RESUMO
Object tracking is an instrumental tool supporting studies of cellular trafficking. There are three challenges in object tracking: the identification of targets; the precise determination of their position and boundaries; and the assembly of correct trajectories. This last challenge is particularly relevant when dealing with densely populated images with low signal-to-noise ratios-conditions that are often encountered in applications such as organelle tracking, virus particle tracking or single-molecule imaging. We have developed a set of methods that can handle a wide variety of signal complexities. They are compiled into a free software package called Diatrack. Here we review its main features and utility in a range of applications, providing a survey of the dynamic imaging field together with recommendations for effective use. The performance of our framework is shown to compare favorably to a wide selection of custom-developed algorithms, whether in terms of localization precision, processing speed or correctness of tracks.
Assuntos
Algoritmos , Transporte Proteico/fisiologia , Imagem Individual de Molécula , Software , Humanos , Microscopia de Fluorescência/métodosRESUMO
Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500-1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Células Eucarióticas/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In higher eukaryotes, the nuclear envelope breaks down during mitosis. It reforms during telophase, and nuclear import is reestablished within <10 min after anaphase onset. It is widely assumed that import functionality simultaneously leads to the exclusion of bulk cytoplasmic proteins. However, nuclear pore complex assembly is not fully completed when import capacity is regained, which raises the question of whether the transport and permeability barrier functions of the nuclear envelope are indeed coupled. In this study, we therefore analyzed the reestablishment of the permeability barrier of the nuclear envelope after mitosis in living cells by monitoring the flux of the reversibly photoswitchable fluorescent protein Dronpa from the cytoplasm into the nucleus after photoactivation. We performed many consecutive flux measurements in the same cell to directly monitor changes in nuclear envelope permeability. Our measurements at different time points after mitosis in individual cells show that contrary to the general view and despite the rapid reestablishment of facilitated nuclear import, the nuclear envelope remains relatively permeable for passive diffusion for the first 2 h after mitosis. Our data demonstrate that reformation of the permeability barrier of nuclear pore complexes occurs only gradually and is uncoupled from regaining active import functionality.
Assuntos
Luz , Membrana Nuclear/metabolismo , Membrana Nuclear/efeitos da radiação , Anáfase/efeitos da radiação , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , Rim/citologia , Permeabilidade/efeitos da radiação , Ratos , Fatores de TempoRESUMO
The RNA export factor Mex67 is essential for the transport of mRNA through the nuclear pore complex (NPC) in yeast, but the molecular mechanism of this export process remains poorly understood. Here, we use quantitative fluorescence microscopy techniques in live budding yeast cells to investigate how Mex67 facilitates mRNA export. We show that Mex67 exhibits little interaction with mRNA in the nucleus and localizes to the NPC independently of mRNA, occupying a set of binding sites offered by FG repeats in the NPC. The ATPase Dbp5, which is thought to remove Mex67 from transcripts, does not affect the interaction of Mex67 with the NPC. Strikingly, we find that the essential function of Mex67 is spatially restricted to the NPC since a fusion of Mex67 to the nucleoporin Nup116 rescues a deletion of MEX67 Thus, Mex67 functions as a mobile NPC component, which receives mRNA export substrates in the central channel of the NPC to facilitate their translocation to the cytoplasm.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Helicases DEAD-box/metabolismo , Deleção de Genes , Microscopia de Fluorescência , Ligação Proteica , Transporte de RNA , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
In eukaryotes, all macromolecules traffic between the nucleus and the cytoplasm through nuclear pore complexes (NPCs), which are among the largest supramolecular assemblies in cells. Although their composition in yeast and metazoa is well characterized, understanding how NPCs are assembled and form the pore through the double membrane of the nuclear envelope and how both processes are controlled still remains a challenge. Here, we summarize what is known about the biogenesis of NPCs throughout the cell cycle with special focus on the membrane reorganization and the regulation that go along with NPC assembly.
Assuntos
Ciclo Celular , Membrana Celular/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Membrana Celular/ultraestrutura , Humanos , Mitose , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/ultraestruturaRESUMO
Chromatin organization is highly dynamic and regulates transcription. Upon transcriptional activation, chromatin is remodeled and referred to as "open," but quantitative and dynamic data of this decompaction process are lacking. Here, we have developed a quantitative high resolution-microscopy assay in living yeast cells to visualize and quantify chromatin dynamics using the GAL7-10-1 locus as a model system. Upon transcriptional activation of these three clustered genes, we detect an increase of the mean distance across this locus by >100 nm. This decompaction is linked to active transcription but is not sensitive to the histone deacetylase inhibitor trichostatin A or to deletion of the histone acetyl transferase Gcn5. In contrast, the deletion of SNF2 (encoding the ATPase of the SWI/SNF chromatin remodeling complex) or the deactivation of the histone chaperone complex FACT lead to a strongly reduced decompaction without significant effects on transcriptional induction in FACT mutants. Our findings are consistent with nucleosome remodeling and eviction activities being major contributors to chromatin reorganization during transcription but also suggest that transcription can occur in the absence of detectable decompaction.
Assuntos
Cromatina/metabolismo , Imageamento Tridimensional , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Acetilação , Bioensaio , Sobrevivência Celular , Loci Gênicos , Histonas/metabolismo , Mutação/genética , Nucleossomos/metabolismo , Fases de Leitura Aberta/genética , Transcrição GênicaRESUMO
We report the observation of chromatin dynamics in living budding yeast (Saccharomyces cerevisiae) cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the GAL locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the GAL locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state. These changes are visible in spite of the large thermally- and biologically-driven heterogeneity in the relative motion of the two loci. Our observations are consistent with current euchromatin vs. heterochromatin models.
RESUMO
The organization and biophysical properties of the cytosol implicitly govern molecular interactions within cells. However, little is known about mechanisms by which cells regulate cytosolic properties and intracellular diffusion rates. Here, we demonstrate that the intracellular environment of budding yeast undertakes a startling transition upon glucose starvation in which macromolecular mobility is dramatically restricted, reducing the movement of both chromatin in the nucleus and mRNPs in the cytoplasm. This confinement cannot be explained by an ATP decrease or the physiological drop in intracellular pH. Rather, our results suggest that the regulation of diffusional mobility is induced by a reduction in cell volume and subsequent increase in molecular crowding which severely alters the biophysical properties of the intracellular environment. A similar response can be observed in fission yeast and bacteria. This reveals a novel mechanism by which cells globally alter their properties to establish a unique homeostasis during starvation.
Assuntos
Núcleo Celular/química , Citoplasma/química , Glucose/metabolismo , Substâncias Macromoleculares/química , Saccharomycetales/fisiologia , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Difusão , Saccharomycetales/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiologiaRESUMO
The organization of the genome is nonrandom and important for correct function. Specifically, the nuclear envelope plays a critical role in gene regulation. It generally constitutes a repressive environment, but several genes, including the GAL locus in budding yeast, are recruited to the nuclear periphery on activation. Here, we combine imaging and computational modeling to ask how the association of a single gene locus with the nuclear envelope influences the surrounding chromosome architecture. Systematic analysis of an entire yeast chromosome establishes that peripheral recruitment of the GAL locus is part of a large-scale rearrangement that shifts many chromosomal regions closer to the nuclear envelope. This process is likely caused by the presence of several independent anchoring points. To identify novel factors required for peripheral anchoring, we performed a genome-wide screen and demonstrated that the histone acetyltransferase SAGA and the activity of histone deacetylases are needed for this extensive gene recruitment to the nuclear periphery.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromossomos Fúngicos/ultraestrutura , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Loci Gênicos , Membrana Nuclear/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , Simulação por Computador , Galactoquinase/genética , Galactoquinase/metabolismo , Galactose/metabolismo , Biblioteca Gênica , Glucose/metabolismo , Histona Desacetilases/metabolismo , Modelos Genéticos , Membrana Nuclear/metabolismo , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismoAssuntos
Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Lamina Tipo A/genética , Mutação , Doenças Neuromusculares/etiologia , Doenças Neuromusculares/genética , Lâmina Nuclear/metabolismo , Lâmina Nuclear/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Progéria/etiologia , Progéria/genéticaRESUMO
Centrosomes are closely associated with the nuclear envelope (NE) throughout the cell cycle and this association is maintained in prophase when they separate to establish the future mitotic spindle. At this stage, the kinetochore constituents CENP-F, NudE, NudEL, dynein, and dynactin accumulate at the NE. We demonstrate here that the N-terminal domain of the nuclear pore complex (NPC) protein Nup133, although largely dispensable for NPC assembly, is required for efficient anchoring of the dynein/dynactin complex to the NE in prophase. Nup133 exerts this function through an interaction network via CENP-F and NudE/EL. We show that this molecular chain is critical for maintaining centrosome association with the NE at mitotic entry and contributes to this process without interfering with the previously described RanBP2-BICD2-dependent pathway of centrosome anchoring. Finally, our study reveals that tethering of centrosomes to the nuclear surface at the G2/M transition contributes, along with other cellular mechanisms, to early stages of bipolar spindle assembly.
Assuntos
Centrossomo/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Poro Nuclear/metabolismo , Prófase , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Linhagem Celular Tumoral , Polaridade Celular , Centrossomo/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Complexo Dinactina , Dineínas/metabolismo , Células HeLa , Humanos , Espaço Intranuclear/metabolismo , Espaço Intranuclear/ultraestrutura , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Antígenos de Histocompatibilidade Menor , Membrana Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Fuso Acromático/metabolismoRESUMO
In metazoa, new nuclear pore complexes (NPCs) form at two different cell cycle stages: at the end of mitosis concomitant with the reformation of the nuclear envelope and during interphase. However, the mechanisms of these assembly processes may differ. In this study, we apply high resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase. We show that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrate that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107-160. This inverse order of recruitment and the overall much slower kinetics compared with postmitotic NPC assembly support the conclusion that the two processes occur by distinct molecular mechanisms.
Assuntos
Interfase , Poro Nuclear/ultraestrutura , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Cinética , Proteínas de Membrana/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , RatosRESUMO
N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas do Envelope Viral/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Retículo Endoplasmático/genética , Glicosilação , Células HeLa , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Microssomos/metabolismo , Sinais de Localização Nuclear/genética , Modificação Traducional de Proteínas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas do Envelope Viral/genéticaRESUMO
During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism.
Assuntos
Células Epiteliais/metabolismo , Mitose/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/genética , Anáfase/genética , Animais , Bioensaio/métodos , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/ultraestrutura , Fase G1/genética , Cinética , Substâncias Macromoleculares/metabolismo , Microscopia Confocal , Modelos Biológicos , Poro Nuclear/genética , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Prófase/genética , Ratos , Fatores de TempoRESUMO
Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(2)-S-Glc(2)), has been determined to 1.5 A resolution. The glycosyl unit in subsite -1 was sterically hindered by Tyr73 and forced into a distorted (2)S(o) conformation. In the enzyme where Tyr73 was mutated to a serine residue, the hindrance was removed and the glycosyl unit in subsite -1 had a relaxed (4)C(1) chair conformation. The relaxed conformation was seen in two complex structures of the mutated enzyme, with cellotetrose (Glc(4)) at 1.64 A and Glc(2)-S-Glc(2) at 1.04 A resolution.