Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity.

In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4PPH-4.1 phosphatase and ATRATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4PPH-4.1, ATRATL-1, and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.

Nematode chromosomes.

The nematode Caenorhabditis elegans has shed light on many aspects of eukaryotic biology, including genetics, development, cell biology, and genomics. A major factor in the success of C. elegans as a model organism has been the availability, since the late 1990s, of an essentially gap-free and well-annotated nuclear genome sequence, divided among 6 chromosomes. In this review, we discuss the structure, function, and biology of C. elegans chromosomes and then provide a general perspective on chromosome biology in other diverse nematode species. We highlight malleable chromosome features including centromeres, telomeres, and repetitive elements, as well as the remarkable process of programmed DNA elimination (historically described as chromatin diminution) that induces loss of portions of the genome in somatic cells of a handful of nematode species. An exciting future prospect is that nematode species may enable experimental approaches to study chromosome features and to test models of chromosome evolution. In the long term, fundamental insights regarding how speciation is integrated with chromosome biology may be revealed.

Sequential peripheral enrichment of H2A.Zac and H3K9me2 during trophoblast differentiation in human embryonic stem cells.

During the transition from pluripotency to a lineage-committed state, chromatin undergoes large-scale changes in structure, involving covalent modification of histone tails, use of histone variants and gene position changes with respect to the nuclear periphery. Here, using high-resolution microscopy and quantitative image analysis, we surveyed a panel of histone modifications for changes in nuclear peripheral enrichment during differentiation of human embryonic stem cells to a trophoblast-like lineage. We found two dynamic modifications at the nuclear periphery, acetylation of histone H2A.Z (H2A.Zac), and dimethylation of histone H3 at lysine 9 (H3K9me2). We demonstrate successive peripheral enrichment of these markers, with H2A.Zac followed by H3K9me2, over the course of 4 days. We find that H3K9me2 increases concomitantly with, but independently of, expression of lamin A, since deletion of lamin A did not affect H3K9me2 enrichment. We further show that inhibition of histone deacetylases causes persistent and increased H2A.Z acetylation at the periphery, delayed H3K9me2 enrichment and failure to differentiate. Our results show a concerted change in the nature of peripheral chromatin occurs upon differentiation into the trophoblast state.

Sycp2 is essential for synaptonemal complex assembly, early meiotic recombination and homologous pairing in zebrafish spermatocytes.

Meiotic recombination is essential for faithful segregation of homologous chromosomes during gametogenesis. The progression of recombination is associated with dynamic changes in meiotic chromatin structures. However, whether Sycp2, a key structural component of meiotic chromatin, is required for the initiation of meiotic recombination is still unclear in vertebrates. Here, we describe that Sycp2 is required for assembly of the synaptonemal complex and early meiotic events in zebrafish spermatocytes. Our genetic screening by N-ethyl-N-nitrosourea mutagenesis revealed that ietsugu (its), a mutant zebrafish line with an aberrant splice site in the sycp2 gene, showed a defect during meiotic prophase I. The its mutation appeared to be a hypomorphic mutation compared to sycp2 knockout mutations generated by TALEN mutagenesis. Taking advantage of these sycp2 hypomorphic and knockout mutant lines, we demonstrated that Sycp2 is required for the assembly of the synaptonemal complex that is initiated in the vicinity of telomeres in wild-type zebrafish spermatocytes. Accordingly, homologous pairing, the foci of the meiotic recombinases Dmc1/Rad51 and RPA, and γH2AX signals were largely diminished in sycp2 knockout spermatocytes. Taken together, our data indicate that Sycp2 plays a critical role in not only the assembly of the synaptonemal complex, but also early meiotic recombination and homologous pairing, in vertebrates.

The histone variant H2A.Z is dynamically expressed in the developing mouse placenta and in differentiating trophoblast stem cells.

The histone variant H2A.Z is important in establishing new chromatin environments necessary for permitting changes in gene expression and thus differentiation in mouse embryonic stem (mES) cells. In this study we show that H2A.Z is highly expressed in the early mouse placenta, and is specifically limited to progenitor-like trophoblast cells. Using in vitro models, we revealed distinct differences in H2A.Z abundance between undifferentiated, differentiating and differentiated mouse trophoblast stem (mTS) cells. Our work supports the hypothesis that in addition to roles in differentiating mES cells, H2A.Z is also involved in the differentiation of extra-embryonic tissues.

Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-ß signaling and astrocyte functions.

Astrocytes modulate neuronal activity and inhibit regeneration. We show that cleaved p75 neurotrophin receptor (p75(NTR)) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of transforming growth factor (TGF)-ß signaling. Cleaved p75(NTR) interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75(NTR) controls astrocyte-neuronal communication in response to profibrotic factors.

Protein phosphatase 4 promotes chromosome pairing and synapsis, and contributes to maintaining crossover competence with increasing age.

Prior to the meiotic divisions, dynamic chromosome reorganizations including pairing, synapsis, and recombination of maternal and paternal chromosome pairs must occur in a highly regulated fashion during meiotic prophase. How chromosomes identify each other's homology and exclusively pair and synapse with their homologous partners, while rejecting illegitimate synapsis with non-homologous chromosomes, remains obscure. In addition, how the levels of recombination initiation and crossover formation are regulated so that sufficient, but not deleterious, levels of DNA breaks are made and processed into crossovers is not understood well. We show that in Caenorhabditis elegans, the highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1, is required independently to carry out four separate functions involving meiotic chromosome dynamics: (1) synapsis-independent chromosome pairing, (2) restriction of synapsis to homologous chromosomes, (3) programmed DNA double-strand break initiation, and (4) crossover formation. Using quantitative imaging of mutant strains, including super-resolution (3D-SIM) microscopy of chromosomes and the synaptonemal complex, we show that independently-arising defects in each of these processes in the absence of PPH-4.1 activity ultimately lead to meiotic nondisjunction and embryonic lethality. Interestingly, we find that defects in double-strand break initiation and crossover formation, but not pairing or synapsis, become even more severe in the germlines of older mutant animals, indicating an increased dependence on PPH-4.1 with increasing maternal age. Our results demonstrate that PPH-4.1 plays multiple, independent roles in meiotic prophase chromosome dynamics and maintaining meiotic competence in aging germlines. PP4's high degree of conservation suggests it may be a universal regulator of meiotic prophase chromosome dynamics.

Application of advanced fluorescence microscopy to the structure of meiotic chromosomes.

Chromosomes undergoing meiosis are defined by a macromolecular protein assembly called the synaptonemal complex which holds homologs together and carries out important meiotic functions. By retaining the molecular specificity, multiplexing ability, and in situ imaging capabilities of fluorescence microscopy, but with vastly increased resolution, 3D-SIM and other superresolution techniques are poised to make significant discoveries about the structure and function of the synaptonemal complex. This review discusses recent developments in this field and poses questions approachable with current and future technology.

HAL-2 promotes homologous pairing during Caenorhabditis elegans meiosis by antagonizing inhibitory effects of synaptonemal complex precursors.

During meiosis, chromosomes align with their homologous pairing partners and stabilize this alignment through assembly of the synaptonemal complex (SC). Since the SC assembles cooperatively yet is indifferent to homology, pairing and SC assembly must be tightly coordinated. We identify HAL-2 as a key mediator in this coordination, showing that HAL-2 promotes pairing largely by preventing detrimental effects of SC precursors (SYP proteins). hal-2 mutants fail to establish pairing and lack multiple markers of chromosome movement mediated by pairing centers (PCs), chromosome sites that link chromosomes to cytoplasmic microtubules through nuclear envelope-spanning complexes. Moreover, SYP proteins load inappropriately along individual unpaired chromosomes in hal-2 mutants, and markers of PC-dependent movement and function are restored in hal-2; syp double mutants. These and other data indicate that SYP proteins can impede pairing and that HAL-2 promotes pairing predominantly but not exclusively by counteracting this inhibition, thereby enabling activation and regulation of PC function. HAL-2 concentrates in the germ cell nucleoplasm and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented. We propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing untimely interactions between SC precursors and chromosomes and allowing sufficient accumulation of precursors for rapid cooperative assembly upon homology verification.

Dynein-dependent processive chromosome motions promote homologous pairing in C. elegans meiosis.

Meiotic chromosome segregation requires homologue pairing, synapsis, and crossover recombination, which occur during meiotic prophase. Telomere-led chromosome motion has been observed or inferred to occur during this stage in diverse species, but its mechanism and function remain enigmatic. In Caenorhabditis elegans, special chromosome regions known as pairing centers (PCs), rather than telomeres, associate with the nuclear envelope (NE) and the microtubule cytoskeleton. In this paper, we investigate chromosome dynamics in living animals through high-resolution four-dimensional fluorescence imaging and quantitative motion analysis. We find that chromosome movement is constrained before meiosis. Upon prophase onset, constraints are relaxed, and PCs initiate saltatory, processive, dynein-dependent motions along the NE. These dramatic motions are dispensable for homologous pairing and continue until synapsis is completed. These observations are consistent with the idea that motions facilitate pairing by enhancing the search rate but that their primary function is to trigger synapsis. This quantitative analysis of chromosome dynamics in a living animal extends our understanding of the mechanisms governing faithful genome inheritance.

Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones.

Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10-30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples.

Fast live simultaneous multiwavelength four-dimensional optical microscopy.

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

Cytoskeletal forces span the nuclear envelope to coordinate meiotic chromosome pairing and synapsis.

During meiosis, each chromosome must pair with its unique homologous partner, a process that usually culminates with the formation of the synaptonemal complex (SC). In the nematode Caenorhabditis elegans, special regions on each chromosome known as pairing centers are essential for both homologous pairing and synapsis. We report that during early meiosis, pairing centers establish transient connections to the cytoplasmic microtubule network. These connections through the intact nuclear envelope require the SUN/KASH domain protein pair SUN-1 and ZYG-12. Disruption of microtubules inhibits chromosome pairing, indicating that these connections promote interhomolog interactions. Dynein activity is essential to license formation of the SC once pairing has been accomplished, most likely by overcoming a barrier imposed by the chromosome-nuclear envelope connection. Our findings thus provide insight into how homolog pairing is accomplished in meiosis and into the mechanisms regulating synapsis so that it occurs selectively between homologs. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

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.

Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy.

BACKGROUND: Telomeres cap chromosome ends and protect the genome. We studied individual telomeres in live human cancer cells. In capturing telomere motions using quantitative imaging to acquire complete high-resolution three-dimensional datasets every second for 200 seconds, telomere dynamics were systematically analyzed. RESULTS: The motility of individual telomeres within the same cancer cell nucleus was widely heterogeneous. One class of internal heterochromatic regions of chromosomes analyzed moved more uniformly and showed less motion and heterogeneity than telomeres. The single telomere analyses in cancer cells revealed that shorter telomeres showed more motion, and the more rapid telomere motions were energy dependent. Experimentally increasing bulk telomere length dampened telomere motion. In contrast, telomere uncapping, but not a DNA damaging agent, methyl methanesulfonate, significantly increased telomere motion. CONCLUSION: New methods for seconds-scale, four-dimensional, live cell microscopic imaging and data analysis, allowing systematic tracking of individual telomeres in live cells, have defined a previously undescribed form of telomere behavior in human cells, in which the degree of telomere motion was dependent upon telomere length and functionality.

N-terminal processing of proteins exported by malaria parasites.

Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signal-mediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this N-terminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence.

Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light.

Three-dimensional structured illumination microscopy and its application to chromosome structure.

This review discusses the exploration of chromosome structure with a recently developed high-resolution microscopy technique, three-dimensional structured illumination microscopy (3dSIM). 3dSIM surpasses the diffraction limit of conventional widefield optical microscopy, increasing the level of detail in images by a factor of 2, while retaining the sample preparation methods, ease of use and flexibility of conventional microscopy. Special attention will be given to the ways in which imaging beyond the diffraction limit can shed light on the structural organization of meiotic chromosomes.

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.