The yeast Red1 protein localizes to the cores of meiotic chromosomes

Abstract

Mutants in the meiosis-specific RED1 gene of S. cerevisiae fail to make any synaptonemal complex (SC) or any obvious precursors to the SC. Using antibodies that specifically recognize the Red1 protein, Red1 has been localized along meiotic pachytene chromosomes. Red1 also localizes to the unsynapsed axial elements present in a zip1 mutant, suggesting that Red1 is a component of the lateral elements of mature SCs. Anti-Red1 staining is confined to the cores of meiotic chromosomes and is not associated with the loops of chromatin that lie outside the SC. Analysis of the spo11 mutant demonstrates that Red1 localization does not depend upon meiotic recombination. The localization of Red1 has been compared with two other meiosis-specific components of chromosomes, Hop1 and Zip1; Zip1 serves as a marker for synapsed chromosomes. Double labeling of wild-type meiotic chromosomes with anti-Zip1 and anti-Red1 antibodies demonstrates that Red1 localizes to chromosomes both before and during pachytene. Double labeling with anti-Hop1 and anti-Red1 antibodies reveals that Hop1 protein localizes only in areas that also contain Red1, and studies of Hop1 localization in a red1 null mutant demonstrate that Hop1 localization depends on Red1 function. These observations are consistent with previous genetic studies suggesting that Red1 and Hop1 directly interact. There is little or no Hop1 protein on pachytene chromosomes or in synapsed chromosomal regions.

Figure 1

Red1 and Zip1 protein localization on wild-type chromosomes. Spread meiotic chromosomes from wild-type cells (BR2495) stained with mouse anti-Red1 antibodies (A, D, and G), rabbit anti-Zip1 antibodies (B, E, and H) and DAPI (C, F, and I). (A–C) Spread pachytene nucleus from 15-h time point. (D–F) Spread nucleus from 10-h time point. (G–I) Spread nucleus from 13-h time point. Categories 1, 2, and 3 are described in the text. Bar, 2 μm.

Figure 2

Red1 protein localization in strains overproducing Red1. Spread meiotic chromosomes from a wild-type cell overproducing Red1 (BS354) stained with rabbit anti-Red1 antibodies (A) and DAPI (B). Bar, 2 μm.

Figure 3

Red1 and histone localization following DNase I digestion of meiotic chromosomes. Wild-type spreads (BR2495) incubated for 1 h at 37°C with 0 U/ml (A–C) or 100 U/ml (D–F) DNase I. Spreads were stained with mouse anti-Red1 (A and D) and rabbit antihistone 2B (B and E) antibodies in addition to DAPI staining (C and F). Bar, 2 μm.

Figure 4

Red1 and Zip1 localization patterns during meiotic prophase progression. At each time point, 100 nuclei were examined and scored for Red1 and Zip1 localization patterns. Categories of staining are described in the text. The percentage of nuclei that stain with antibodies is always <100% because not all cells enter meiosis and those cells that undergo sporulation do not proceed through meiosis in synchrony.

Figure 5

Red1 and Hop1 protein localization on wild-type chromosomes. Spread meiotic chromosomes from wild-type cells (BR2495) stained with mouse anti-Red1 antibodies (A, E, and I), rabbit anti-Hop1 antibodies (B, F, and J) and DAPI (D, H, and L). (A–D) Spread nucleus from 10-h time point with category 1 staining pattern. (E–H) Spread nucleus from 13-h time point with category 2 staining pattern. (I–L) Spread pachytene nucleus from 15-h time point with category 3 staining pattern. (C, G, and K) Fusion of anti-Red1 and anti-Hop1 staining patterns; areas of overlap appear as yellow spots. Bar, 2 μm.

Figure 6

Red1 and Hop1 localization patterns during meiotic prophase progression. At each time point, 100 nuclei were examined and scored for Red1 and Hop1 localization. Categories of staining are described in the text.

Figure 7

Hop1 and Zip1 protein localization on wild-type chromosomes. Spread zygotene nucleus from a wild-type cell (BR2495) from 13-h time point stained with rabbit anti-Hop1 antibodies (A) and mouse anti-Zip1 antibodies (B). Bar, 2 μm.

Figure 8

Red1 and Hop1 localization on zip1 chromosomes. Spread meiotic chromosomes from a zip1 cell (MY129) stained with mouse anti-Red1 antibodies (A), rabbit anti-Hop1 antibodies (B), and DAPI (C). Bar, 2 μm.

Figure 9

Zip1 localization on red1 chromosomes. Spread meiotic chromosomes from red1 cells (MY231) stained with rabbit antiZip1 antibodies (A) and DAPI (B). Bar, 2 μm.

Figure 10

Red1 and Hop1 localization on spo11 chromosomes. Spread meiotic chromosomes from spo11 cells (S2888) stained with mouse anti-Red1 antibodies (A and D), rabbit anti-Hop1 antibodies (B and E), and DAPI (C and F). Bar, 2 μm.

Figure 11

Summary of immunofluorescence and electron micrographic stages during meiotic prophase.