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1.
Philos Trans A Math Phys Eng Sci ; 379(2199): 20200144, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-33896204

RESUMO

We report that high-density single-molecule super-resolution microscopy can be achieved with a conventional epifluorescence microscope set-up and a mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM) is an extension of single-molecule localization microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as 'blinking'), detected and localized. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that stimulated emission depletion and LFSM can be performed on the same biological sample using a simple commercial mounting medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.


Assuntos
Microscopia de Fluorescência/instrumentação , Imagem Individual de Molécula/instrumentação , Anfíbios , Animais , Cromossomos/química , Cromossomos/ultraestrutura , Desenho de Equipamento , Corantes Fluorescentes , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Compostos Orgânicos , Estudo de Prova de Conceito , Imagem Individual de Molécula/métodos , Complexo Sinaptonêmico/química , Complexo Sinaptonêmico/ultraestrutura , Xantenos
2.
PLoS Genet ; 17(3): e1009205, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33730019

RESUMO

During sexual reproduction the parental homologous chromosomes find each other (pair) and align along their lengths by integrating local sequence homology with large-scale contiguity, thereby allowing for precise exchange of genetic information. The Synaptonemal Complex (SC) is a conserved zipper-like structure that assembles between the homologous chromosomes, bringing them together and regulating exchanges between them. However, the molecular mechanisms by which the SC carries out these functions remain poorly understood. Here we isolated and characterized two mutations in the dimerization interface in the middle of the SC zipper in C. elegans. The mutations perturb both chromosome alignment and the regulation of genetic exchanges. Underlying the chromosome-scale phenotypes are distinct alterations to the way SC subunits interact with one another. We propose a model whereby the SC brings homologous chromosomes together through two activities: obligate zipping that prevents assembly on unpaired chromosomes; and a tendency to extend pairing interactions along the entire length of the chromosomes.


Assuntos
Pareamento Cromossômico , Troca Genética , Meiose/genética , Complexo Sinaptonêmico , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Imunofluorescência , Imuno-Histoquímica , Masculino , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Complexo Sinaptonêmico/metabolismo , Complexo Sinaptonêmico/ultraestrutura
3.
Exp Cell Res ; 399(2): 112455, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33400935

RESUMO

During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Meiose/fisiologia , Complexo Sinaptonêmico/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Cromátides/ultraestrutura , Pareamento Cromossômico/fisiologia , DNA/química , DNA/ultraestrutura , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Complexo Sinaptonêmico/fisiologia , Complexo Sinaptonêmico/ultraestrutura
4.
PLoS Biol ; 18(8): e3000817, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813728

RESUMO

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative, and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 functionally distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister-chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging 3 per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister-chromatid loops. Indeed, immunofluorescence/fluorescence in situ hybridization (immuno-FISH) assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.


Assuntos
Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Cromossomos/ultraestrutura , Meiose , Complexo Sinaptonêmico/ultraestrutura , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos/metabolismo , Análise Citogenética , Hibridização in Situ Fluorescente , Fase S/genética , Complexo Sinaptonêmico/metabolismo , Coesinas
5.
Methods Mol Biol ; 2061: 181-196, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31583660

RESUMO

An improved method of preparing two-dimensional surface spreads of synaptonemal complexes (SCs) in higher plants for examination by electron microscopy is described. This protocol produces clear, well-spread preparations of SCs and unpaired axial cores from a range of meiotic prophase I stages (leptotene to pachytene) from meiocytes of different plant species. Synaptonemal complex (SC) analyses have been widely used in plant cytogenetic studies to address the process of meiotic chromosome synapses, because of the high-resolution allowed by electron microscopy. Although the real role of SC is still enigmatic, its presence and structural conservation in the vast majority of organisms reflect the importance of this protein structure in the meiotic process.


Assuntos
Meiose , Microscopia Eletrônica , Plantas/genética , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/ultraestrutura , Microscopia Eletrônica/métodos
6.
Sci Rep ; 9(1): 16102, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31695079

RESUMO

The synaptonemal complex is a multiprotein complex, which mediates the synapsis and recombination between homologous chromosomes during meiosis. The complex is comprised of two lateral elements and a central element connected by perpendicular transverse filaments (TFs). A 3D model based on actual morphological data of the SC is missing. Here, we applied electron tomography (ET) and manual feature extraction to generate a quantitative 3D model of the murine SC. We quantified the length (90 nm) and width (2 nm) of the TFs. Interestingly, the 80 TFs/µm are distributed asymmetrically in the central region of the SC challenging available models of SC organization. Furthermore, our detailed 3D topological analysis does not support a bilayered organization of the central region as proposed earlier. Overall, our quantitative analysis is relevant to understand the functions and dynamics of the SC and provides the basis for analyzing multiprotein complexes in their morphological context using ET.


Assuntos
Pareamento Cromossômico , Cromossomos/genética , Meiose , Animais , Cromossomos/ultraestrutura , Tomografia com Microscopia Eletrônica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/ultraestrutura , Testículo/citologia
7.
Exp Cell Res ; 383(2): 111587, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31454492

RESUMO

A typical nucleolus structure is shaped by three components. A meshwork of fine fibers forming the fibrillar center (FC) is surrounded by densely packed fibers forming the dense fibrillar component (DFC). Meanwhile, wrapping the FC and DFC is the granular component (GC). During the mitotic prophase, the nucleolus undergoes disassembling of its components. On the contrary, throughout the first meiotic prophase that occurs in the cells of the germ line, small nucleoli are assembled into one nucleolus by the end of the prophase. These nucleoli are transcriptionally active, suggesting that they are fully functional. Electron microscopy analysis has suggested that these nucleoli display their three main components but a typical organization has not been observed. Here, by immunolabeling and electron microscopy, we show that the nucleolus has its three main components. The GC is interlaced with the DFC and is not as well defined as previously thought during leptotene and zygotene stage.


Assuntos
Nucléolo Celular/ultraestrutura , Prófase/fisiologia , Espermatócitos/citologia , Espermatócitos/ultraestrutura , Animais , Nucléolo Celular/fisiologia , Masculino , Meiose/fisiologia , Microscopia Eletrônica , Ratos , Complexo Sinaptonêmico/ultraestrutura , Testículo/citologia , Testículo/ultraestrutura
8.
PLoS Genet ; 15(5): e1008072, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31150390

RESUMO

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.


Assuntos
Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiose , Microtúbulos/metabolismo , Proteína Fosfatase 1/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Centrômero/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos de Insetos/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica , Microtúbulos/ultraestrutura , Oócitos/citologia , Oócitos/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Separase/genética , Separase/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Complexo Sinaptonêmico/metabolismo , Complexo Sinaptonêmico/ultraestrutura , Coesinas
9.
J Math Biol ; 77(5): 1341-1362, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29922920

RESUMO

We show that an inhomogeneous Bernoulli site percolation process running upon a fullerene's dual [Formula: see text] can be used for representing bivalents attached to the nuclear envelope in mouse Mus M. Domesticus 2n = 40 meiotic spermatocytes during pachytene. It is shown that the induced clustering generated by overlapping percolation domains correctly reproduces the probability distribution observed in the experiments (data) after fine tuning the parameters.


Assuntos
Cromossomos/genética , Meiose , Modelos Genéticos , Espermatócitos/ultraestrutura , Animais , Cromossomos/ultraestrutura , Simulação por Computador , Heterocromatina/genética , Heterocromatina/ultraestrutura , Masculino , Conceitos Matemáticos , Meiose/genética , Camundongos , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Estágio Paquíteno/genética , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/ultraestrutura
10.
J Cell Sci ; 131(5)2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29420300

RESUMO

Sexual dimorphism has been used to describe morphological differences between the sexes, but can be extended to any biologically related process that varies between males and females. The synaptonemal complex (SC) is a tripartite structure that connects homologous chromosomes in meiosis. Here, aided by super-resolution microscopy techniques, we show that the SC is subject to sexual dimorphism, in mouse germ cells. We have identified a significantly narrower SC in oocytes and have established that this difference does not arise from a different organization of the lateral elements nor from a different isoform of transverse filament protein SYCP1. Instead, we provide evidence for the existence of a narrower central element and a different integration site for the C-termini of SYCP1, in females. In addition to these female-specific features, we speculate that post-translation modifications affecting the SYCP1 coiled-coil region could render a more compact conformation, thus contributing to the narrower SC observed in females.


Assuntos
Proteínas Nucleares/genética , Oócitos/ultraestrutura , Espermatócitos/ultraestrutura , Complexo Sinaptonêmico/ultraestrutura , Animais , Proteínas de Ligação a DNA , Feminino , Masculino , Meiose/genética , Camundongos , Proteínas Nucleares/química , Oócitos/metabolismo , Gravidez , Conformação Proteica , Caracteres Sexuais , Espermatócitos/metabolismo , Complexo Sinaptonêmico/genética , Testículo/metabolismo , Testículo/ultraestrutura
11.
Proc Natl Acad Sci U S A ; 114(33): E6857-E6866, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28760978

RESUMO

The synaptonemal complex (SC), a structure highly conserved from yeast to mammals, assembles between homologous chromosomes and is essential for accurate chromosome segregation at the first meiotic division. In Drosophila melanogaster, many SC components and their general positions within the complex have been dissected through a combination of genetic analyses, superresolution microscopy, and electron microscopy. Although these studies provide a 2D understanding of SC structure in Drosophila, the inability to optically resolve the minute distances between proteins in the complex has precluded its 3D characterization. A recently described technology termed expansion microscopy (ExM) uniformly increases the size of a biological sample, thereby circumventing the limits of optical resolution. By adapting the ExM protocol to render it compatible with structured illumination microscopy, we can examine the 3D organization of several known Drosophila SC components. These data provide evidence that two layers of SC are assembled. We further speculate that each SC layer may connect two nonsister chromatids, and present a 3D model of the Drosophila SC based on these findings.


Assuntos
Drosophila melanogaster/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia Eletrônica/métodos , Complexo Sinaptonêmico/ultraestrutura , Animais , Feminino , Microscopia Imunoeletrônica/métodos
12.
Genetics ; 207(1): 103-114, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28710064

RESUMO

Segregation of homologous chromosomes during meiosis depends on their ability to reorganize within the nucleus, discriminate among potential partners, and stabilize pairwise associations through assembly of the synaptonemal complex (SC). Here we report a high-resolution time-course analysis of these key early events during Caenorhabditis elegans meiosis. Labeled nucleotides are incorporated specifically into the X chromosomes during the last 2 hr of S phase, a property we exploit to identify a highly synchronous cohort of nuclei. By tracking X-labeled nuclei through early meiotic prophase, we define the sequence and duration of chromosome movement, nuclear reorganization, pairing at pairing centers (PCs), and SC assembly. Appearance of ZYG-12 foci (marking attachment of PCs to the nuclear envelope) and onset of active mobilization occur within an hour after S-phase completion. Movement occurs for nearly 2 hr before stable pairing is observed at PCs, and autosome movement continues for ∼4 hr thereafter. Chromosomes are tightly clustered during a 2-3 hr postpairing window, during which the bulk of SC assembly occurs; however, initiation of SC assembly can precede evident chromosome clustering. SC assembly on autosomes begins immediately after PC pairing is detected and is completed within ∼3.5 hr. For the X chromosomes, PC pairing is contemporaneous with autosomal pairing, but autosomes complete synapsis earlier (on average) than X chromosomes, implying that X chromosomes have a delay in onset and/or a slower rate of SC assembly. Additional evidence suggests that transient association among chromosomes sharing the same PC protein may contribute to partner discrimination.


Assuntos
Caenorhabditis elegans/genética , Pareamento Cromossômico , Prófase Meiótica I , Complexo Sinaptonêmico/genética , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Complexo Sinaptonêmico/ultraestrutura , Cromossomo X/genética
13.
Chromosoma ; 126(5): 615-631, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28101670

RESUMO

Homologous chromosomes exchange genetic information through recombination during meiosis, a process that increases genetic diversity, and is fundamental to sexual reproduction. In an attempt to shed light on the dynamics of mammalian recombination and its implications for genome organization, we have studied the recombination characteristics of 112 individuals belonging to 28 different species in the family Bovidae. In particular, we analyzed the distribution of RAD51 and MLH1 foci during the meiotic prophase I that serve, respectively, as proxies for double-strand breaks (DSBs) which form in early stages of meiosis and for crossovers. In addition, synaptonemal complex length and meiotic DNA loop size were estimated to explore how genome organization determines DSBs and crossover patterns. We show that although the number of meiotic DSBs per cell and recombination rates observed vary between individuals of the same species, these are correlated with diploid number as well as with synaptonemal complex and DNA loop sizes. Our results illustrate that genome packaging, DSB frequencies, and crossover rates tend to be correlated, while meiotic chromosomal axis length and DNA loop size are inversely correlated in mammals. Moreover, axis length, DSB frequency, and crossover frequencies all covary, suggesting that these correlations are established in the early stages of meiosis.


Assuntos
Cromossomos de Mamíferos/ultraestrutura , Meiose , Recombinação Genética , Ruminantes/genética , Complexo Sinaptonêmico/ultraestrutura , Animais , Cromossomos de Mamíferos/metabolismo , Quebras de DNA de Cadeia Dupla , Masculino , Camundongos , Proteína 1 Homóloga a MutL , Rad51 Recombinase , Ruminantes/metabolismo , Complexo Sinaptonêmico/metabolismo
14.
Cytogenet Genome Res ; 150(2): 77-85, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27997882

RESUMO

Human infertility is often classified as idiopathic in both males and females. Meiotic errors may account for at least part of these cases. As the synaptonemal complex (SC, a meiosis-specific protein scaffold) is essential for successful meiosis progression, in this paper, we analyzed the mutations in genes coding for SC components described in infertile patients to assess to what extent alterations in the SC can be related to human infertility. So far, mutations in SYCP3 and SYCE1 genes have been reported. While most SYCP3 mutations are heterozygous mutations with dominant-negative effect on the region encoding the C-terminal coiled coil of the protein, SYCE1 mutations are homozygous, which is consistent with a recessive inheritance. Similarities and differences between males and females as well as between mice and humans have been found and are discussed herein. The results suggest that a low percentage of human infertility cases may be explained by mutations in genes coding for SC components. The characterization of these mutations, together with available information from the study of knockout mice, will enable a deeper understanding of the underlying molecular bases for some of the cases of idiopathic infertility.


Assuntos
Fertilidade/genética , Mutação , Complexo Sinaptonêmico/genética , Animais , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Complexo Sinaptonêmico/ultraestrutura
15.
PLoS One ; 11(8): e0161379, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27548613

RESUMO

The synaptonemal complex protein 1 (SYCP1) is the main structural element of transverse filaments (TFs) of the synaptonemal complex (SC), which is a meiosis-specific complex structure formed at the synapse of homologue chromosomes to hold them together. The N-terminal domain of SYCP1 is known to be located within the central elements (CEs), whereas the C-terminal domain is located toward lateral elements (LEs). SYCP1 is a well-known meiosis marker that is also known to be a prognostic marker in the early stage of several cancers including breast, gliomas, and ovarian cancers. The structure of SC, especially the TF structure formed mainly by SYCP1, remains unclear without any structural information. To elucidate a molecular basis of SC formation and function, we first solved the crystal structure of C-terminal coiled-coil domain of SYCP1. The coiled-coil domain of SYCP1 forms asymmetric, anti-parallel dimers in solution.


Assuntos
Proteínas Nucleares/química , Complexo Sinaptonêmico/ultraestrutura , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Proteínas de Ligação a DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Meiose , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo
16.
Cell ; 166(4): 1028-1040, 2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27397506

RESUMO

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.


Assuntos
Técnicas Citológicas/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Bacteriófagos/ultraestrutura , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Técnicas Citológicas/instrumentação , Complexo de Golgi/ultraestrutura , Masculino , Camundongos , Microscopia de Fluorescência/instrumentação , Imagem Individual de Molécula/instrumentação , Espermatócitos/ultraestrutura , Complexo Sinaptonêmico/ultraestrutura
17.
Chromosome Res ; 24(3): 393-405, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27294972

RESUMO

Holocentric chromosomes occur in a number of independent eukaryotic lineages, and they form holokinetic kinetochores along the entire poleward chromatid surfaces. Due to this alternative chromosome structure, Luzula elegans sister chromatids segregate already in anaphase I followed by the segregation of the homologues in anaphase II. However, not yet known is the localization and dynamics of cohesin and the structure of the synaptonemal complex (SC) during meiosis. We show here that the α-kleisin subunit of cohesin localizes at the centromeres of both mitotic and meiotic metaphase chromosomes and that it, thus, may contribute to assemble the centromere in L. elegans. This localization and the formation of a tripartite SC structure indicate that the prophase I behaviour of L. elegans is similar as in monocentric species.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Magnoliopsida/genética , Meiose/genética , Prófase Meiótica I/genética , Proteínas de Plantas/metabolismo , Complexo Sinaptonêmico/ultraestrutura , Autoantígenos/genética , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteína Centromérica A , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Cinetocoros/metabolismo , Magnoliopsida/metabolismo , Proteínas de Plantas/genética , Análise de Sequência de DNA , Coesinas
18.
Nat Struct Mol Biol ; 23(5): 369-77, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27142324

RESUMO

The synaptonemal complex (SC) is a meiosis-specific scaffold that links homologous chromosomes from end to end during meiotic prophase and is required for the formation of meiotic crossovers. Assembly of SC components is regulated by a combination of associated nonstructural proteins and post-translational modifications, such as SUMOylation, which together coordinate the timing between homologous chromosome pairing, double-strand-break formation and recombination. In addition, transcriptional and translational control mechanisms ensure the timely disassembly of the SC after crossover resolution and before chromosome segregation at anaphase I.


Assuntos
Meiose , Complexo Sinaptonêmico/metabolismo , Animais , Pareamento Cromossômico , Segregação de Cromossomos , Humanos , Processamento de Proteína Pós-Traducional , Complexo Sinaptonêmico/química , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/ultraestrutura
19.
Exp Cell Res ; 344(1): 22-29, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27090018

RESUMO

The synaptonemal complex (SC) is a proteinaceous structure that holds the homologous chromosomes in close proximity while they exchange genetic material in a process known as meiotic recombination. This meiotic recombination leads to genetic variability in sexually reproducing organisms. The ultrastructure of the SC is studied by electron microscopy and it is observed as a tripartite structure. Two lateral elements (LE) separated by a central region (CR) confer its classical tripartite organization. The LEs are the anchoring platform for the replicated homologous chromosomes to properly exchange genetic material with one another. An accurate assembly of the LE is indispensable for the proper completion of meiosis. Ultrastructural studies suggested that the LE is organized as a multilayered unit. However, no validation of this model has been previously provided. In this ultrastructural study, by using mice with different genetic backgrounds that affect the LE width, we provide further evidence that support a multilayered organization of the LE. Additionally, we provide data suggesting additional roles of the different cohesin complex components in the structure of the LEs of the SC.


Assuntos
Complexo Sinaptonêmico/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA , Masculino , Meiose , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Complexo Sinaptonêmico/ultraestrutura , Coesinas
20.
J Cell Sci ; 129(11): 2239-49, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27103161

RESUMO

The synaptonemal complex transiently stabilizes pairing interactions between homologous chromosomes during meiosis. Assembly of the synaptonemal complex is mediated through integration of opposing transverse filaments into a central element, a process that is poorly understood. We have, here, analyzed the localization of the transverse filament protein SYCP1 and the central element proteins SYCE1, SYCE2 and SYCE3 within the central region of the synaptonemal complex in mouse spermatocytes using immunoelectron microscopy. Distribution of immuno-gold particles in a lateral view of the synaptonemal complex, supported by protein interaction data, suggest that the N-terminal region of SYCP1 and SYCE3 form a joint bilayered central structure, and that SYCE1 and SYCE2 localize in between the two layers. We find that disruption of SYCE2 and TEX12 (a fourth central element protein) localization to the central element abolishes central alignment of the N-terminal region of SYCP1. Thus, our results show that all four central element proteins, in an interdependent manner, contribute to stabilization of opposing N-terminal regions of SYCP1, forming a bilayered transverse-filament-central-element junction structure that promotes synaptonemal complex formation and synapsis.


Assuntos
Complexo Sinaptonêmico/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/metabolismo , Proteínas de Ligação a DNA , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estágio Paquíteno , Ligação Proteica , Complexo Sinaptonêmico/ultraestrutura
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