(B–E) COS7-cells (from African green monkeys—no endogenous CDC14Bretro gene) transfected with CDC14B-DsRed fusion constructs (i.e., CDC14B signals in red) are shown. Microtubules were stained using an anti-beta tubulin antibody (green) and the nucleus was stained using DAPI (blue). Merged images: CDC14B1 (nucleoli) and −2 (nuclear speckles) localize to the nucleus (violet) and CDC14B3 and CDC14Bpar co-localize with microtubules (yellow). We obtained very similar results when using HeLa or LN229 (glioblastoma) cell lines (both carrying an endogenous CDC14Bretro gene, Figure S1). Thus, there appear to be no cell line–specific effects with respect to the observed subcellular localization patterns. Scale bars (white) = 10 μm.
Figure 2.
Spatial Expression Patterns of the CDC14B Parental and Retrogene Variants
Shaded areas indicate expression in a given tissue. “ND” indicates that expression was not determined. Note that weak amplification products of human CDC14Bretro were obtained for the following tissues in some of the cDNA panels tested: kidney, lung, ovary, thymus, and thyroid. This suggests that it may be expressed at low levels in these (and perhaps other) tissues in some individuals.
Figure 3.
Coding Sequence Evolution of CDC14Bretro/CDC14Bpar and Subcellular Localization of Encoded Proteins
(A) Phylogenetic tree based on the CDC14B coding sequences (mouse CDC14Bpar sequence is used as an outgroup, see Materials and Methods for details). Approximate divergence times in millions of years (Mya) are shown (estimates based on [15]). Maximum likelihood dN/dS values and the estimated number of nonsynonymous over synonymous substitutions (in parentheses) for each branch are indicated (estimated numbers of substitutions are rounded to the nearest integer). Internal nodes: Duplication event (node D), Before positive selection (node B, sequence from last common great ape ancestor), and After positive selection (node A, last common African ape ancestor). The branch showing positive selection is highlighted in red.
Abbreviations: human CDC14Bretro, HuRetro; chimpanzee CDC14Bretro, ChRetro; gorilla CDC14Bretro, GoRetro; orangutan CDC14Bretro, OrRetro; gibbon CDC14Bretro, GiRetro; human CDC14Bpar, HuPar.
(B) CDC14Bretro from node A in interphase. COS7 cells transfected with a CDC14Bretro-GFP fusion construct (i.e., CDC14B signals in green) are shown. Microtubules are stained in red (anti-beta tubulin antibody). Nuclear DNA was stained with DAPI (blue).
(C) Human CDC14Bretro in interphase. (D) Human CDC14Bretro during mitosis. (E) Human CDC14Bretro during cytokinesis. (F) CDC14Bretro from node B in interphase. (G) Human CDC14Bpar in interphase. (H) CDC14Bpar during mitosis. (I) CDC14Bpar during cytokinesis.
Only merged images are shown; unmerged images are shown in Figure S3. Similar results were obtained when using either human HeLa or LN229 cell lines (Figure S4). Scale bars = 10 μm.
Figure 4.
Alignment of Extant and Reconstructed CDC14Bretro and CDC14Bpar sequences
N and C termini are highlighted in light and dark blue, respectively. The 12 sites where amino acid changes arose in the common African ape ancestor are highlighted in orange. The amino acid motif KKIR, previously described to be crucial for nuclear localization of the parental variants CDC14B1 and CDC14B2, is highlighted in green. Sites crucial for the phosphatase function of CDC14B [17,35] are highlighted in dark red. The sequence N terminal of the new initial methionine in humans and chimpanzees is marked in gray, as this part of the sequence is likely not translated in these species. The same abbreviations as in the legend of Figure 3 were used to designate CDC14B sequences from the different species.
Figure 5.
Subcellular Localization of CDC14Bretro from Humans and Node A
(A) Human CDC14Bretro in interphase. COS7 cells transfected with two different CDC14Bretro-GFP fusion constructs (green) and labeled with an anti-GRP94 antibody (red, a marker of the endoplasmic reticulum) and with DAPI (blue).
(B) CDC14Bretro from node A (see Figure 3) in interphase.
(C) Human CDC14Bretro during mitosis.
Similar results were obtained when using either human HeLa or LN229 cell lines (Figure S8). We note that in addition to the ER, CDC14Bretro from African apes localizes to vesicles that show no co-staining with any of the cellular markers tested (see Materials and Methods) more than 24 h after transfection. Scale bars = 10 μm.
Figure 6.
Subcellular Localization of Hybrid Constructs BAB and ABA
COS7 cells transfected with hybrid fusion construct BAB and ABA (i.e., CDC14B signals in green). Microtubules stained in red (anti-beta tubulin antibody). Nuclear DNA was stained with DAPI (blue). Scale bars = 10 μm.
Figure 7.
Microtubule Stabilization Capacity of CDC14Bretro and Parental CDC14B Variants
Cells transfected with CDC14Bretro/CDC14B were treated with the depolymerizing agent nocodazole (see Materials and Methods for details). All experiments were repeated five times. Representative images of COS7 cells treated with nocodazole 24 h after transfection with human CDC14Bretro and human CDC14Bpar (fused to DsRed), in which microtubules (labeled in green) are stabilized (right image, yellow/green filaments; yellow indicates CDC14Bpar-microtubule co-localization) or degraded (left image) are shown.
Statistical analysis reveals significant stabilization differences between the different CDC14B proteins (one-way ANOVA, p < 10−4). Relevant pairwise comparisons of proteins that show significant stabilization differences (p < 0.01, Tukey's Post Hoc test): (i) CDC14Bretro from humans, chimpanzee, gorilla, node A versus CDC14Bretro from orangutan, gibbon, node B, CDC14Bpar, and CDC14B3; (ii) CDC14B1 and CDC14B2 versus CDC14Bpar/CDC14B3; (iii) node B versus BAB/ABA hybrids; (iv) BAB versus ABA; (v) CDC14Bretro from orangutan and gibbon versus CDC14Bretro from node B and CDC14Bpar. Scale bars = 10 μm.