Nejvíce citovaný článek - PubMed ID 32429054
Super-Resolution Microscopy Reveals Diversity of Plant Centromere Architecture
In most studied eukaryotes, chromosomes are monocentric, with centromere activity confined to a single region. However, the rush family (Juncaceae) includes species with both monocentric (Juncus) and holocentric (Luzula) chromosomes, where centromere activity is distributed along the entire chromosome length. Here, we combine chromosome-scale genome assembly, epigenetic analysis, immuno-FISH and super-resolution microscopy to study the transition to holocentricity in Luzula sylvatica. We report repeat-based holocentromeres with an irregular distribution of features along the chromosomes. Luzula sylvatica holocentromeres are predominantly associated with two satellite DNA repeats (Lusy1 and Lusy2), while CENH3 also binds satellite-free gene-poor regions. Comparative repeat analysis suggests that Lusy1 plays a crucial role in centromere function across most Luzula species. Furthermore, synteny analysis between L. sylvatica (n = 6) and Juncus effusus (n = 21) suggests that holocentric chromosomes in Luzula could have arisen from chromosome fusions of ancestral monocentric chromosomes, accompanied by the expansion of CENH3-associated satellite repeats.
- MeSH
- centromera * genetika MeSH
- chromozomy rostlin * genetika MeSH
- DNA rostlinná genetika MeSH
- genom rostlinný MeSH
- hybridizace in situ fluorescenční MeSH
- molekulární evoluce MeSH
- repetitivní sekvence nukleových kyselin genetika MeSH
- satelitní DNA * genetika MeSH
- syntenie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA rostlinná MeSH
- satelitní DNA * MeSH
The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.
The segregation of chromosomes depends on the centromere. Most species are monocentric, with the centromere restricted to a single region per chromosome. In some organisms, the monocentric organization changed to holocentric, in which the centromere activity is distributed over the entire chromosome length. However, the causes and consequences of this transition are poorly understood. Here, we show that the transition in the genus Cuscuta was associated with dramatic changes in the kinetochore, a protein complex that mediates the attachment of chromosomes to microtubules. We found that in holocentric Cuscuta species, the KNL2 genes were lost; the CENP-C, KNL1, and ZWINT1 genes were truncated; the centromeric localization of CENH3, CENP-C, KNL1, MIS12, and NDC80 proteins was disrupted; and the spindle assembly checkpoint (SAC) degenerated. Our results demonstrate that holocentric Cuscuta species lost the ability to form a standard kinetochore and do not employ SAC to control the attachment of microtubules to chromosomes.
- Klíčová slova
- Cuscuta, centromere, holocentric, kinetochore, monocentric,
- MeSH
- centromera genetika MeSH
- Cuscuta * MeSH
- kinetochory * MeSH
- mikrotubuly metabolismus MeSH
- segregace chromozomů MeSH
- struktury chromozomu MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Centromeres in the legume genera Pisum and Lathyrus exhibit unique morphological characteristics, including extended primary constrictions and multiple separate domains of centromeric chromatin. These so-called metapolycentromeres resemble an intermediate form between monocentric and holocentric types, and therefore provide a great opportunity for studying the transitions between different types of centromere organizations. However, because of the exceedingly large and highly repetitive nature of metapolycentromeres, highly contiguous assemblies needed for these studies are lacking. Here, we report on the assembly and analysis of a 177.6 Mb region of pea (Pisum sativum) chromosome 6, including the 81.6 Mb centromere region (CEN6) and adjacent chromosome arms. Genes, DNA methylation profiles, and most of the repeats were uniformly distributed within the centromere, and their densities in CEN6 and chromosome arms were similar. The exception was an accumulation of satellite DNA in CEN6, where it formed multiple arrays up to 2 Mb in length. Centromeric chromatin, characterized by the presence of the CENH3 protein, was predominantly associated with arrays of three different satellite repeats; however, five other satellites present in CEN6 lacked CENH3. The presence of CENH3 chromatin was found to determine the spatial distribution of the respective satellites during the cell cycle. Finally, oligo-FISH painting experiments, performed using probes specifically designed to label the genomic regions corresponding to CEN6 in Pisum, Lathyrus, and Vicia species, revealed that metapolycentromeres evolved via the expansion of centromeric chromatin into neighboring chromosomal regions and the accumulation of novel satellite repeats. However, in some of these species, centromere evolution also involved chromosomal translocations and centromere repositioning.
- MeSH
- centromera genetika MeSH
- chromatin genetika MeSH
- hrách setý * genetika MeSH
- lidé MeSH
- lidské chromozomy, pár 6 * MeSH
- satelitní DNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chromatin MeSH
- satelitní DNA MeSH
The documentation of plant growth and development requires integrative and scalable approaches to investigate and spatiotemporally resolve various dynamic processes at different levels of plant body organization. The present update deals with vigorous developments in mesoscopy, microscopy and nanoscopy methods that have been translated to imaging of plant subcellular compartments, cells, tissues and organs over the past 3 years with the aim to report recent applications and reasonable expectations from current light-sheet fluorescence microscopy (LSFM) and super-resolution microscopy (SRM) modalities. Moreover, the shortcomings and limitations of existing LSFM and SRM are discussed, particularly for their ability to accommodate plant samples and regarding their documentation potential considering spherical aberrations or temporal restrictions prohibiting the dynamic recording of fast cellular processes at the three dimensions. For a more comprehensive description, advances in living or fixed sample preparation methods are also included, supported by an overview of developments in labeling strategies successfully applied in plants. These strategies are practically documented by current applications employing model plant Arabidopsis thaliana (L.) Heynh., but also robust crop species such as Medicago sativa L. and Hordeum vulgare L. Over the past few years, the trend towards designing of integrative microscopic modalities has become apparent and it is expected that in the near future LSFM and SRM will be bridged to achieve broader multiscale plant imaging with a single platform.
Repeat-rich regions of higher plant genomes are usually associated with constitutive heterochromatin, a specific type of chromatin that forms tightly packed nuclear chromocenters and chromosome bands. There is a large body of cytogenetic evidence that these chromosome regions are often composed of tandemly organized satellite DNA. However, comparatively little is known about the sequence arrangement within heterochromatic regions, which are difficult to assemble due to their repeated nature. Here, we explore long-range sequence organization of heterochromatin regions containing the major satellite repeat CUS-TR24 in the holocentric plant Cuscuta europaea. Using a combination of ultra-long read sequencing with assembly-free sequence analysis, we reveal the complex structure of these loci, which are composed of short arrays of CUS-TR24 interrupted frequently by emerging simple sequence repeats and targeted insertions of a specific lineage of LINE retrotransposons. These data suggest that the organization of satellite repeats constituting heterochromatic chromosome bands can be more complex than previously envisioned, and demonstrate that heterochromatin organization can be efficiently investigated without the need for genome assembly.
- Klíčová slova
- Fluorescence in situ hybridization, Heterochromatin, Holocentric chromosomes, LINE elements, Oxford Nanopore sequencing, Satellite DNA,
- Publikační typ
- časopisecké články MeSH
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
- Klíčová slova
- Hordeum vulgare, chromatin, deconvolution microscopy, metaphase chromosome, nanoscopy, photoactivated localization microscopy, stimulated emission depletion microscopy, structured illumination microscopy, topoisomerase II, wide-field microscopy,
- MeSH
- chromozomy rostlin chemie genetika metabolismus MeSH
- DNA-topoisomerasy typu II metabolismus MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční mikroskopie metody MeSH
- indoly chemie MeSH
- ječmen (rod) cytologie genetika MeSH
- konfokální mikroskopie metody MeSH
- metafáze genetika MeSH
- reprodukovatelnost výsledků MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- DAPI MeSH Prohlížeč
- DNA-topoisomerasy typu II MeSH
- fluorescenční barviva MeSH
- indoly MeSH
