Microtubule-organizing center formation at telomeres induces meiotic telomere clustering

Abstract

During meiosis, telomeres cluster and promote homologous chromosome pairing. Telomere clustering requires the interaction of telomeres with the nuclear membrane proteins SUN (Sad1/UNC-84) and KASH (Klarsicht/ANC-1/Syne homology). The mechanism by which telomeres gather remains elusive. In this paper, we show that telomere clustering in fission yeast depends on microtubules and the microtubule motors, cytoplasmic dynein, and kinesins. Furthermore, the γ-tubulin complex (γ-TuC) is recruited to SUN- and KASH-localized telomeres to form a novel microtubule-organizing center that we termed the "telocentrosome." Telocentrosome formation depends on the γ-TuC regulator Mto1 and on the KASH protein Kms1, and depletion of either Mto1 or Kms1 caused severe telomere clustering defects. In addition, the dynein light chain (DLC) contributes to telocentrosome formation, and simultaneous depletion of DLC and dynein also caused severe clustering defects. Thus, the telocentrosome is essential for telomere clustering. We propose that telomere-localized SUN and KASH induce telocentrosome formation and that subsequent microtubule motor-dependent aggregation of telocentrosomes via the telocentrosome-nucleated microtubules causes telomere clustering.

Figure 1.

Telomere clustering defects in dynein and kinesin-8 mutants. (A) The meiotic process in fission yeast. (B) Telomere and SPB locations in wild-type (Wt) and dhc1Δ dlc1Δ mutant zygotes with a single nucleus. White lines indicate cell shapes. (C and D) Telomere distribution in dynein, dynactin, and kinesin-8 mutant zygotes with a single nucleus.

Figure 2.

Telomere clustering depends on microtubules. (A) The dynamics of telomeres (Telo) and microtubules (Mt) before cell conjugation. A left photo shows an examined cell, which is undergoing cell conjugation (white dashed box). (B) Schematic showing mat gene-induced haploid meiosis. (C) Meiotic progression (top graphs) and telomere clustering (bottom graphs) in haploid meiosis. Arrows show the addition of DMSO (left) or 50 µg/ml MBC (right). The data shown are from a single representative experiment out of two repeats. More than 100 cells were examined at each time point. Photos indicate the location of telomeres (Taz1) and SPB (Sid4). nuc, nucleus. (D and E) Dynamics of microtubules and telomeres in a haploid meiotic cell after MBC removal. MBC was removed ∼5 h after nitrogen depletion. In E, enlarged images are shown at the bottom row. Arrows show the telomere movement. White lines indicate cell shapes.

Figure 3.

Telomere-localized factors before cell conjugation. (A) Colocalization of various factors with telomeres (Taz1) or Sad1. (B–D) Colocalization patterns of the various factors in cells containing multiple Sad1 signals before cell conjugation. (E) Intracellular localization of a Dhc1 fragment lacking a motor domain before cell conjugation. The schematic indicates the Dhc1 architecture as deduced from amino acid sequence homology (Mocz and Gibbons, 2001) and the region containing the Dhc1 fragment. Numbers indicate amino acid positions. White lines in images indicate cell shapes. Mt, microtubule; Wt, wild type.

Figure 4.

Defective telomere clustering and reduced spore viability and recombination in the mto1Δ mutant and defective telomere interaction of Alp4 and microtubules in the mto1Δ, kms1Δ, and dhc1Δ dlc1Δ zygotes. (A) Telomere clustering defects in mto1Δ zygotes. (B) Reduced spore viability and meiotic recombination in the mto1Δ mutant. n, number of spores or tetrads that were examined. Graphs of gene conversion show mean ade⁺ frequencies. Error bars indicate SEM (n = 3). (C) Sad1 and telomere localization and their colocalization frequency. (D) Alp4 and Sad1 localization and their colocalization frequency. Arrowheads indicate Alp4-free (white) and -diminished (magenta) Sad1 dots. (E) Colocalization frequency of Mto1 and Sad1. (F) Microtubule organization (Mt) and Sad1 location (magenta arrowheads). Green arrowheads, SPB-originated MTOCs. An obvious MTOC was not observed in the mto1Δ mutant. White lines in images indicate cell shapes. Wt, wild type.

Figure 5.

Defective Alp4 telomere localization in haploid mto1Δ and dlc1Δ mutants and the telomere clustering mechanism. (A) Representative Alp4 localization in relation to telomeres (Taz1) and the SPB (Sid4). White lines indicate cell shapes. (B) Telomere (Telo) clustering in haploid meiotic cells (top graphs) and Alp4 telomere localization in MBC-treated cells (bottom graphs). The experiment of mto1Δ mutant was completed once. The data of other strains are from a single experiment out of two (wild type [Wt] and dhc1Δ) or three (dlc1Δ and dhc1Δ dlc1Δ) repeats. More than 100 cells were examined at each time point. (C) A proposed model for the telomere clustering mechanism. MT, microtubule.