Inna N. Golubovskaya

Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional wide-field microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

A bouquet of chromosomes

During meiotic prophase, telomeres attach to the inner nuclear envelope and cluster to form the so-called meiotic bouquet. Although this has been observed in almost all organisms studied, its precise function remains elusive. The coincidence of telomere clustering and initiation of chromosome synapsis has led to the hypothesis that the bouquet facilitates homologous chromosome pairing and synapsis. However, recent mutant analysis suggests that the bouquet is not absolutely required for either homologous pairing or synapsis but that it makes both processes much faster and more efficient. The initiation of bouquet formation is independent of the initiation of recombination. However, the progression through recombination and synapsis may be required for exit from the bouquet stage. Little is known about the mechanism of telomere clustering but recent studies show that it is an active process.

Alleles of afd1 dissect REC8 functions during meiotic prophase I

REC8 is a master regulator of chromatin structure and function during meiosis. Here, we dissected the functions of absence of first division (afd1), a maize rec8/α-kleisin homolog, using a unique afd1 allelic series. The first observable defect in afd1 mutants is the inability to make a leptotene chromosome. AFD1 protein is required for elongation of axial elements but not for their initial recruitment, thus showing that AFD1 acts downstream of ASY1/HOP1. AFD1 is associated with the axial and later the lateral elements of the synaptonemal complex. Rescuing 50% of axial element elongation in the weakest afd1 allele restored bouquet formation demonstrating that extent of telomere clustering depends on axial element elongation. However, rescuing bouquet formation was not sufficient for either proper RAD51 distribution or homologous pairing. It provides the basis for a model in which AFD1/REC8 controls homologous pairing through its role in axial element elongation and the subsequent distribution of the recombination machinery independent of bouquet formation.

A REC8-dependent plant Shugoshin is required for maintenance of centromeric cohesion during meiosis and has no mitotic functions.

During meiosis, sequential release of sister chromatid cohesion (SSC) during two successive nuclear divisions allows the production of haploid gametes from diploid progenitor cells. Release of SSC along chromosome arms allows first a reductional segregation of homologs, and, subsequently, release of centromeric cohesion at anaphase II allows the segregation of chromatids. The Shugoshin (SGO) protein family plays a major role in the protection of centromeric cohesion in Drosophila and yeast. We have isolated a maize mutant that displays premature loss of centromeric cohesion at anaphase I. We showed that this phenotype is due to the absence of ZmSGO1 protein, a maize shugoshin homolog. We also show that ZmSGO1 is localized to the centromeres. The ZmSGO1 protein is not found on mitotic chromosomes and has no obvious mitotic function. On the basis of these results, we propose that ZmSGO1 specifically maintains centromeric cohesion during meiosis I and therefore suggest that SGO1 core functions during meiosis are conserved across kingdoms and in large-genome species. However, in contrast to other Shugoshins, we observed an early and REC8-dependent recruitment of ZmSGO1 in maize, suggesting that control of SGO1 recruitment to chromosomes is different in plants than in other model organisms.

Altered Nuclear Distribution of Recombination Protein RAD51 in Maize Mutants Suggests the Involvement of RAD51 in Meiotic Homology Recognition

The recombination protein RAD51 is a component of the meiotic recombination pathway and has been proposed to play a role in the homology search, a process by which homologous chromosomes find each other before they pair in the prophase of meiosis. To study the relationship between recombination and chromosome pairing, we examined the distribution of RAD51 foci on meiotic chromosomes in maize mutants with defects in chromosome pairing. The patterns of RAD51 distribution showed dramatic variation among the meiotic mutants. The mutants generally exhibited significant decreases in the number of RAD51 foci at zygotene, corresponding to the degree of their pairing defects. These results provide evidence for a key role of RAD51 structures in the homology search.

Functional Analysis of Maize RAD51 in Meiosis and Double-Strand Break Repair

In Saccharomyces cerevisiae, Rad51p plays a central role in homologous recombination and the repair of double-strand breaks (DSBs). Double mutants of the two Zea mays L. (maize) rad51 homologs are viable and develop well under normal conditions, but are male sterile and have substantially reduced seed set. Light microscopic analyses of male meiosis in these plants reveal reduced homologous pairing, synapsis of nonhomologous chromosomes, reduced bivalents at diakinesis, numerous chromosome breaks at anaphase I, and that >33% of quartets carry cells that either lack an organized nucleolus or have two nucleoli. This indicates that RAD51 is required for efficient chromosome pairing and its absence results in nonhomologous pairing and synapsis. These phenotypes differ from those of an Arabidopsis rad51 mutant that exhibits completely disrupted chromosome pairing and synapsis during meiosis. Unexpectedly, surviving female gametes produced by maize rad51 double mutants are euploid and exhibit near-normal rates of meiotic crossovers. The finding that maize rad51 double mutant embryos are extremely susceptible to radiation-induced DSBs demonstrates a conserved role for RAD51 in the repair of mitotic DSBs in plants, vertebrates, and yeast.

Maize AMEIOTIC1 is essential for multiple early meiotic processes and likely required for the initiation of meiosis.

Molecular mechanisms that initiate meiosis have been studied in fungi and mammals, but little is known about the mechanisms directing the meiosis transition in other organisms. To elucidate meiosis initiation in plants, we characterized and cloned the ameiotic1 (am1) gene, which affects the transition to meiosis and progression through the early stages of meiotic prophase in maize. We demonstrate that all meiotic processes require am1, including expression of meiosis-specific genes, establishment of the meiotic chromosome structure, meiosis-specific telomere behavior, meiotic recombination, pairing, synapsis, and installation of the meiosis-specific cytoskeleton. As a result, in most am1 mutants premeiotic cells enter mitosis instead of meiosis. Unlike the genes involved in initiating meiosis in yeast and mouse, am1 also has a second downstream function, whereby it regulates the transition through a novel leptotene–zygotene checkpoint, a key step in early meiotic prophase. The am1 gene encodes a plant-specific protein with an unknown biochemical function. The AM1 protein is diffuse in the nucleus during the initiation of meiosis and then binds to chromatin in early meiotic prophase I when it regulates the leptotene–zygotene progression.

Maize multiple archesporial cells 1 (mac1), an ortholog of rice TDL1A, modulates cell proliferation and identity in early anther development

To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A. Contrary to prior studies in rice and Arabidopsis in which mac1-like genes were inferred to act late to suppress trans-differentiation of somatic tapetal cells into meiocytes, we find that mac1 anthers contain excess archesporial (AR) cells that proliferate at least twofold more rapidly than normal prior to tapetal specification, suggesting that MAC1 regulates cell proliferation. mac1 transcript is abundant in immature anthers and roots. By immunolocalization, MAC1 protein accumulates preferentially in AR cells with a declining radial gradient that could result from diffusion. By transient expression in onion epidermis, we demonstrate experimentally that MAC1 is secreted, confirming that the predicted signal peptide domain in MAC1 leads to secretion. Insights from cytology and double-mutant studies with ameiotic1 and absence of first division1 mutants confirm that MAC1 does not affect meiotic cell fate; it also operates independently of an epidermal, Ocl4-dependent pathway that regulates proliferation of subepidermal cells. MAC1 both suppresses excess AR proliferation and is responsible for triggering periclinal division of subepidermal cells. We discuss how MAC1 can coordinate the temporal and spatial pattern of cell proliferation in maize anthers.

PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.

Interlock Formation and Coiling of Meiotic Chromosome Axes During Synapsis

The meiotic prophase chromosome has a unique architecture. At the onset of leptotene, the replicated sister chromatids are organized along an axial element. During zygotene, as homologous chromosomes pair and synapse, a synaptonemal complex forms via the assembly of a transverse element between the two axial elements. However, due to the limitations of light and electron microscopy, little is known about chromatin organization with respect to the chromosome axes and about the spatial progression of synapsis in three dimensions. Three-dimensional structured illumination microscopy (3D-SIM) is a new method of superresolution optical microscopy that overcomes the 200-nm diffraction limit of conventional light microscopy and reaches a lateral resolution of at least 100 nm. Using 3D-SIM and antibodies against a cohesin protein (AFD1/REC8), we resolved clearly the two axes that form the lateral elements of the synaptonemal complex. The axes are coiled around each other as a left-handed helix, and AFD1 showed a bilaterally symmetrical pattern on the paired axes. Using the immunostaining of the axial element component (ASY1/HOP1) to find unsynapsed regions, entangled chromosomes can be easily detected. At the late zygotene/early pachytene transition, about one-third of the nuclei retained unsynapsed regions and 78% of these unsynapsed axes were associated with interlocks. By late pachytene, no interlocks remain, suggesting that interlock resolution may be an important and rate-limiting step to complete synapsis. Since interlocks are potentially deleterious if left unresolved, possible mechanisms for their resolution are discussed in this article.