Figure 6.
The interaction of TCL1A and CDC20 takes place via defined motifs and impairs CDC20’s interaction with its negative regulators MAD2 and PLK1. (A) PLA of CLL-like MEC1 cells that were synchronized in mitosis (9 μM RO-3306 for 20 hours, 1 hour after release). TCL1A interacts with CDC20, MAD2, and CDK1 during mitosis. Quantification of PLA foci per cell is displayed in supplemental Figure 7A. (B) Images were taken using an SP8 confocal microscope (Leica) PLA for TCL1A and CDC20 was performed in MEC1 and HG3 CLL-like cells (synchronized as in panel A), and PLA foci per cell were quantified in mitotic and nonmitotic cells. Significantly more foci were counted in mitotic cells compared with nonmitotic cells. Negative control for MEC1: no primary antibody staining (Backgr.). Negative control for HG3: EV-transfected cells (TCL1A-negative HG3-EV) stained with primary antibodies. Boxes display medians with 25th to 75th percentiles and whiskers minimum and maximum; N = 25 cells (significances by one-way analysis of variance). Representative immunofluorescent images are in supplemental Figure 7C. (C) Split yellow fluorescent protein (YFP)–reporter principle of the protein complementation assay (PCA). Signal emission in living cells was induced upon noncovalent complementation of the N/C-terminal YFP components if the TCL1A-bait and the ‘candidate’ protein interact for >0.5 seconds. As a negative control, a construct encoding an ATG codon fused with the N-terminal part of YFP (ATG-YFP) was used to exclude fluorescence derived from spontaneous transient rejoining of split YFP parts. Images were taken on an Axio Scope.A1 microscope at original magnification ×100. (D) HeLa cells were cotransfected with TCL1A-YFPN and CDC20-YFPC constructs and the TCL1A-CDC20 interaction was visualized by the YFP signal (green). Cytoskeleton and DNA were visualized by rhodamine-phalloidin (red) and Hoechst (blue), respectively. Prominent signals corresponding to the specific TCL1A-CDC20 interaction accumulated at the mitotic plate and dispersed during anaphase. (E) Five constructs of indicated CDC20 variants were generated by PCR-mediated site-directed mutagenesis (top) and were used in co-IPs in HEK293 cells to interrogate sequence restrictions of the CDC20-TCL1A interaction (bottom). RCRY4A and del(97-169) nearly completely abolished the interaction. (F) The CDC20-TCL1A complex was predicted based on the published experimental protein structures of CDC20 (pdb ID 4GGA) and TCL1A (pdb ID 1JSG). Predictive tools to propose putative interfaces (CPORT)36 were used and the outputs exploited in the High Ambiguity Driven protein-protein DOCKing 2.2 (HADDOCK 2.2) algorithm for modeling biomolecular complexes.37 This in silico modeling predicted 2 potential models, which differ in the amino acids (aa) of TCL1A involved in this interaction. The residues E40, K42, and Y96 of TCL1A are expected to stabilize the TCL1A-CDC20 interaction in model A, whereas E9, R93, and Y96 would promote this interaction in model B. Detailed HADDOCK statistics are summarized in supplemental Tables 12 and 13. (G) TCL1A mutants used in co-IP studies (top). They represent model A (EKYSAE, in red) with the aa substitutions E40S, K42A, and Y96E, and model B (ERYKDE, blue) with E9K, R93D, and Y96E. CDC20 co-IP of lysates from JVM3 cells stably overexpressing TCL1A-FLAG, TCL1A-EKYSAE-FLAG, or TCL1A-ERYKDE-FLAG (bottom). Cells were synchronized before in G2 by 9 μM RO-3306 for 20 hours. In the last 5 hours, 10 μM MG132 was added to reduce protein degradation of the 2 mutants. The EKYSAE (model A) nearly abolished the TCL1A-CDC20 interaction. (H) PLA for CDC20 and MAD2 was performed in MEC1-shTCL1A (TCL1A knockdown) vs -shCtrl cells as well as in HG3-EV vs -TCL1A (TCL1A introduction) cells. Significantly less PLA foci were counted per cell in the TCL1A-expressing/-high lines, suggesting an impaired CDC20-MAD2 interaction in the presence of TCL1A. Boxes display medians with 25th to 75th percentiles and whiskers minimum and maximum; N = 40 cells (significances as per Student t test). Images were taken using an SP8 confocal microscope. (I) JVM3 ± TCL1A cells were synchronized in G2 by 9 μM RO-3306 for 20 hours and released in full medium. CDC20-IP was performed in lysates at indicated time points to determine CDC20-PLK1 interaction. IgG control: pooled lysates of JVM3EV and JVM3TCL1A. CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; MIM, MAD2-interacting motif; neg, negative.

The interaction of TCL1A and CDC20 takes place via defined motifs and impairs CDC20’s interaction with its negative regulators MAD2 and PLK1. (A) PLA of CLL-like MEC1 cells that were synchronized in mitosis (9 μM RO-3306 for 20 hours, 1 hour after release). TCL1A interacts with CDC20, MAD2, and CDK1 during mitosis. Quantification of PLA foci per cell is displayed in supplemental Figure 7A. (B) Images were taken using an SP8 confocal microscope (Leica) PLA for TCL1A and CDC20 was performed in MEC1 and HG3 CLL-like cells (synchronized as in panel A), and PLA foci per cell were quantified in mitotic and nonmitotic cells. Significantly more foci were counted in mitotic cells compared with nonmitotic cells. Negative control for MEC1: no primary antibody staining (Backgr.). Negative control for HG3: EV-transfected cells (TCL1A-negative HG3-EV) stained with primary antibodies. Boxes display medians with 25th to 75th percentiles and whiskers minimum and maximum; N = 25 cells (significances by one-way analysis of variance). Representative immunofluorescent images are in supplemental Figure 7C. (C) Split yellow fluorescent protein (YFP)–reporter principle of the protein complementation assay (PCA). Signal emission in living cells was induced upon noncovalent complementation of the N/C-terminal YFP components if the TCL1A-bait and the ‘candidate’ protein interact for >0.5 seconds. As a negative control, a construct encoding an ATG codon fused with the N-terminal part of YFP (ATG-YFP) was used to exclude fluorescence derived from spontaneous transient rejoining of split YFP parts. Images were taken on an Axio Scope.A1 microscope at original magnification ×100. (D) HeLa cells were cotransfected with TCL1A-YFPN and CDC20-YFPC constructs and the TCL1A-CDC20 interaction was visualized by the YFP signal (green). Cytoskeleton and DNA were visualized by rhodamine-phalloidin (red) and Hoechst (blue), respectively. Prominent signals corresponding to the specific TCL1A-CDC20 interaction accumulated at the mitotic plate and dispersed during anaphase. (E) Five constructs of indicated CDC20 variants were generated by PCR-mediated site-directed mutagenesis (top) and were used in co-IPs in HEK293 cells to interrogate sequence restrictions of the CDC20-TCL1A interaction (bottom). RCRY4A and del(97-169) nearly completely abolished the interaction. (F) The CDC20-TCL1A complex was predicted based on the published experimental protein structures of CDC20 (pdb ID 4GGA) and TCL1A (pdb ID 1JSG). Predictive tools to propose putative interfaces (CPORT)36 were used and the outputs exploited in the High Ambiguity Driven protein-protein DOCKing 2.2 (HADDOCK 2.2) algorithm for modeling biomolecular complexes.37 This in silico modeling predicted 2 potential models, which differ in the amino acids (aa) of TCL1A involved in this interaction. The residues E40, K42, and Y96 of TCL1A are expected to stabilize the TCL1A-CDC20 interaction in model A, whereas E9, R93, and Y96 would promote this interaction in model B. Detailed HADDOCK statistics are summarized in supplemental Tables 12 and 13. (G) TCL1A mutants used in co-IP studies (top). They represent model A (EKYSAE, in red) with the aa substitutions E40S, K42A, and Y96E, and model B (ERYKDE, blue) with E9K, R93D, and Y96E. CDC20 co-IP of lysates from JVM3 cells stably overexpressing TCL1A-FLAG, TCL1A-EKYSAE-FLAG, or TCL1A-ERYKDE-FLAG (bottom). Cells were synchronized before in G2 by 9 μM RO-3306 for 20 hours. In the last 5 hours, 10 μM MG132 was added to reduce protein degradation of the 2 mutants. The EKYSAE (model A) nearly abolished the TCL1A-CDC20 interaction. (H) PLA for CDC20 and MAD2 was performed in MEC1-shTCL1A (TCL1A knockdown) vs -shCtrl cells as well as in HG3-EV vs -TCL1A (TCL1A introduction) cells. Significantly less PLA foci were counted per cell in the TCL1A-expressing/-high lines, suggesting an impaired CDC20-MAD2 interaction in the presence of TCL1A. Boxes display medians with 25th to 75th percentiles and whiskers minimum and maximum; N = 40 cells (significances as per Student t test). Images were taken using an SP8 confocal microscope. (I) JVM3 ± TCL1A cells were synchronized in G2 by 9 μM RO-3306 for 20 hours and released in full medium. CDC20-IP was performed in lysates at indicated time points to determine CDC20-PLK1 interaction. IgG control: pooled lysates of JVM3EV and JVM3TCL1A. CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; MIM, MAD2-interacting motif; neg, negative.

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