Bqt2p is essential for initiating telomere clustering upon pheromone sensing in fission yeast

The telomere bouquet, i.e., telomere clustering on the nuclear envelope (NE) during meiotic prophase, is thought to promote homologous chromosome pairing. Using a visual screen, we identified bqt2/im295, a mutant that disrupts telomere clustering in fission yeast. Bqt2p is required for linking telomeres to the meiotic spindle pole body (SPB) but not for attachment of telomeres or the SPB to the NE. Bqt2p is expressed upon pheromone sensing and colocalizes thereafter to Sad1p, an SPB protein. This localization only depends on Bqt1p, not on other identified proteins required for telomere clustering. Upon pheromone sensing, generation of Sad1p foci next to telomeres depends on Bqt2p. However, depletion of Bqt2p from the SPB is dispensable for dissolving the telomere bouquet at the end of meiotic prophase. Therefore, telomere bouquet formation requires Bqt2p as a linking component and is finely regulated during meiotic progression.     

Meiotic proteins bqt1 and bqt2 tether telomeres to form the bouquet arrangement of chromosomes

In many organisms, meiotic chromosomes are bundled at their telomeres to form a "bouquet" arrangement. The bouquet formation plays an important role in homologous chromosome pairing and therefore progression of meiosis. As meiotic telomere clustering occurs in response to mating pheromone signaling in fission yeast, we looked for factors essential for bouquet formation among genes induced under mating pheromone signaling. This genome-wide search identified two proteins, Bqt1 and Bqt2, that connect telomeres to the spindle-pole body (SPB; the centrosome equivalent in fungi). Neither Bqt1 nor Bqt2 alone functions as a connector, but together the two proteins form a bridge between Rap1 (a telomere protein) and Sad1 (an SPB protein). Significantly, when both Bqt1 and Bqt2 are ectopically expressed in mitotic cells, they also form a bridge between Rap1 and Sad1. Thus, a complex including Bqt1 and Bqt2 is essential for connecting telomeres to the SPB.     

A Taz1- and Microtubule-Dependent Regulatory Relationship between Telomere and Centromere Positions in Bouquet Formation Secures Proper Meiotic Divisions

During meiotic prophase, telomeres cluster, forming the bouquet chromosome arrangement, and facilitate homologous chromosome pairing. In fission yeast, bouquet formation requires switching of telomere and centromere positions. Centromeres are located at the spindle pole body (SPB) during mitotic interphase, and upon entering meiosis, telomeres cluster at the SPB, followed by centromere detachment from the SPB. Telomere clustering depends on the formation of the microtubule-organizing center at telomeres by the linker of nucleoskeleton and cytoskeleton complex (LINC), while centromere detachment depends on disassembly of kinetochores, which induces meiotic centromere formation. However, how the switching of telomere and centromere positions occurs during bouquet formation is not fully understood. Here, we show that, when impaired telomere interaction with the LINC or microtubule disruption inhibited telomere clustering, kinetochore disassembly-dependent centromere detachment and accompanying meiotic centromere formation were also inhibited. Efficient centromere detachment required telomere clustering-dependent SPB recruitment of a conserved telomere component, Taz1, and microtubules. Furthermore, when artificial SPB recruitment of Taz1 induced centromere detachment in telomere clustering-defective cells, spindle formation was impaired. Thus, detachment of centromeres from the SPB without telomere clustering causes spindle impairment. These findings establish novel regulatory mechanisms, which prevent concurrent detachment of telomeres and centromeres from the SPB during bouquet formation and secure proper meiotic divisions.     

Meiotic telomere clustering requires actin for its formation and cohesin for its resolution

In diploid organisms, meiosis reduces the chromosome number by half during the formation of haploid gametes. During meiotic prophase, telomeres transiently cluster at a limited sector of the nuclear envelope (bouquet stage) near the spindle pole body (SPB). Cohesin is a multisubunit complex that contributes to chromosome segregation in meiosis I and II divisions. In yeast meiosis, deficiency for Rec8 cohesin subunit induces telomere clustering to persist, whereas telomere cluster-SPB colocalization is defective. These defects are rescued by expressing the mitotic cohesin Scc1 in rec8delta meiosis, whereas bouquet-stage exit is independent of Cdc5 pololike kinase. An analysis of living Saccharomyces cerevisiae meiocytes revealed highly mobile telomeres from leptotene up to pachytene, with telomeres experiencing an actin- but not microtubule-dependent constraint of mobility during the bouquet stage. Our results suggest that cohesin is required for exit from actin polymerization-dependent telomere clustering and for linking the SPB to the telomere cluster in synaptic meiosis.     

Csm4-dependent telomere movement on nuclear envelope promotes meiotic recombination

During meiotic prophase, chromosomes display rapid movement, and their telomeres attach to the nuclear envelope and cluster to form a “chromosomal bouquet.” Little is known about the roles of the chromosome movement and telomere clustering in this phase. In budding yeast, telomere clustering is promoted by a meiosis-specific, telomere-binding protein, Ndj1. Here, we show that a meiosis-specific protein, Csm4, which forms a complex with Ndj1, facilitates bouquet formation. In the absence of Csm4, Ndj1-bound telomeres tether to nuclear envelopes but do not cluster, suggesting that telomere clustering in the meiotic prophase consists of at least two distinct steps: Ndj1-dependent tethering to the nuclear envelope and Csm4-dependent clustering/movement. Similar to Ndj1, Csm4 is required for several distinct steps during meiotic recombination. Our results suggest that Csm4 promotes efficient second-end capture of a double-strand break following a homology search, as well as resolution of the double-Holliday junction during crossover formation. We propose that chromosome movement and associated telomere dynamics at the nuclear envelope promotes the completion of key biochemical steps during meiotic recombination.     

Absence of yKu/Hdf1 but not myosin-like proteins alters chromosome dynamics during prophase I in yeast

Abstract Meiosis is central to the formation of haploid gametes or spores in that it segregates homologous chromosomes and halves the chromosome number. A prerequisite of this genome bisection is the pairing of homologous chromosomes during the first meiotic prophase. When budding yeast cells are induced to undergo meiosis, this has profound consequences for nuclear structure: after premeiotic DNA replication centromeres disperse, while telomeres move about the nuclear periphery and temporarily cluster during the leptotene/zygotene transition (bouquet stage) of the prophase to first meiotic division. In vegetative cells, Hdf1p (yKu) and the myosin-like proteins Mlp1p and Mlp2p have been suggested to contribute to the organization of silent chromatin, tethering of telomeres to the nuclear periphery, DNA repair, and telomere maintenance. Here, we investigated by molecular cytology whether yKu and Mlp proteins contribute to telomere and chromosome dynamics in meiosis. It was found that mlp1 Δ mlp2 Δ double-mutant cells undergo centromere dispersion, telomere clustering, homologue pairing, and sporulation like wild type. On the other hand, cells deficient for yKu underwent meiosis-specific chromosomal events with a delay, while they eventually sporulated like wild type. These results suggest that the absence of yKu not only affects vegetative nuclear architecture ( Laroche et al., 1998 ) but also interferes with the ordered occurrence of chromosome dynamics during first meiotic prophase.     

Fission yeast mutants affecting telomere clustering and meiosis-specific spindle pole body integrity

In meiotic prophase of many eukaryotic organisms, telomeres attach to the nuclear envelope and form a polarized configuration called the bouquet. Bouquet formation is hypothesized to facilitate homologous chromosome pairing. In fission yeast, bouquet formation and telomere clustering occurs in karyogamy and persists throughout the horsetail stage. Here we report the isolation and characterization of six mutants from our screen for meiotic mutants. These mutants show defective telomere clustering as demonstrated by mislocalization of Swi6::GFP, a heterochromatin-binding protein, and Taz1p::GFP, a telomere-specific protein. These mutants define four complementation groups and are named dot1 to dot4-defective organization of telomeres. dot3 and dot4 are allelic to mat1-Mm and mei4, respectively. Immunolocalization of Sad1, a protein associated with the spindle pole body (SPB), in dot mutants showed an elevated frequency of multiple Sad1-nuclei signals relative to wild type. Many of these Sad1 foci were colocalized with Taz1::GFP. Impaired SPB structure and function were further demonstrated by failure of spore wall formation in dot1, by multiple Pcp1::GFP signals (an SPB component) in dot2, and by abnormal microtubule organizations during meiosis in dot mutants. The coincidence of impaired SPB functions with defective telomere clustering suggests a link between the SPB and the telomere cluster.     

Rap1-independent telomere attachment and bouquet formation in mammalian meiosis

Attachment of telomeres to the nuclear envelope (NE) and their clustering in a chromosomal bouquet during meiotic prophase I is an evolutionary conserved event that promotes chromosome pairing and recombination. In fission yeast, bouquet formation fails when the telomeric protein Rap1 is absent or when the telomeric protein Taz1 fails to recruit Rap1 to telomeres. The mammalian Rap1 orthologue is a component of the shelterin complex and localises to telomeres through an interaction with a Taz1-like telomeric DNA binding factor, TRF2. Here, we investigated the role of mammalian Rap1 in meiotic telomere attachment and clustering by analysing spermatogenesis in Rap1-deficient mice. The results establish that the meiotic three-dimensional nuclear architecture and recombination are not affected by the absence of Rap1. Furthermore, Rap1-deficient meiotic telomeres assemble the SUN1 nuclear membrane protein, attach to the NE, and undergo bouquet formation indistinguishable from the wild-type setting. Thus, the role of Rap1 in meiosis is not conserved between fission yeast and mammals, suggesting that mammals have alternative modes for connecting telomeres to SUN proteins on the meiotic nuclear envelope.     

Meiotic chromosome pairing is promoted by telomere-led chromosome movements independent of bouquet formation

Chromosome pairing in meiotic prophase is a prerequisite for the high fidelity of chromosome segregation that haploidizes the genome prior to gamete formation. In the budding yeast Saccharomyces cerevisiae, as in most multicellular eukaryotes, homologous pairing at the cytological level reflects the contemporaneous search for homology at the molecular level, where DNA double-strand broken ends find and interact with templates for repair on homologous chromosomes. Synapsis (synaptonemal complex formation) stabilizes pairing and supports DNA repair. The bouquet stage, where telomeres have formed a transient single cluster early in meiotic prophase, and telomere-promoted rapid meiotic prophase chromosome movements (RPMs) are prominent temporal correlates of pairing and synapsis. The bouquet has long been thought to contribute to the kinetics of pairing, but the individual roles of bouquet and RPMs are difficult to assess because of common dependencies. For example, in budding yeast RPMs and bouquet both require the broadly conserved SUN protein Mps3 as well as Ndj1 and Csm4, which link telomeres to the cytoskeleton through the intact nuclear envelope. We find that mutants in these genes provide a graded series of RPM activity: wild-type>mps3-dCC>mps3-dAR>ndj1Δ>mps3-dNT = csm4Δ. Pairing rates are directly correlated with RPM activity even though only wild-type forms a bouquet, suggesting that RPMs promote homologous pairing directly while the bouquet plays at most a minor role in Saccharomyces cerevisiae. A new collision trap assay demonstrates that RPMs generate homologous and heterologous chromosome collisions in or before the earliest stages of prophase, suggesting that RPMs contribute to pairing by stirring the nuclear contents to aid the recombination-mediated homology search.     

Meiotic telomere protein Ndj1p is required for meiosis-specific telomere distribution, bouquet formation and efficient homologue pairing

We have investigated the requirements for NDJ1 in meiotic telomere redistribution and clustering in synchronized cultures of Saccharomyces cerevisiae. On induction of wild-type meiosis, telomeres disperse from premeiotic aggregates over the nuclear periphery, and then cluster near the spindle pole body (bouquet arrangement) before dispersing again. In ndj1Delta meiocytes, telomeres are scattered throughout the nucleus and fail to form perinuclear meiosis-specific distribution patterns, suggesting that Ndj1p may function to tether meiotic telomeres to the nuclear periphery. Since ndj1Delta meiocytes fail to cluster their telomeres at any prophase stage, Ndj1p is the first protein shown to be required for bouquet formation in a synaptic organism. Analysis of homologue pairing by two-color fluorescence in situ hybridization with cosmid probes to regions on III, IX, and XI revealed that disruption of bouquet formation is associated with a significant delay (>2 h) of homologue pairing. An increased and persistent fraction of ndj1Delta meiocytes with Zip1p polycomplexes suggests that chromosome polarization is important for synapsis progression. Thus, our observations support the hypothesis that meiotic telomere clustering contributes to efficient homologue alignment and synaptic pairing. Under naturally occurring conditions, bouquet formation may allow for rapid sporulation and confer a selective advantage.     

The meiotic bouquet promotes homolog interactions and restricts ectopic recombination in Schizosaccharomyces pombe

Chromosome architecture undergoes extensive, programmed changes as cells enter meiosis. A highly conserved change is the clustering of telomeres at the nuclear periphery to form the "bouquet" configuration. In the fission yeast Schizosaccharomyces pombe the bouquet and associated nuclear movement facilitate initial interactions between homologs. We show that Bqt2, a meiosis-specific protein required for bouquet formation, is required for wild-type levels of homolog pairing and meiotic allelic recombination. Both gene conversion and crossing over are reduced and exhibit negative interference in bqt2Delta mutants, reflecting reduced homolog pairing. While both the bouquet and nuclear movement promote pairing, only the bouquet restricts ectopic recombination (that between dispersed repetitive DNA). We discuss mechanisms by which the bouquet may prevent deleterious translocations by restricting ectopic recombination.     

Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing

The preconditions and early steps of meiotic chromosome pairing were studied by fluorescence in situ hybridization (FISH) with chromosome- specific DNA probes to mouse and human testis tissue sections. Premeiotic pairing of homologous chromosomes was not detected in spermatogonia of the two species. FISH with centromere- and telomere- specific DNA probes in combination with immunostaining (IS) of synaptonemal complex (SC) proteins to testis sections of prepuberal mice at days 4-12 post partum was performed to study sequentially the meiotic pairing process. Movements of centromeres and then telomeres to the nuclear envelope, and of telomeres along the nuclear envelope leading to the formation of a chromosomal bouquet were detected during mouse prophase. At the bouquet stage, pairing of a mouse chromosome-8- specific probe was observed. SC-IS and simultaneous telomere FISH revealed that axial element proteins appear as large aggregates in mouse meiocytes when telomeres are attached to the nuclear envelope. Axial element formation initiates during tight telomere clustering and transverse filament-IS indicated the initiation of synapsis during this stage. Comparison of telomere and centromere distribution patterns of mouse and human meiocytes revealed movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process. Chromosome painting in human spermatogonia revealed compacted, largely mutually exclusive chromosome territories. The territories developed into long, thin threads at the onset of meiotic prophase. Based on these results a unified model of the pairing process is proposed.     

A novel fission yeast gene, kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1 ⁺ gene is involved in SPB function. However, the kms1 ⁺ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins.     

The desynaptic (dy) and desynaptic1 (dsy1) mutations in maize (Zea mays L) cause distinct telomere-misplacement phenotypes during meiotic prophase

During meiotic prophase, telomeres actively attach themselves to the nuclear envelope and cluster in an arrangement called the bouquet. The bouquet is unique to meiosis, highly conserved, and thought to facilitate homologous chromosome synapsis. Analy sis of three-dimensional fluorescence in situ hybridization (3-D FISH) image data has been employed to characterize the bouquet in fixed pollen mother cells of maize (Zea mays L.). In order to examine the function of the bouquet further, several meiotic mutants were screened for telomeric defects using 3-D FISH as an assay. Two mutants, desynaptic (dy) and desynaptic1 (dsy1), were found to exhibit novel telomere-misplacement phenotypes. In both cases, the telomere-associated mutant phenotypes occurred prior to what was previously reported as the earliest affected stage. Three alleles of the desynaptic1 mutation (dsy1-1, dsy1-9101, and dsy1-9307) resulted in a partial bouquet phenotype at the zygotene stage of meiotic prophase. By contrast, dy nuclei contained apparently normal bouquets, but then resulted in a premature intranuclear localization of telomeres at the pachytene stage, when telomeres normally disperse but remain attached to the nuclear envelope. The dsy1 mutation is known to impair the fidelity and progression of homologous synapsis, whereas the dy mutation is known to reduce recombination rates. If the telomere misplacements are primary defects of these mutants, then these data would be consistent with the hypothesis that meiotic telomeres have at least two separable functions, one involving proper homologous chromosome synapsis at the bouquet stage and another involving post-bouquet cross-over control.     

A cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast.

Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by astral microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at astral microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.     

Atm Inactivation Results in Aberrant Telomere Clustering during Meiotic Prophase

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.     

Rapid telomere movement in meiotic prophase is promoted by NDJ1, MPS3, and CSM4 and is modulated by recombination

Haploidization of the genome in meiosis requires that chromosomes be sorted exclusively into pairs stabilized by synaptonemal complexes (SCs) and crossovers. This sorting and pairing is accompanied by active chromosome positioning in meiotic prophase in which telomeres cluster near the spindle pole to form the bouquet before dispersing around the nuclear envelope. We now describe telomere-led rapid prophase movements (RPMs) that frequently exceed 1 microm/s and persist throughout meiotic prophase. Bouquet formation and RPMs depend on NDJ1, MPS3, and a new member of this pathway, CSM4, which encodes a meiosis-specific nuclear envelope protein required specifically for telomere mobility. RPMs initiate independently of recombination but differ quantitatively in mutants that fail to complete recombination, suggesting that RPMs respond to recombination status. Together with recombination defects described for ndj1, our observations suggest that RPMs and SCs balance the disruption and stabilization of recombinational interactions, respectively, to regulate crossing over.     

The telomere bouquet controls the meiotic spindle

Bouquet formation, in which telomeres gather to a small region of the nuclear membrane in early meiosis, has been observed in diverse eukaryotes, but the function of the bouquet has remained a mystery. Here, we demonstrate that the telomere bouquet plays a crucial role in controlling the behavior of the fission yeast microtubule-organizing center (known as the spindle pole body or SPB) and the meiotic spindle. Using mutations that specifically disrupt the bouquet, we analyze chromosome, SPB, and spindle dynamics throughout meiosis. If the bouquet fails to form, the SPB becomes fragmented at meiosis I, leading to monopolar, multiple, and mislocalized spindles. Correct SPB and spindle behavior require not only the SPB recruitment of telomere proteins but also that the proteins are properly bound to telomeric DNA. This discovery illuminates an unanticipated level of communication between chromosomes and the spindle apparatus that may be widely conserved among eukaryotes.     

Centromere and telomere redistribution precedes homologue pairing and terminal synapsis initiation during prophase I of cattle spermatogenesis

Alterations in nuclear topology associated with meiotic chromosome pairing were studied in premeiotic cells and spermatocytes I of adult bovine males. To this end, we performed FISH with chromosome, pericentromeric satellite-DNA and telomere-specific probes in combination with immunostaining of synaptonemal complex proteins (SCP3, SCP1) on testis tissue sections. Nuclei of premeiotic cells (spermatogonia) exhibited a scattered telomere distribution while pericentromeres formed a few intranuclear clusters. We observed that the chromosome pairing process in cattle prophase I is preceded by repositioning of centromeres and telomeres to the nuclear periphery during preleptotene. Clustering of chromosome ends (bouquet formation) was observed during the transition from leptonema to zygonema and coincided with pairing of a sub-centromeric marker of bovine chromosomes 7. Dissolution of bouquet topology during zygonema left perinuclear telomeres scattered over the nuclear periphery at pachynema. SCP3 staining in frozen tissue sections revealed the appearance of this axial element protein in intranuclear aggregates during preleptotene, followed by extensive axial element formation during leptotene. Synapsis as revealed by SCP1 staining initiated peripherally at earliest zygotene, at this stage nuclei still contained numerous SCP3 clusters. Our observations reveal prominent non-homologous satellite-DNA associations in spermatogonia and indicate the conservation of topological features of the meiotic chromosome pairing process among mammals. The comparison of telomere dynamics in mouse and cattle prophase I suggests that a larger number of chromosomes prolongs the duration of the bouquet stage.     

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three‐dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre‐meiotic G₂ and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.