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First published online 28 April 2009
doi: 10.1242/jcs.047738

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A splice variant of cyclin D2 regulates cardiomyocyte cell cycle through a novel protein aggregation pathway

Qian Sun^*, Feixiong Zhang^*, Karim Wafa, Timothy Baptist and Kishore B. S. Pasumarthi

Department of Pharmacology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, NS, B3H 1X5 Canada

View larger version (34K): [in this window] [in a new window]   Fig. 1. (A) Deduced amino acid sequence of the D2SV cDNA cloned from the mouse heart RNA. The novel 20 amino acid C-terminus (CT) is underlined. The + signs above the CT indicate the conserved amino acid residues. (B) Comparison of functional domains of cyclin D1, D2 and D2SV. Binding sites for the members of retinoblastoma protein family (Rb), p21, CDK4, Dmp1 and estrogen receptor (ER) as well as the phosphorylation site of GSK-3β at Thr286 are indicated. Numbers indicate amino acid residues. Sequence homologies between different domains of D2SV and members of the cyclin D family (cycD1, D2 and D3) are also indicated. (C) Western blot analysis of HEK293 cells transfected with an empty pcDNA3.1 vector (Con) and pcDNA3.1-D2SV construct (D2SV) using polyclonal antibodies raised against the C-terminus of D2SV protein. Arrows indicate high molecular mass D2SV immunoreactive bands.

View larger version (87K): [in this window] [in a new window]   Fig. 2. Characterization of endogenous D2SV expression in embryonic and adult mouse hearts. (A,B) Low magnification views of E11.5 embryo transverse sections simultaneously processed for D2SV immunostaining (A) and nuclear stain (B). Note the abundance of D2SV protein in the myocardial region compared with the surrounding tissue. (C) A control E11.5 section incubated with secondary antibodies but without the D2SV antibody. (D) A merged image of an E14.5 myocardial region processed for D2SV and nuclear staining. Arrows indicate trabecular zone and asterisks indicate compact zone myocytes. (E) Confocal microscopy indicates the presence of cytoplasmic D2SV micro-aggregates in E11.5 myocardial cells. (F-H) Low magnification (F) and high magnification (G,H) views of adult mouse heart ventricular sections simultaneously processed for D2SV immunostaining (F,G) and nuclear stain (H). Scale bars: 100 µm (A-C,D), 20 µm (E), 25 µm (F) and 10 µm (G,H).

View larger version (63K): [in this window] [in a new window]   Fig. 3. (A-E) Relationship between endogenous D2SV expression and cardiomyocyte cell cycle activity. (A) Sections of E14.5 heart showing phosphohistone H3 immunostaining (green) in D2SV-expressing (red, arrows) and -nonexpressing (arrowheads) cardiomyocytes. (B-D) Endogenous D2SV expression and [3H]thymidine incorporation. D2SV expression was identified by anti-D2SV immunostaining (B), nuclei were visualized with Hoechst stain (C) and thymidine in the nucleus (silver grains) was visualized using bright-field optics (D). (E) [3H]thymidine labeling index is determined as the percentage of D2SV-positive or -negative myocytes that also show thymidine incorporation. *P<0.005; n=3 experiments, 500 cells/group. (F,G) Non-denatured protein samples from HEK293 cells transfected with an empty vector (Con) or the D2SV construct (D2SV) were fractionated on either a 5% basic-native PAGE gel (F) or on a 12.5% denaturing SDS-PAGE gel (G). Note the absence of the low molecular mass D2SV band under complete non-denaturing conditions (F) but not under partially denaturing conditions (G). Scale bars: 20 µm.

View larger version (67K): [in this window] [in a new window]   Fig. 4. Subcellular localization of D2SV (A-F,J) and cycD2 (G-I) proteins in transfected E14.5 ventricular myocyte cultures. (A-I) Cells were processed for immunostaining with antibodies specific for Myc (A,G), D2SV (D,J) and sarcomeric myosin (E). Nuclei were counterstained with Hoechst stain (B,H). (J) Confocal imaging was used to generate a mid-plane optical section (0.5 µm) of a transfected cell co-stained for D2SV (Alexa Fluor 488) and membrane-specific ConA-Alexa Fluor 647 stains. Scale bars: 20 µm. (K) Quantification of protein aggregation in E14.5 myocytes expressing D2SV or cycD2. *P<0.05, D2SV versus cycD2, n=3-5 experiments, 300 cells per group.

View larger version (53K): [in this window] [in a new window]   Fig. 5. (A-C) Photomicrographs depicting [3H]thymidine labeling assay. Transfected E14.5 myocytes were identified by anti-Myc immunostaining (A), nuclei were visualized with Hoechst stain (B) and thymidine in the nucleus (silver grains) was visualized using bright-field optics (C). Scale bar: 20 µm. (D) The labeling index, determined as the percentage of Myc-positive myocytes that also show the presence of silver grains. *P<0.005; D2SV or NLS-D2SV versus other groups, n=3-6 experiments/group, 300-600 cells/group.

View larger version (54K): [in this window] [in a new window]   Fig. 6. (A-H) Photomicrographs depicting localization of D2SV and CDK4 (A-C) and cycD2 and CDK4 (D-F) in E14.5 cultures co-transfected with the D2SVMyc or cycD2Myc and CDK4 constructs. Note the presence of colocalized protein aggregates in the cytoplasm of myocytes transfected with D2SV and CDK4 (A-C) but not those co-transfected with cycD2 and CDK4 (D-F). Scale bars: 20 µm. (G) Quantification of DNA synthesis in transfected cardiomyocyte cultures. *P<0.005; cycD2+CDK4 versus other groups, n=3-6 experiments/group, 300 cells per group.

View larger version (73K): [in this window] [in a new window]   Fig. 7. Subcellular trafficking of D2SV protein aggregates. E14.5 myocyte cultures were transfected with constructs pcDNA-D2SVMyc (A-F; J-L) or pEGFP-D2SV (G-I) and stained with markers as indicated. (A-C) Transfected myocytes were processed for Myc (A) and protein disulphide isomerase (PDI, an ER marker; B) immunostaining and nuclei were stained with Hoechst (in the merged image, C). (D-F) Myocytes were simultaneously processed for Myc (D) and giantin (Golgi marker; E) immunostaining. (G-I) D2SV-transfected cells were identified using an EGFP tag (G) and lysosomes were visualized using Lysotracker Red DND-99 stain (H) in live cultures using a confocal microscope. (J-L) Cells were simultaneously processed for Myc immunostaining (J) and TCR-GFP (K, ER stress reporter). Scale bars: 20 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 8. (A) Faucher and Pliska hydrophobicity plot of D2SV protein sequence. Note the high degree of hydrophobicity in the CT region (amino acids 136-156) as indicated by a higher index on y-axis. (B-E) E14.5 myocytes were transfected with the D2SVMyc construct (B,C) or a construct lacking the CT region (D2SVCTMyc; D,E), and processed for Myc immunostaining (B,D) and nuclei were counterstained with Hoechst stain (C,E). Scale bars: 20 µm. (F,G) Quantification of protein aggregation in cells transfected with D2SV (F) and D2SVCT (G). (F) *P<0.05 for cytoplasmic aggregates versus all other groups, (G) #P<0.05 for nuclear aggregates versus all other groups, n=3 experiments/group, 300 transfected cells per group. Nuc, nuclear aggregates; Perinuc, perinuclear aggregates; Cyto, cytoplasmic aggregates; No, no aggregates.

View larger version (44K): [in this window] [in a new window]   Fig. 9. (A-F) Increased levels of dynamitin expression result in loss of D2SV aggregation. Dynamitin (Dyn) and D2SV were visualized by anti-Myc (A,D) and anti-D2SV (B,E) immunostaining, respectively. (G) Quantification of protein aggregation in E14.5 cells transfected with D2SV and dynamitin. Cells were transfected with 1 µg of D2SV and 1 µg of pcDNA3.1 (Con) or 1 µg of Myc-tagged dynamitin plasmids. *P<0.05 for D2SV+Con versus D2SV+dynamitin in both aggregation-positive and no aggregation groups; n=3 experiments/group, 300 transfected cells per group. (H-M) Increased levels of CHOP expression result in loss of protein aggregation and promote nuclear localization of D2SV and cycD2. Signals were visualized by anti-Myc (H,K), anti-D2SV (I) and anti-cycD2 (L) antibodies. Nuclei were counterstained with Hoechst stain (J,M). (N) Quantification of protein aggregation in cells co-transfected with D2SV and CHOP plasmids. Cells were transfected with 1 µg of D2SV and 1 µg of pcDNA3.1 (Con) or varying concentrations (0.1-1 µg) of Myc-tagged CHOP plasmids. For all D2SV+CHOP groups, total plasmid DNA was kept to 2 µg by supplementing with pcDNA3.1 plasmid. *P<0.05 for D2SV+CHOP 1:0.5 and 1:1 groups versus all other groups, n=3 experiments, 300 transfected cells per group. Scale bars: 20 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 10. (A-L) Overexpression of D2SV can sequester endogenous cyclin D2 (endo D2; A-C) and cyclin B1 (endo B1; D-F) or transfected cycD1 (tf D1; G-I) and cycB1 (tf B1; J-L) into aggregates. Transfected E14.5 cells were processed for immunostaining with antibodies specific for Myc (A,D,G,J), cycD2 (B), cycB1 (E,K) and cycD1 (H). Nuclei were counterstained with Hoechst stain (see merged images, C,F,I,L). The presence of colocalized protein aggregates in merged images in D, H and L is indicated by arrows. The arrowheads in A-C point to a D2SV aggregate that does not co-aggregate with endogenous D2. Scale bars: 20 µm. (M) Interaction of endogenous D2SV in whole embryo lysates with endogenous cell cycle proteins, IP, immunoprecipitation; Super, supernatant after IP; ab, antibody; LC, IgG light chain; HC, IgG heavy chain. (N) Co-expression of D-type or B-type cyclins with D2SV can increase DNA synthesis similar to the levels observed with vector-transfected or non-transfected cells. *P<0.005; D2SV versus other groups, n=3-6 experiments/group, 300–600 cells per group.

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