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First published online 23 January 2003
doi: 10.1242/jcs.00330

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Dynamic relocalization of the chromosomal passenger complex proteins inner centromere protein (INCENP) and aurora-B kinase during male mouse meiosis

¹ Departamento de Biología, Edificio de Biológicas, Universidad Autónoma de Madrid, E-28049 Madrid, Spain
² Institute of Cell and Molecular Biology, University of Edinburgh, Swann Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland, UK

View larger version (68K): [in a new window]   Fig. 1. Immunoblot analysis of HeLa cell extracts (lane 1) and mouse testes extracts (lane 2) probed with pAb1186 serum. The positions of molecular mass markers are indicated by numbers, and the arrowhead indicates the INCENP bands.

View larger version (65K): [in a new window]   Fig. 2. Expression of INCENP in spermatogonial mitosis (INCENP, green; ACA serum, red; DAPI, blue). (A,B) Partial projection of two focal planes throughout a spermatogonial interphase showing bright INCENP signals at heterochromatic chromocentres (arrows) highlighted with DAPI. ACA signals are located inside chromocentres. (C-E) Partial projection of three focal planes throughout a spermatogonial prophase. Bright fuzzy INCENP signals are located at centromeres (arrow in C,E) where pairs of ACA dots, representing sister kinetochores (arrows in D), are found. Inserts show a larger magnification of the arrowed centromere. A faint INCENP labelling is also found on condensing chromosomes. (F,G) Partial projection of five focal planes throughout an end-on view of a spermatogonial metaphase. A faint INCENP labelling is found on chromosome arms, whereas bright fuzzy signals are found at centromeric regions. The insert shows a centromeric region with its two sister kinetochores. (H,I) Partial projection of two focal planes throughout a spermatogonial anaphase. INCENP only labels some thin threads (arrows) at the spindle midzone. (J,K) Partial projection of two focal planes throughout a spermatogonial telophase where INCENP is present at two small and very bright signals (arrowheads) at both sides of the midbody. Bar, 10 µm.

View larger version (96K): [in a new window]   Fig. 3. INCENP distribution in early prophase I spermatocytes (INCENP, green; ACA serum, red; DAPI, blue). (A,B) Partial projection of 21 focal planes throughout an early prophase I spermatocyte. No INCENP signals are discerned; instead only ACA signals representing unpaired centromeres are seen. (C,D) Partial projection of 15 focal planes throughout an early prophase I spermatocyte. Some short and discontinuous INCENP threads (arrows) are observed in the nucleus. (E,F) Partial projection of three focal planes throughout a pachytene spermatocyte. Long INCENP threads (arrows) showing a heterogeneous thickness are detected in the nucleus. The sex body (XY) shows a faint INCENP labelling (arrowhead). (G) Projection of two focal planes of an INCENP-labelled thread at pachytene. The thread presents narrower regions (arrowheads) along its length. (H) Side view of the proximal end of an INCENP thread at pachytene. The single ACA signal, representing synapsed homologous centromeres, surrounds the end of the thread. (I,J) End-on view of the proximal end of an INCENP thread at pachytene. The spot representing the INCENP signal is lateral to the ACA signal. (K) Partial projection of three focal planes throughout the pachytene sex body that appears slightly labelled with INCENP. The unpaired sex centromeres (yellow arrowheads), and the INCENP-labelled pseudoautosomal region (arrow), are observed. (L-N) Two focal planes throughout a late pachytene spermatocyte. Chromocentres appear labelled (arrows), whereas INCENP labelling at nuclear threads is fainter. The sex body (XY) appears faintly labelled (white arrowhead in M). The centromeres of both sex chromosomes (yellow arrowheads in M) are separated. (A-F,L-N) Bar in N, 10 µm. (G,K) Bar in G, 5 µm. (H-J) Bar in H, 2 µm.

View larger version (104K): [in a new window]   Fig. 5. Double immunolabelling of INCENP and SCP3 in either squashed (A-O,R,S) or spread (P,Q) prophase I spermatocytes (INCENP, green; SCP3, red; regions of colocalization, yellow). (A-C) Single focal plane of a zygotene spermatocyte. Some short INCENP threads colocalize with SCP3 threads where homologous chromosomes have synapsed. (D-F) Partial projection of two focal planes throughout a pachytene spermatocyte. The long INCENP threads colocalize with SCP3-labelled synaptonemal complexes (SCs). (G-L) Selected pachytene SC fragments where the two lateral elements (LEs) (arrowheads) are clearly individualised. The INCENP threads, presenting narrow regions along their lengths (arrows in G), are located in between the LEs. (M-O) End of a pachytene SC showing that the INCENP thread also appears in between LEs (white arrowheads). (P,Q) Selected sex bivalent from a spread of an early pachytene spermatocyte. INCENP only labels the pseudoautosomal region. The unsynapsed sex axial elements (X,Y) are indicated. (R,S) Selected diplotene bivalent. A short INCENP thread (arrows) colocalizes with the region where LEs are still synapsed. Chromocentres show a bright INCENP labelling. (A-F) Bar in C, 5 µm. (G-O) Bar in I, 0.7 µm. (P,Q) Bar in Q, 2 µm. (R-S) Bar in S, 3.3 µm.

View larger version (82K): [in a new window]   Fig. 6. Double immunolabelling of INCENP and SCP1 in prophase I squashed spermatocytes (INCENP, green; SCP1, red; DAPI, blue). (A-D) Partial projection of four focal planes throughout a zygotene spermatocyte. Short INCENP threads colocalize with short SCP1 segments representing SC portions with central element where homologous chromosomes have synapsed. (E-H) Partial projection of two focal planes throughout a pachytene spermatocyte. INCENP threads and SC central elements along autosomal bivalents colocalize. The peudoautosomal region in the sex bivalent (XY) is labelled by both proteins (arrows). (I-L) Partial projection of three focal planes throughout an early diplotene spermatocyte. INCENP threads (arrows in I) and remaining SC central elements in desynapsing autosomal bivalents (arrows in J) colocalize. Note that the INCENP threads are fainter than the corresponding SCP1 fragments. Chromocentres (arrowhead) are brightly labelled only with INCENP. (M-P) Partial projection of two focal planes throughout a late diplotene spermatocyte. Short INCENP threads (arrows in M) are very faint in comparison with the corresponding SCP1 fragments (arrows in N). Chromocentres (arrowhead) are brightly labelled with INCENP. A faint INCENP labelling of the sex body (XY) is also observed. Bar in P, 10 µm.

View larger version (104K): [in a new window]   Fig. 4. INCENP expression in diplotene and diakinesis spermatocytes (INCENP, green; ACA serum, red; DAPI, blue). (A-C) Two partial projections from the same diplotene spermatocyte. INCENP labels the chromocentres, which appear very bright (arrowheads in B,C), the sex body (XY), which is fainter, and the short and faint nuclear threads (arrows). The unsynapsed sex centromeres (yellow arrowheads) are observed in the sex body. (D,E) Side view of the insertion of the proximal ends of two INCENP threads into the nuclear envelope in an early diplotene spermatocyte. The centromeric heterochromatin of these two threads appears joined as a single bright chromocentre. (F,G) End-on view of the insertion of five autosomal centromeric regions into the nuclear envelope in an early diplotene spermatocyte. Note that the INCENP-labelled heterochromatic regions are larger than the ACA signals representing synapsed homologous centromeres. (H,I) Partial projection of four focal planes throughout a late diplotene spermatocyte. No INCENP threads are observed. INCENP labels intensely the chromocentres, and dimly the sex body (XY) where the centromeres (yellow arrowheads) are indicated. (J,K) Partial projection of three focal planes throughout a diakinesis spermatocyte. INCENP signals partially colocalize with ACA ones. Inserts show a high magnification of two centromeres. (A-C,H-K) Bar in K, 10 µm. (D-G) Bar in G, 5 µm.

View larger version (103K): [in a new window]   Fig. 7. INCENP distribution in metaphase I spermatocytes (INCENP, green; ACA serum, red in B, C, E, F, H, I, K and L; SCP3, red in N and O; DAPI, blue). (A-F) Two partial projections, each one from three focal planes, throughout the same metaphase I spermatocyte. INCENP labels the centromeres, as detected with the ACA serum. A diffuse faint labelling is observed on bivalents, and a bright signal is discerned at the interchromatid domain (arrowheads) in the sex (XY) bivalent. (G-L) Two selected metaphase I autosomal bivalents. INCENP labels a T-shaped structure beneath the kinetochore labelling obtained with the ACA serum, although both signals partially colocalize. (M-O) Selected metaphase I centromere. INCENP and SCP3 colocalize at a T-shaped structure. (A-F) Bar in F, 10 µm. (G-L) Bar in L, 2 µm. (M-O) Bar in O, 0.5 µm.

View larger version (75K): [in a new window]   Fig. 8. INCENP expression from anaphase I up to interkinesis (INCENP, green; ACA serum, red in I; DAPI, blue). (A-C) Two focal planes throughout an early anaphase I spermatocyte. The only INCENP signals are at centromeres (arrows). (D-E) Single focal plane of a late anaphase I spermatocyte. A bright INCENP labelling is detected at the spindle midzone. Some faint and dispersed INCENP labelling is observed at centromeres. Since a short exposure time was used to accurately visualize the midzone signal, labelling at centromeres is very low. (F,G) Single focal plane of a telophase I spermatocyte. INCENP only labels the spindle midzone (arrowheads). (H-J) Partial projection of two focal planes throughout an interkinesis spermatocyte. A bright INCENP labelling is observed at chromocentres (arrows). Bar, 10 µm.

View larger version (82K): [in a new window]   Fig. 9. INCENP labelling from metaphase II up to telophase II (INCENP, green; ACA serum, red; DAPI, blue). (A-C) Partial projection of five focal planes throughout a metaphase II spermatocyte. INCENP signals appear at centromeres and are concentrated in two spots (arrowheads) partially colocalizing with sister kinetochores as revealed with the ACA serum. (D-G) Selected metaphase II chromosomes. INCENP is concentrated below sister kinetochores and at an irregular connecting strand that joins them. (H,I) Early anaphase II chromosome. INCENP is concentrated at sister kinetochores (arrowheads), and some faint interrupted labelling at the connecting strand (arrow) is visible. (J,K) Partial projection of two focal planes throughout an anaphase II spermatocyte. A diffuse INCENP labelling at centromeres (arrows) is observed. (L,M) A single focal plane of a late anaphase II spermatocyte. The INCENP labelling is discerned as two parallel plates (arrowheads) at the midbody. (A-C,J-M) Bar in M, 5 µm. (D-I) Bar in I, 2 µm.

View larger version (90K): [in a new window]   Fig. 10. INCENP labelling at telophase II poles and round spermatids (INCENP, green; ACA serum, red; DAPI, blue). (A-C) Partial projection of two focal planes throughout an endon view of an early telophase II pole. INCENP signals colocalize with kinetochores (arrows). (D-F) Partial projection of two focal planes throughout an end-on view of a late telophase II pole. INCENP signals appear as diffuse and faintly labelled clouds surrounding kinetochores. (G,H) Single plane of an early round spermatid. INCENP localizes at a centrally located and large brightly labelled chromocentre containing centromeres. (I,J) Single plane of a late round spermatid where only centromere signals are observed. Bar, 5 µm.

View larger version (87K): [in a new window]   Fig. 11. Double immunolabelling of INCENP and aurora-B kinase (A-P), or aurora-B and phosphorylated histone H3 (pH3) (Q-X), in squashed spermatocytes (INCENP, green in A-P; aurora-B kinase, red in A-P, green in Q-X; pH3, red in Q-X; DAPI, blue). (A-D) Partial projection of three focal planes throughout a diplotene spermatocyte. Chromocentres and short and faint nuclear threads (arrows) are labelled with INCENP, but not with aurora-B. (E-H) Partial projection of two focal planes throughout a late diplotene spermatocyte. Chromocentres (arrowheads) are labelled by both INCENP and aurora-B. The sex body (XY) appears faintly labelled with INCENP. (I-L) Selected metaphase I centromere. INCENP and aurora-B colocalize at a T-shaped structure. (M-P) Selected metaphase II centromere. The connecting strand traversing the centromere shows colocalization of INCENP and aurora-B. (Q-T) Late diplotene spermatocyte. Chromocentres only appear labelled with aurora-B. (U-X) Late diplotene spermatocyte. Chromocentres and the sex body (XY) are labelled with aurora-B and pH3. (A-H,Q-X) Bar in D, 10 µm. (I-P) Bar in P, 2 µm.

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