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

First published online 25 July 2006
doi: 10.1242/jcs.03054

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend 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 Golubovskaya, I. N. Articles by Cande, W. Z. Search for Related Content PubMed PubMed Citation Articles by Golubovskaya, I. N. Articles by Cande, W. Z.

Alleles of afd1 dissect REC8 functions during meiotic prophase I

Inna N. Golubovskaya^1,2,*, Olivier Hamant^1,*, Ljuda Timofejeva³, Chung-Ju Rachel Wang¹, David Braun^4,, Robert Meeley⁵ and W. Zacheus Cande^1,

¹ Department of Molecular and Cell Biology, University California, Berkeley, CA 94720, USA
² N. I. Vavilov Institute of Plant Breeding, St Petersburg, 190000, Russia
³ Department of Gene Technology, Tallinn University of Technology, 19086, Estonia
⁴ Department of Plant and Microbial Biology, University California, Berkeley, CA 94720, USA
⁵ Pioneer Hi-Bred International, 7300 NW 62nd Ave, Johnston, IA 50131-1004, USA

View larger version (57K): [in a new window]   Fig. 1. afd1 encodes a REC8 homolog. (A) Partial sequence of AFD1 in the wild-type and afd1-1 mutant. In afd1-1, a G to A point mutation in the donor site of intron 16 (^) introduces a stop codon (*). (B) Position of the Mu insertions in afd1-2, afd1-3 and afd1-4. (C) RT-PCR analysis of the afd1 gene compared with smc3 of root tips, leaves from 7-day-old and 21-day-old plants, ear at meiosis stage, tassel at meiosis stage, old-tassel-containing pollen. (D) RT-PCR analysis of the afd1 gene compared with smc3 in the tassel from wild-type and afd1 alleles for 30 and 35 PCR cycles. (E) Quantification of the RT-PCR signal shown in D as a percentage of the wild-type signal intensity. (F) AFD1 immunolocalization (green in merged) in wild-type and afd1 meiocytes stained with DAPI (red). Individual channels are shown in black and white for clarity. In the wild type, we observed a strong AFD1 signal between paired homologous chromosomes. In afd1 mutants, we enhanced the signal to detect any signal on the chromosomes, thereby increasing the background signal. No staining was detected on the afd1-1 and afd1-2 chromosomes. In afd1-3, patchy signals were detected in the nucleus (arrowheads). In afd1-4, the AFD1 signal was diffuse and lines could be detected suggesting preferential colocalization with chromosomes. Bars, 5 µm.

View larger version (121K): [in a new window]   Fig. 2. AFD1 localizes to the axial and lateral elements. (A) Localization of AFD1 in a wild-type nucleus stained with DAPI (red) and anti-AFD1 antibody (green) at zygotene, showing an AFD1 signal on both synapsed and unsynapsed chromosome regions. Arrowheads indicate unsynapsed regions. The three panels on the right are magnifications of one region. Individual channels are shown in black and white for clarity. (B,C) Localization of AFD1 and ASY1/HOP1 in wild-type nuclei stained with DAPI (red), anti-AFD1 (green), and anti-ASY1/HOP1 antibodies (blue). Individual channels are shown in black and white for clarity. A projection view of a zygotene nucleus (4% paraformaldehyde fixation) is shown in B. AFD1 and ASY1/HOP1 signals are equally bright on unsynapsed chromosome regions. The ASY1/HOP1 signal is much dimmer where chromosomes are synapsed (arrow). (C) A projection view of a 0.4-µm section of the pachytene nucleus (2% paraformaldehyde fixation). AFD1 and ASY1/HOP1 colocalize and occur in between synapsed homologous chromosomes as two strands, marking the lateral elements. This pattern of immunostaining resembles silver-nitrate staining of lateral elements in SCs. Bars, 5 µm.

View larger version (111K): [in a new window]   Fig. 3. Leptotene chromosome structure is rescued in weak afd1 alleles. Wild-type and afd1 nuclei stained with DAPI at leptotene (A-D), zygotene (E-H) and pachytene (I-L). Note the presence of chromosome threads at leptotene in the wild type, afd1-3 and afd1-4, and their absence in afd1-1. Bar, 5 µm.

View larger version (101K): [in a new window]   Fig. 4. AE elongation is partially rescued in the weak afd1 alleles. (A-J) TEM analysis of synaptonemal spreads from wild-type and afd1 meiocytes. SC in the wild type at pachytene (A) with a close-up (F) showing two lateral elements (LE) and a central element (CE). The stained round patches correspond to kinetochores, the large circle is the nucleolus. Short synaptic structures in afd1-1 (B) during early prophase I with a close-up (G) showing their multilayered composition. Short synaptic structures in afd1-2 (C) with a close-up (H) showing their multilayered and entangled shape. Short synaptic structures surrounded with elongated shaggy filaments in afd1-3 (D,I). Elongated AEs in afd1-4 with synaptic structures (SS), and regions where two axial elements are coalligned similar to normal synapsis (E,J). (K-N) Non homologous synapsis in afd1-4 as shown by synapsis occurring within the same AE (red; K,L) and between different partners (red and green; M,N). (O-R) Localization of ASY1 in afd1-mutant nuclei. Patches of staining could correspond to the synaptic structures observed by TEM. Elongated AEs are clearly visible in afd1-3 and afd1-4. (S-V) Synaptic structures (S,U) and ASY1/HOP1 immunostaining (T,V) in afd1-1 and afd1-4, showing that AEs and ASY1/HOP1 are still present in late pachytene-early diplotene. Bars in A-E and K-V, 5 µm, in F-J, 1 µm.

View larger version (102K): [in a new window]   Fig. 5. Bouquet formation is rescued in afd1-4, but homologous pairing and RAD51 distribution are impaired in all the afd1 alleles. (A-D) Homologous pairing in wild-type and afd1 nuclei during pachytene using FISH with 5S rRNA probe (green) and DAPI staining (red). (A) Paired 5S rRNA focus in the wild type. (B-D) Unpaired and doubled 5S rRNA foci in afd1-1, afd1-3, afd1-4. (E-H) Bouquet formation in wild-type and afd1 mutant nuclei at zygotene shown by FISH with telomere probe (green). (E) Bouquet in wild type. (F,G) Dispersed telomeres in afd1-1 and afd1-3 nuclei. (H) Bouquet in afd1-4 nuclei. (I-L) RAD51 foci (green) morphology and distribution during pachytene monitored by immunofluorescence. (I) Multiple round RAD51 foci in wild-type nuclei during pachytene; some foci are paired (arrowheads). (J-L) Cluster of RAD51 foci in afd1-1, afd1-3 and afd1-4. Bar, 5 µm.

View larger version (24K): [in a new window]   Fig. 6. Summary of afd1 phenotypes. In the wild type, AEs are recruited and elongate along the entire chromosome. Telomere clustering requires elongated AEs. RAD51 is recruited and forms paired foci during recognition of homologous chromosomes. The bouquet, RAD51 and other unknown factors contribute to homologous pairing and synapsis. In afd1-1, AEs are recruited but their elongation is arrested. This impacts on both bouquet formation, RAD51 polymerization, homologous pairing and synapsis. In afd1-4, AEs are recruited and 50% of normal AEs elongation occurs. These AEs are sufficiently long to allow bouquet formation in 20% of the meiocytes. However, RAD51 polymerization is impaired and homologous pairing and synapsis does not occur. Therefore, homologous pairing and synapsis do not solely rely on the presence of elongated AEs and the bouquet. On the basis of these data, we propose that AFD1/REC8 controls the extent of AE elongation prior to the proper distribution of the recombination machinery, leading to pairing of homologous chromosomes independently of bouquet formation.