AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

Abstract

Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.

Keywords: mitotic arrest deficient; translocated in promoter region.

Figure 1.

Abnormal nuclear morphology, CCFs and lagging chromosomes in AKAP95-depleted cells. (A) Quantification of nuclei with indicated phenotypes in DAPI-stained HeLa cells siRNA-treated with oligonucleotides to AKAP95 (siAKAP95), an unrelated protein (siUnrProt) and with a random sequence (siControl). Data (mean +/− SEM) from 4 independent experiments (N = 600) analyzed by Student's t test. **P < 0.01, ***P < 0.001 compared with siControl. Corresponding HeLa nuclei images (bottom). Scale bar 5 μm. (B). Time lapse imaging of AKAP95-depleted and control HeLa^H2B-GFP cells starting from NEBD and with image acquisition every 5 minutes. Chromosome segregation defects and micronuclei (arrowheads). (C) Quantification of cells as in B with indicated chromosome segregation pattern. Data (mean +/− SEM) from 4 independent experiments (N = 28).

Figure 2.

Identification of in vivo binding partners of AKAP95. (A) Schematic representation of Myc-BirA*-AKAP95. (B) Western blot analysis using anti-myc antibody and HRP-labeled streptavidin of lysates from Myc-BirA*-AKAP95 expressing- and control U2OS cells cultured as indicated. (C) Immunolocalization of Myc-BirA*-AKAP95 (anti-myc; red) and protein biotinylation (FITC-labeled streptavidin; green) in stable Myc-BirA*–AKAP95 U2OS cells cultured as indicated. Immunolocalization of AKAP95 in U2OS cells (bottom). Scale bar, 5 μm. (D) Gene ontology analysis and nuclear component classification of proteins identified by AKAP95 BioID. (E) Western blot analysis using indicated antibodies of the samples from Myc-BirA*-AKAP95-expressing and control U2OS cells that were subjected to BioID.

Figure 3.

AKAP95 targets TPR to the mitotic spindle area. (A) Western blot analysis using indicated antibodies of immunoprecipitates from mitotic HeLa cell lysates. (B) Immunolocalization of AKAP95 and TPR at indicated mitotic stages in unsynchronized HeLa cells. Insets: DAPI staining. Plot profiles show for both channels the variation in fluorescence intensity along the indicated line. (C) Confocal microscopy 3D image projections from PLA experiments between TPR and AKAP95. Positive control: PLA between TPR using 2 antibodies from different species. (D) Quantification of cells as in C with indicated number of foci per nucleus from 3 representative experiments (N = 36). (E) Immunolocalization of AKAP95 and TPR at mitosis in HeLa cells as indicated. Insets: DAPI staining. Scale bar = 5 μm. Plot profiles show the variation in TPR fluorescence intensity along the indicated line. (F) Western blot analysis using indicated antibodies (top) and quantification of micronuclei (lower) in HeLa cells treated as indicated. Quantification from 3 independent experiments (mean +/− SEM, N = 83).

Figure 4.

AKAP95-depleted HeLa cells have a compromised SAC. (A) Immunofluorescence analysis with indicated antibodies of AKAP95-depleted and control HeLa cells treated with nocodazole. Indicated areas are shown after deconvolution, magnification and merge of anti-centromere HCT-0100 and MAD1 fluorescence signals. Scale bar 5 μm. (B) Fluorescence intensity quantification of MAD1 relative to HCT-0100 signals at KTs from cells in A (mean +/− SEM, N = 100, analyzed by Student's t test ***P < 0.001). (C) Time lapse imaging of AKAP95-depleted and control HeLa^H2B-GFP cells. Scale bar 5 μm. Quantification of absolute time from NEBD to anaphase onset (mean +/− SEM, N = 20, analyzed by Student's t test. **P < 0.01). (D) Flow analysis of AKAP95-depleted and control HeLa cells treated with nocodazole for the indicated period and stained with anti-phospho-Histone H3. (E) Quantification of the mitotic cell populations from D from 1 representative experiment.