RESUMO
Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.
Assuntos
Oócitos , Proteínas , Gravidez , Animais , Feminino , Oócitos/metabolismo , Proteínas/metabolismo , Embrião de Mamíferos/metabolismo , Citoesqueleto , Ribossomos , Desenvolvimento Embrionário , MamíferosRESUMO
Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes1-4. There are at least two NPC assembly pathways-one during the exit from mitosis and one during nuclear growth in interphase-but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Humanos , Interfase , Mitose , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Espectrometria de FluorescênciaRESUMO
Most human embryos are aneuploid. Aneuploidy frequently arises during the early mitotic divisions of the embryo, but its origin remains elusive. Human zygotes that cluster their nucleoli at the pronuclear interface are thought to be more likely to develop into healthy euploid embryos. Here, we show that the parental genomes cluster with nucleoli in each pronucleus within human and bovine zygotes, and clustering is required for the reliable unification of the parental genomes after fertilization. During migration of intact pronuclei, the parental genomes polarize toward each other in a process driven by centrosomes, dynein, microtubules, and nuclear pore complexes. The maternal and paternal chromosomes eventually cluster at the pronuclear interface, in direct proximity to each other, yet separated. Parental genome clustering ensures the rapid unification of the parental genomes on nuclear envelope breakdown. However, clustering often fails, leading to chromosome segregation errors and micronuclei, incompatible with healthy embryo development.
Assuntos
Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Aneuploidia , Animais , Bovinos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos/metabolismo , Fertilização/genética , Humanos , Masculino , Microtúbulos/metabolismo , Mitose , Oócitos/metabolismo , Espermatozoides/metabolismo , Zigoto/metabolismoRESUMO
The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.
Assuntos
Fator de Ligação a CCCTC/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Dosagem de Genes , Expressão Gênica , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromátides/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Recuperação de Fluorescência Após Fotodegradação/métodos , Fase G1/genética , Genoma Humano/genética , Células HeLa , Humanos , Espectrometria de Massas/métodos , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , CoesinasRESUMO
Essential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, the timing of interactions and changes in cellular structure are all crucial to ensure the correct assembly, function and regulation of protein complexes1-4. Imaging of live cells can reveal protein distributions and dynamics but experimental and theoretical challenges have prevented the collection of quantitative data, which are necessary for the formulation of a model of mitosis that comprehensively integrates information and enables the analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries. Here we generate a canonical model of the morphological changes during the mitotic progression of human cells on the basis of four-dimensional image data. We use this model to integrate dynamic three-dimensional concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy5. The approach taken here to generate a dynamic protein atlas of human cell division is generic; it can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types, and can be conceptually transferred to other cellular functions.
Assuntos
Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Mitose , Edição de Genes , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imageamento Tridimensional , Microscopia de Fluorescência , Imagem Molecular , Fatores de TempoRESUMO
At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.
Assuntos
Segregação de Cromossomos , Embrião de Mamíferos/embriologia , Herança Materna/genética , Herança Paterna/genética , Polos do Fuso/metabolismo , Zigoto/metabolismo , Anáfase , Animais , Blastômeros/citologia , Núcleo Celular/metabolismo , Feminino , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismoRESUMO
The ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables quantitative understanding of protein dynamics at the physiological level. Genome-editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, thereby opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool for investigating spatial and temporal protein dynamics; however, it lacks the required quantitative power to make the kind of absolute and comparable measurements required for systems analysis. In contrast, fluorescence correlation spectroscopy (FCS) provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius, and oligomerization but lacks the capability for high-throughput application in 4D spatial and temporal imaging. Here we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high throughput. These data are processed to yield a calibration curve relating the fluorescence intensities (FIs) of image voxels to the absolute protein abundance. The calibration curve allows the conversion of the arbitrary FIs to protein amounts for all voxels of 4D imaging stacks. Using our workflow, users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared with other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 d.
Assuntos
Células/química , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Proteínas/análise , Proteoma/análise , Proteômica/métodos , Coloração e Rotulagem/métodos , Automação Laboratorial/métodos , Edição de Genes/métodos , Análise Espaço-TemporalRESUMO
The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing, and dynamic localization during mitosis by fluorescence correlation spectroscopy (FSC)-calibrated live-cell imaging and superresolution microscopy. Although â¼35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, â¼195,000 Condensin I complexes dynamically bind in two steps: prometaphase and early anaphase. The two Condensins rarely colocalize at the chromatid axis, where Condensin II is centrally confined, but Condensin I reaches â¼50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of â¼450 kb at the chromatid axis, whose size is then reduced by Condensin I binding to â¼90 kb in prometaphase and â¼70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.
Assuntos
Adenosina Trifosfatases/genética , Segregação de Cromossomos/genética , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Mitose/genética , Complexos Multiproteicos/genética , Anáfase/genética , Cromátides/genética , Edição de Genes , HumanosRESUMO
Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.
Assuntos
Amiloide/química , Proteína Huntingtina/genética , Mutação , Peptídeos/química , Sistema Livre de Células , Éxons , Humanos , Proteína Huntingtina/química , Microscopia de Força Atômica , Microscopia Eletrônica , Modelos Moleculares , Agregados Proteicos , Estrutura Secundária de ProteínaRESUMO
Multinucleated cells are important in many organisms, but the mechanisms governing the movements of nuclei sharing a common cytoplasm are not understood. In the hyphae of the plant pathogenic fungus Ashbya gossypii, nuclei move back and forth, occasionally bypassing each other, preventing the formation of nuclear clusters. This is essential for genetic stability. These movements depend on cytoplasmic microtubules emanating from the nuclei that are pulled by dynein motors anchored at the cortex. Using three-dimensional stochastic simulations with parameters constrained by the literature, we predict the cortical anchor density from the characteristics of nuclear movements. The model accounts for the complex nuclear movements seen in vivo, using a minimal set of experimentally determined ingredients. Of interest, these ingredients power the oscillations of the anaphase spindle in budding yeast, but in A. gossypii, this system is not restricted to a specific nuclear cycle stage, possibly as a result of adaptation to hyphal growth and multinuclearity.
Assuntos
Núcleo Celular/fisiologia , Eremothecium/fisiologia , Microtúbulos/fisiologia , Actinas/metabolismo , Anáfase/fisiologia , Núcleo Celular/metabolismo , Simulação por Computador , Citoplasma/metabolismo , Dineínas/metabolismo , Eremothecium/citologia , Eremothecium/metabolismo , Células Gigantes/metabolismo , Células Gigantes/fisiologia , Hifas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fuso Acromático/metabolismo , Fuso Acromático/fisiologiaRESUMO
Oscillations occur in a wide variety of cellular processes, for example in calcium and p53 signaling responses, in metabolic pathways or within gene-regulatory networks, e.g. the circadian system. Since it is of central importance to understand the influence of perturbations on the dynamics of these systems a number of experimental and theoretical studies have examined their robustness. The period of circadian oscillations has been found to be very robust and to provide reliable timing. For intracellular calcium oscillations the period has been shown to be very sensitive and to allow for frequency-encoded signaling. We here apply a comprehensive computational approach to study the robustness of period and amplitude of oscillatory systems. We employ different prototype oscillator models and a large number of parameter sets obtained by random sampling. This framework is used to examine the effect of three design principles on the sensitivities towards perturbations of the kinetic parameters. We find that a prototype oscillator with negative feedback has lower period sensitivities than a prototype oscillator relying on positive feedback, but on average higher amplitude sensitivities. For both oscillator types, the use of Michaelis-Menten instead of mass action kinetics in all degradation and conversion reactions leads to an increase in period as well as amplitude sensitivities. We observe moderate changes in sensitivities if replacing mass conversion reactions by purely regulatory reactions. These insights are validated for a set of established models of various cellular rhythms. Overall, our work highlights the importance of reaction kinetics and feedback type for the variability of period and amplitude and therefore for the establishment of predictive models.
Assuntos
Relógios Biológicos/fisiologia , Sinalização do Cálcio/fisiologia , Fenômenos Fisiológicos Celulares , Ritmo Circadiano/fisiologia , Modelos Biológicos , Oscilometria/métodos , Animais , Simulação por Computador , Humanos , CinéticaRESUMO
The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Membrana Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/genética , Animais , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Células HeLa , Humanos , Microscopia de Fluorescência , Membrana Nuclear/química , Membrana Nuclear/ultraestrutura , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestruturaRESUMO
Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.
Assuntos
Segregação de Cromossomos , Cromossomos Humanos/metabolismo , Antígeno Ki-67/metabolismo , Mitose , Modelos Biológicos , Tensoativos/química , Fenômenos Biomecânicos , Compartimento Celular , Cromatina/metabolismo , Cromossomos Humanos/química , Humanos , Antígeno Ki-67/química , Antígeno Ki-67/genética , Membrana Nuclear/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , Solventes/química , Fuso Acromático/metabolismo , Eletricidade EstáticaRESUMO
Faithful chromosome segregation, during meiosis, is of critical importance to prevent aneuploidy in the resulting embryo. In mammalian oocytes, the segregation of homologous chromosomes takes place with the spindle located at the cell's periphery. The spindle is often assembled close to the centre of the cell, which necessitates the actin network for spindle transport to the cell cortex. In this study, we investigate how the segregation of chromosomes is coordinated with the positioning of the metaphase I spindle. We develop different assays to perturb the spindle's position and to delay its relocation to the cell periphery. We find that anaphase is delayed until the spindle is positioned in close proximity with the oocyte cortex. We further show that the metaphase arrest is dependent on a functional actin network, in addition to the spindle assembly checkpoint. Our work provides the first evidence for the existence of a functional spindle position checkpoint.
Assuntos
Actinas/metabolismo , Divisão Celular Assimétrica , Pontos de Checagem da Fase M do Ciclo Celular , Meiose , Oócitos/citologia , Fuso Acromático/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Segregação de Cromossomos , Proteínas de Fluorescência Verde , Metáfase , Camundongos , Microscopia Confocal , Microtúbulos/metabolismoRESUMO
Targeting of inner nuclear membrane (INM) proteins is essential for nuclear architecture and function, yet its mechanism remains poorly understood. Here, we established a new reporter that allows real-time imaging of membrane protein transport from the ER to the INM using Lamin B receptor and Lap2ß as model INM proteins. These reporters allowed us to characterize the kinetics of INM targeting and establish a mathematical model of this process and enabled us to probe its molecular requirements in an RNA interference screen of 96 candidate genes. Modeling of the phenotypes of genes involved in transport of these INM proteins predicted that it critically depended on the number and permeability of nuclear pores and the availability of nuclear binding sites, but was unaffected by depletion of most transport receptors. These predictions were confirmed with targeted validation experiments on the functional requirements of nucleoporins and nuclear lamins. Collectively, our data support a diffusion retention model of INM protein transport in mammalian cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Imagem Molecular , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Membrana Nuclear/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptor de Lamina BRESUMO
The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Modelos Moleculares , Multimerização Proteica , Citoesqueleto de Actina/ultraestrutura , Animais , Fenômenos Biomecânicos , Biologia Computacional , Simulação por Computador , Humanos , Técnicas In VitroRESUMO
Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B. We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.
Assuntos
Aurora Quinase B/genética , Proteínas de Fluorescência Verde/genética , Imagem Molecular , Plasmídeos/metabolismo , Transfecção/métodos , Transgenes , Aurora Quinase B/metabolismo , Sequência de Bases , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Mitose , Dados de Sequência Molecular , Plasmídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.
Assuntos
Núcleo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fuso Acromático/metabolismo , Núcleo Celular/ultraestrutura , Simulação por Computador , Proteínas Nucleares/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestruturaRESUMO
We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.
Assuntos
Citoesqueleto/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Eremothecium/metabolismo , Eremothecium/ultraestrutura , Hifas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto/ultraestrutura , Hifas/ultraestrutura , Microtúbulos/ultraestrutura , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
We present a multiscale, spatially distributed model of lung and airway behaviour with the goal of furthering the understanding of airway hyper-responsiveness and asthma. The model provides an initial computational framework for linking events at the cellular and molecular levels, such as Ca(2+) and crossbridge dynamics, to events at the level of the entire organ. At the organ level, parenchymal tissue is modelled using a continuum approach as a compressible, hyperelastic material in three dimensions, with expansion and recoil of lung tissue due to tidal breathing. The governing equations of finite elasticity deformation are solved using a finite element method. The airway tree is embedded in this tissue, where each airway is modelled with its own airway wall, smooth muscle and surrounding parenchyma. The tissue model is then linked to models of the crossbridge mechanics and their control by Ca(2+) dynamics, thus providing a link to molecular and cellular mechanisms in airway smooth muscle cells. By incorporating and coupling the models at these scales, we obtain a detailed, computational multiscale model incorporating important physiological phenomena associated with asthma.