QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 17 February 2004
doi: 10.1242/jcs.00947

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Parra, M. T. Articles by Suja, J. A. Search for Related Content PubMed PubMed Citation Articles by Parra, M. T. Articles by Suja, J. A.

Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I

¹ Departamento de Biología, Edificio de Biológicas, Universidad Autónoma de Madrid, 28049 Madrid, Spain
² Department of Cell and Developmental Biology, Theodor Boveri Institute, University of Würzburg, 97074 Würzburg, Germany
³ Departamento de Genética, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain

View larger version (96K): [in a new window]   Fig. 1. SCP3 accumulates on the LEs during late diplotene, is partially lost from LEs during diakinesis, but still persists at centromeres and arms in prometaphase I bivalents. SCP3 is labelled in green; Rad21 in red in B,E; centromeres in red in G,H,K,M,N, and DNA in blue. (A-C) Superimposition of five focal planes throughout a pachytene spermatocyte showing colocalization of SCP3 and Rad21 at SCs. (D-F) Superimposition of nine focal planes throughout an early diplotene spermatocyte showing colocalization of SCP3 and Rad21 at desynapsed LEs. (G-I) Partial projections from different focal planes throughout a late diplotene nucleus where some accumulations of SCP3 (red arrowheads) are found on some desynapsed LEs. Some small SCP3 agglomerates (white arrowhead in H) are also observed separated from LEs. The sex AEs are indicated (XY). The inset in G is a magnification of the boxed area showing the SCP3 labelling at an homologous centromere. (J) Superimposition of different focal planes throughout an early diakinesis nucleus where a large number of SCP3 accumulations (red arrowheads) are observed along thin desynapsed LEs (white arrows). Some SCP3 agglomerates (white arrowheads) are detected in the nucleoplasm. (K,L) Single focal plane throughout a late diakinesis nucleus showing a high SCP3 labelling dispersed in the nucleoplasm where some large SCP3 agglomerates (white arrowheads) are present. An SCP3 accumulation (red arrowhead) is observed in continuity with a thin desynapsed LE (white arrows). The inset in K is a magnification of the boxed area showing the SCP3 labelling at a homologous centromere. The inset in L shows three SCP3 accumulations along unsynapsed sex AEs. (M,N) Two focal planes of a prometaphase I spermatocyte. The nucleoplasm shows a faint labelling and some large SCP3 agglomerates (arrowheads). SCP3 persists at centromeres and appears as wiggly lines (arrows) inside bivalents. The inset in M is a magnification of the boxed area showing the SCP3 labelling at a homologous centromere. Scale bar: 10 µm.

View larger version (81K): [in a new window]   Fig. 2. SCP3 is highly enriched at homologous centromeres (arrowheads), but is still present at the interchromatid domain (arrows) in metaphase I bivalents. SCP3 is labelled in green and DNA in blue. (A,C) Partial projections from different focal planes throughout the same metaphase I spermatocyte. The SCP3 labelling in the sex bivalent (XY) is observed in C. A pachytene (P) SC fragment (SC) is apparent in the upper right corner in C. Scale bar: 10 µm.

View larger version (106K): [in a new window]   Fig. 3. Selected metaphase I autosomal (A-D,I) and sex (E,H) bivalents, and sex univalents (F,G,J). SCP3 is labelled in green, centromeres in red in A-G, Rad21 in red in H-J, and DNA in blue. In the autosomal bivalents SCP3 appears highly enriched at homologous centromeres (red arrowheads in A). A fainter SCP3 labelling is also found at the interchromatid domain (red arrows in D). Note however that this labelling does not reach the distal chromosome ends (red arrows in B), and that it is interrupted at chiasma sites (white arrows in C). In the sex bivalent the SCP3 labelling at the centromere of the X chromosome (X) (red arrowhead in E) is bigger than that found at the centromere of the Y chromosome (Y). The SCP3 labelling at the interchromatid domain of the sex chromosomes is larger and more continuous (red arrows in E) than that observed in autosomal bivalents independently of whether they appear as a bivalent (E,H) or as univalents (F,G,J). In E the chiasma site in one sex bivalent is indicated by an arrow and a large SCP3 agglomerate (indicated by an arrowhead) lies in the cytoplasm near the sex bivalent. (H,I) Rad21 colocalizes with SCP3 in sex (H) and autosomal (I) bivalents, and for instance in the univalent X chromosome (J). Scale bar: 2 µm.

View larger version (69K): [in a new window]   Fig. 4. SCP3 is located beneath associated sister kinetochores at the inner domain of metaphase I centromeres, showing a complex `double cornet'-like arrangement, and colocalizing with the cohesin Rad21. SCP3 is labelled in green, and centromeres (ACA), CENP-A, CENP-E, INCENP, Rap1 and Rad21 are labelled in red. In merged images colocalization regions appear in yellow. SCP3 appears as a T- or Y-like structure in centromeres viewed from the side (A,C,E,F,H,I), but as two closely associated rings when centromeres are viewed from the top (B,D,G,J). SCP3 appears beneath the ACA and CENP-A signals with which there is some degree of colocalization (A,C). The ACA, and the pair of CENP-A dots representing sister kinetochores, appear surrounded by the SCP3 rings (B,D). CENP-E appears as a single plate separated from the underlying SCP3 signal (E). By contrast, SCP3 colocalizes at the inner centromere domain with INCENP (F) and Rad21 (I). In this sense, Rad21 is the only protein appearing as two closely associated rings colocalizing with SCP3 when centromeres are viewed from the top (J). Sister proximal telomeric sequences, as detected with the anti-Rap1 antibody, are detected as a pair of dots partially colocalizing with the middle region of the T-shaped SCP3 signal (H). Scale bar: 1 µm.

View larger version (111K): [in a new window]   Fig. 5. The ultrastructure of the inner centromere domain is revealed by the Os-PPD technique. (A) Section of a metaphase I spermatocyte. Condensed bivalents show very low contrast, while centromeres show high contrast. Bundles of kinetochore microtubules (MTs) are present. A semithin section of a metaphase I spermatocyte viewed by phase-contrast microscopy is shown in the inset. (B,C) Enlarged centromeres facing opposite poles where the outer (black arrowheads) and middle electron-lucent (white arrowheads) kinetochore plates are visible. Beneath kinetochores (K), a highly contrasted T-shaped region, representing the inner centromere domain (In), is observed. Scale bar: (A) 2.5 µm; (B,C) 0.5 µm.

View larger version (235K): [in a new window]   Fig. 6. Sister kinetochores are closely associated during metaphase I. (A) Section of a metaphase I spermatocyte. Centromeres (arrowheads) are more contrasted than the condensed chromatin at the arms. A pair of centrioles (Ce) is visible at a cell pole. (B-G) Six serial sections of a metaphase I centromere. Sister kinetochores are not resolved, but a continuous outer kinetochore plate (black arrowheads), where kinetochore microtubules end, is discernible. The inner kinetochore plate is visible in some sections (white arrowhead in B). Scale bars: (A) 3 µm; (B-G) 600 nm.

View larger version (143K): [in a new window]   Fig. 7. Sister kinetochores are individualized in anaphase I. (A-C) Three serial sections of an anaphase I chromosome. (D-F) High magnification of kinetochores in A-C. Two outer kinetochore plates, corresponding to sister kinetochores are evident. The outer (black arrowhead) and middle electron-lucent (white arrowhead) kinetochore plates can be seen. Note that both sister kinetochores interact with microtubules. Scale bars: (A-C) 300 nm; (D-F) 150 nm.

View larger version (90K): [in a new window]   Fig. 8. SCP3 and Rad21 are lost from chromosome arms during the metaphase I/anaphase I transition, but are retained at centromeres until telophase I. SCP3 is labelled in green, centromeres in red in E, F, H, I, Rad21 in red in K, and DNA in blue. (A-C) Partial projections from different focal planes throughout an anaphase I spermatocyte. SCP3 has been lost from chromosome arms but is still highly enriched at homologous centromeres. A large SCP3 agglomerate (arrowhead in A) is found in the cytoplasm. (D-F) Top view of an anaphase I cell pole. SCP3 rings (arrowheads) are observed at some centromeres where the two sister kinetochores lye side by side. (G-I) Partial projection from two focal planes of a telophase I cell pole. SCP3 is usually found as small bars that may appear between the slightly separated sister kinetochores (white arrowheads) or displaced from them (blue arrowheads). (J-L) Single focal plane of a telophase I cell pole. Rad21 and SCP3 colocalize at bars. Scale bars: (A-C) 2.5 µm; (D-L) 5 µm.

View larger version (65K): [in a new window]   Fig. 9. SCP3 persists at chromocentres during interkinesis, but is not present at centromeres of prometaphase II or metaphase II chromosomes. SCP3 is labelled in green, centromeres in red in A,C,D,E,G,H,I, Rad21 in red in K, and DNA in blue. (A,B) Single focal plane of an interkinesis nucleus where SCP3 is found as elongated bars at chromocentres but displaced from pairs of sister kinetochores. (C,D) Single focal plane throughout a prometaphase II spermatocyte. SCP3 is not present at centromeres. Sister kinetochores are separated at the centromere of an unaligned chromosome (arrow; and see inset). (E,F) Partial projection of two focal planes throughout a metaphase II spermatocyte. SCP3 is not present at centromeres where sister kinetochores are facing opposite poles. (G,H) Selected metaphase II chromosomes. Sister kinetochores occupy opposite locations at the centromere. (I,J) Partial projection of three focal planes throughout an early anaphase II spermatocyte. No SCP3 labelling is found between segregating chromatids. (K,L) Single focal plane of a metaphase II spermatocyte. Rad21 is not present at all. Scale bars: (A-F) 5 µm; (G,H) 2 µm; (I-L) 3 µm.

View larger version (26K): [in a new window]   Fig. 10. Schematic representation of the distribution of SCP3 and Rad21 from pachytene up to telophase I (A); their localization and that of other proteins at metaphase I centromeres (B); and their three-dimensional localization relative to sister kinetochores (C). (A) One chromosome is depicted in light grey while its homologue is depicted in darker grey. Centromere labelling is red, and SCP3/Rad21 is green. Regions of overlap are in yellow. From middle diplotene up to metaphase I there is a continuous loss of SCP3/Rad21 from chromosome arms, and an accumulation at centromeres, that in turn change their structure. During diakinesis SCP3/Rad21 also appear as accumulations on desynapsed LEs and as large agglomerates in the nucleoplasm. SCP3 and Rad21 remain at the interchromatid domain of metaphase I bivalents as small patches that are lost during the metaphase I/anaphase I transition. A population of SCP3/Rad21 persist at anaphase I centromeres but is released when sister kinetochores separate during telophase I. (B) A metaphase I centromere. Different proteins are aligned among them in side and top views. Trilaminar sister kinetochores are tightly associated at the surface of the centromere, and associated with microtubules emanating from the same pole. A single dashed line aligned with sister kinetochores has been included to facilitate the analysis of the relative distribution of these proteins. (C) Interpretation showing how sister kinetochores (red) are associated and supported by the `double-cornet'-shaped inner centromere domain containing SCP3, SCP2, Rad21, INCENP and aurora-B.