RESUMO
Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.
Assuntos
Cromatina/ultraestrutura , Cromossomos/ultraestrutura , Metáfase , Microscopia Eletrônica de Varredura/métodos , Prófase , Linhagem Celular Transformada , Nucléolo Celular/química , Nucléolo Celular/ultraestrutura , Cromatina/química , Cromossomos/química , Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Epitélio Pigmentado da Retina/química , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestruturaRESUMO
Whether gene repositioning to the nuclear periphery during differentiation adds another layer of regulation to gene expression remains controversial. Here, we resolve this by manipulating gene positions through targeting the nuclear envelope transmembrane proteins (NETs) that direct their normal repositioning during myogenesis. Combining transcriptomics with high-resolution DamID mapping of nuclear envelope-genome contacts, we show that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression. Retargeting a NET39 fragment to nucleoli correspondingly repositioned a target gene, indicating a direct tethering mechanism. Being able to manipulate gene position independently of other changes in differentiation revealed that repositioning contributes â to â of a gene's normal repression in myogenesis. Together, these NETs affect 37% of all genes changing expression during myogenesis, and their combined knockdown almost completely blocks myotube formation. This unequivocally demonstrates that NET-directed gene repositioning is critical for developmental gene regulation.
Assuntos
Posicionamento Cromossômico , Regulação da Expressão Gênica no Desenvolvimento , Canais Iônicos/genética , Proteínas de Membrana/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Animais , Diferenciação Celular , Linhagem Celular , Regulação para Baixo , Humanos , Canais Iônicos/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Interferência de RNA , TransfecçãoRESUMO
Disruption of axon-glia interactions in the peripheral nervous system has emerged as a major cause of arthrogryposis multiplex congenita (AMC), a condition characterized by multiple congenital postural abnormalities involving the major joints. Several genes crucially important to the biology of Schwann cells have now been implicated with AMC. One such gene is LGI4 which encodes a secreted glycoprotein. LGI4 is expressed and secreted by Schwann cells and binds its receptor ADAM22 on the axonal membrane to drive myelination. Homozygous mutations in LGI4 or ADAM22 results in severe congenital hypomyelination and joint contractures in mice. Recently bi-allelic LGI4 loss of function mutations has been described in three unrelated families with severe AMC. Two individuals in a fourth, non-consanguineous family were found to be compound heterozygous for two LGI4 missense mutations. It is not known how these missense mutations affect the biology of LGI4. Here we investigated whether these missense mutations affected the secretion of the protein, its ADAM22 binding capacity, or its myelination-promoting function. We demonstrate that the mutations largely affect the progression of the mutant protein through the endomembrane system resulting in severely reduced expression. Importantly, binding to ADAM22 and myelination-promoting activity appear largely unaffected, suggesting that treatment with chemical chaperones to improve secretion of the mutant proteins might prove beneficial.
Assuntos
Artrogripose , Animais , Artrogripose/genética , Artrogripose/metabolismo , Axônios/metabolismo , Humanos , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células de Schwann/metabolismoRESUMO
The requirement for condensin in chromosome formation in somatic cells remains unclear, as imperfectly condensed chromosomes do form in cells depleted of condensin by conventional methodologies. In order to dissect the roles of condensin at different stages of vertebrate mitosis, we have established a versatile cellular system that combines auxin-mediated rapid degradation with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells in the presence and absence of condensin. We analyzed the outcome by live- and fixed-cell microscopy methods, including serial block face scanning electron microscopy with digital reconstruction. Following rapid depletion of condensin, chromosomal defects were much more obvious than those seen after a slow depletion of condensin. The total mitotic chromatin volume was similar to that in control cells, but a single mass of mitotic chromosomes was clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments where the auxin concentration was titrated showed that different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.This article has an associated First Person interview with the first author of the paper.
Assuntos
Adenosina Trifosfatases/genética , Cromatina/ultraestrutura , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/genética , Mitose/genética , Complexos Multiproteicos/genética , Adenosina Trifosfatases/metabolismo , Animais , Galinhas , Cromatina/genética , Cromatina/metabolismo , Aberrações Cromossômicas , Segregação de Cromossomos/genética , Cromossomos/genética , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ácidos Indolacéticos/farmacologia , Microscopia Eletrônica de Varredura , Complexos Multiproteicos/metabolismo , Proteólise/efeitos dos fármacosRESUMO
During cell division, interactions between microtubules and chromosomes are mediated by the kinetochore, a proteinaceous structure located at the primary constriction of chromosomes. In addition to the centromere histone centromere protein A (CENP-A), 15 other members of the constitutive centromere associated network (CCAN) participate in the formation of a chromatin-associated scaffold that supports kinetochore structure. We performed a targeted screen analyzing unfolded centrochromatin from CENP-depleted chromosomes. Our results revealed that CENP-C and CENP-S are critical for the stable folding of mitotic kinetochore chromatin. Multipeak fitting algorithms revealed the presence of an organized pattern of centrochromatin packing consistent with arrangement of CENP-A-containing nucleosomes into up to five chromatin "subunits"-each containing roughly 20-30 nucleosomes. These subunits could be either layers of a boustrophedon or small loops of centromeric chromatin.
Assuntos
Cinetocoros/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Desdobramento de Proteína , Animais , Linhagem Celular , Centrômero/química , Proteína Centromérica A/química , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Galinhas , Cromatina/química , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Cinetocoros/metabolismo , Microscopia de Fluorescência , Mitose , Mutação , Proteínas Recombinantes de Fusão , VertebradosRESUMO
Kinetochore fibres (K-fibres) of the spindle apparatus move chromosomes during mitosis. These fibres are discrete bundles of parallel microtubules (MTs) that are crosslinked by inter-MT 'bridges' that are thought to improve fibre stability during chromosomal movement. The identity of these bridges is unknown. Clathrin is a multimeric protein that has been shown to stabilise K-fibres during early mitosis by a mechanism independent of its role in membrane trafficking. In this study, we show that clathrin at the mitotic spindle is in a transforming acidic colied-coil protein 3 (TACC3)/colonic, hepatic tumour overexpressed gene (ch-TOG)/clathrin complex. The complex is anchored to the spindle by TACC3 and ch-TOG. Ultrastructural analysis of clathrin-depleted K-fibres revealed a selective loss of a population of short inter-MT bridges and a general loss of MTs. A similar loss of short inter-MT bridges was observed in TACC3-depleted K-fibres. Finally, immunogold labelling confirmed that inter-MT bridges in K-fibres contain clathrin. Our results suggest that the TACC3/ch-TOG/clathrin complex is an inter-MT bridge that stabilises K-fibres by physical crosslinking and by reducing rates of MT catastrophe.
Assuntos
Clatrina/metabolismo , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Aurora Quinases , Clatrina/genética , Células HeLa , Humanos , Immunoblotting , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Mitose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fuso Acromático/genéticaRESUMO
Voltage-gated sodium channels cluster in macromolecular complexes at nodes of Ranvier to promote rapid nerve impulse conduction in vertebrate nerves. Node assembly in peripheral nerves is thought to be initiated at heminodes at the extremities of myelinating Schwann cells, and fusion of heminodes results in the establishment of nodes. Here we show that assembly of 'early clusters' of nodal proteins in the murine axonal membrane precedes heminode formation. The neurofascin (Nfasc) proteins are essential for node assembly, and the formation of early clusters also requires neuronal Nfasc. Early clusters are mobile and their proteins are dynamically recruited by lateral diffusion. They can undergo fusion not only with each other but also with heminodes, thus contributing to the development of nodes in peripheral axons. The formation of early clusters constitutes the earliest stage in peripheral node assembly and expands the repertoire of strategies that have evolved to establish these essential structures.
Assuntos
Interneurônios/metabolismo , Proteína Nodal/metabolismo , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/metabolismo , Feminino , Gânglios Espinais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/metabolismo , Condução Nervosa , Sistema Nervoso Periférico , Células de Schwann/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
The rapid evolution of super-resolution light microscopy has narrowed the gap between light and electron microscopy, allowing the imaging of molecules and cellular structures at high resolution within their normal cellular and tissue context. Multimodal imaging approaches such as correlative light electron microscopy (CLEM) combine these techniques to create a tool with unique imaging capacity. However, these approaches are typically reserved for specialists, and their application to the analysis of neural tissue is challenging. Here we present SuperCLEM, a relatively simple approach that combines super-resolution fluorescence light microscopy (FLM), 3D electron microscopy (3D-EM) and rendering into 3D models. We demonstrate our workflow using neuron-glia cultures from which we first acquire high-resolution fluorescent light images of myelinated axons. After resin embedding and re-identification of the region of interest, serially aligned EM sections are acquired and imaged using a serial block face scanning electron microscope (SBF-SEM). The FLM and 3D-EM datasets are then combined to render 3D models of the myelinated axons. Thus, the SuperCLEM imaging pipeline is a useful new tool for researchers pursuing similar questions in neuronal and other complex tissue culture systems.
RESUMO
The centromere is located at the primary constriction of condensed chromosomes where it acts as a platform regulating chromosome segregation. The histone H3 variant CENP-A is the foundation for kinetochore formation. CENP-A directs the formation of a highly dynamic molecular neighborhood whose temporal characterization during mitosis remains a challenge due to limitations in available techniques. BioID is a method that exploits a "promiscuous" biotin ligase (BirA118R or BirA*) to identify proteins within close proximity to a fusion protein of interest. As originally described, cells expressing BirA* fusions were exposed to high biotin concentrations for 24 h during which the ligase transferred activated biotin (BioAmp) to other proteins within the immediate vicinity. The protein neighborhood could then be characterized by streptavidin-based purification and mass spectrometry. Here we describe a further development to this technique, allowing CENP-A interactors to be characterized within only a few minutes, in an in vitro reaction in lysed cells whose physiological progression is "frozen." This approach, termed in vitro BioID (ivBioID), has the potential to study the molecular neighborhood of any structural protein whose interactions change either during the cell cycle or in response to other changes in cell physiology.
Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Mapeamento de Interação de Proteínas/métodos , Biotinilação , Células HeLa , Humanos , Espectrometria de Massas , MitoseRESUMO
The chromosome periphery is a complex network of proteins and RNA molecules (many derived from nucleoli) that covers the outer surface of chromosomes and whose function remains mysterious. Although it was first described over 130 years ago, technological advances and the recent discovery that Ki-67 acts as an organiser of this region have allowed the chromosome periphery to be dissected in previously unattainable detail, leading to a revival of interest in this obscure chromosomal compartment. Here, we review the most recent advances into the composition, structure and function of the chromosome periphery, discuss possible roles of Ki-67 during mitosis and consider why this structure is likely to remain the focus of ongoing attention in the future.
Assuntos
Cromossomos/metabolismo , Antígeno Ki-67/metabolismo , Mitose , Ribonucleoproteínas/metabolismo , Animais , Nucléolo Celular/metabolismo , Humanos , Antígeno Ki-67/genética , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1-3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Técnicas de Inativação de Genes , Ácidos Indolacéticos/metabolismo , Cinetocoros/metabolismo , Mitose , Animais , Morte Celular , Linhagem Celular , Sobrevivência Celular , Galinhas , Metáfase , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteólise , ProteômicaRESUMO
Kinetochore fibers (K-fibers) of the mitotic spindle are force-generating units that power chromosome movement during mitosis. K-fibers are composed of many microtubules that are held together throughout their length. Here, we show, using 3D electron microscopy, that K-fiber microtubules (MTs) are connected by a network of MT connectors. We term this network 'the mesh'. The K-fiber mesh is made of linked multipolar connectors. Each connector has up to four struts, so that a single connector can link up to four MTs. Molecular manipulation of the mesh by overexpression of TACC3 causes disorganization of the K-fiber MTs. Optimal stabilization of K-fibers by the mesh is required for normal progression through mitosis. We propose that the mesh stabilizes K-fibers by pulling MTs together and thereby maintaining the integrity of the fiber. Our work thus identifies the K-fiber meshwork of linked multipolar connectors as a key integrator and determinant of K-fiber structure and function.
Assuntos
Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Fuso Acromático/metabolismo , Tomografia com Microscopia Eletrônica , Expressão Gênica , Células HeLa , Humanos , Imageamento Tridimensional , Proteínas Associadas aos Microtúbulos/metabolismo , MitoseRESUMO
When the nucleolus disassembles during open mitosis, many nucleolar proteins and RNAs associate with chromosomes, establishing a perichromosomal compartment coating the chromosome periphery. At present nothing is known about the function of this poorly characterised compartment. In this study, we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells. Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin. Following siRNA depletion of Ki-67, NIFK, B23, nucleolin, and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes. Correlative light and electron microscopy (CLEM) images suggest a near-complete loss of the entire perichromosomal compartment. Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells.DOI: http://dx.doi.org/10.7554/eLife.01641.001.
Assuntos
Nucléolo Celular/metabolismo , Cromossomos Humanos/metabolismo , Antígeno Ki-67/metabolismo , Mitose , Proteína Fosfatase 1/metabolismo , Nucléolo Celular/ultraestrutura , Cromossomos Humanos/ultraestrutura , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígeno Ki-67/genética , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de Proteína , Transdução de Sinais , Transfecção , NucleolinaRESUMO
Electron microscopy (EM) has dominated high-resolution cellular imaging for over 50 years, thanks to its ability to resolve on nanometer-scale intracellular structures such as the microtubules of the mitotic spindle. It is advantageous to view the cell of interest prior to processing the sample for EM. Correlative light-electron microscopy (CLEM) is a technique that allows one to visualize cells of interest by light microscopy (LM) before being transferred to EM for ultrastructural examination. Here, we describe how CLEM can be applied as an effective tool to study the spindle apparatus of mitotic cells. This approach allows transfected cells of interest, in desirable stages of mitosis, to be followed from LM to EM. CLEM has often been considered as a technically challenging and laborious technique. In this chapter, we provide step-by-step pictorial guides that allow successful CLEM to be achieved. In addition, we explain how it is possible to vary the sectioning plane, allowing spindles and microtubules to be analyzed from different angles, and the outputs that can be obtained from these methods when applied to the study of kinetochore fiber ultrastructure.
Assuntos
Cinetocoros/ultraestrutura , Microscopia Eletrônica/métodos , Microtúbulos/ultraestrutura , Fuso Acromático/ultraestrutura , Linhagem Celular Tumoral , Células HeLa , Humanos , MitoseRESUMO
Accurate chromosome segregation during mitosis is achieved by the kinetochore fibers (K-fibers) of the spindle apparatus. These fibers are bundles of microtubules (MTs) connected by non-motor bridges. We recently identified a TACC3/ch-TOG/clathrin complex that constitutes the shortest class of inter-MT bridge in K-fibers. TACC3 anchors the complex to MTs and this is dependent on phosphorylation by Aurora A kinase. Here we show that inhibition of Aurora A kinase using MLN8237 results in (1) loss of clathrin and TACC3 from spindles, (2) destabilization of K-fibers and (3) loss of inter-MT bridges. These results are similar to those in cells depleted of clathrin or TACC3; suggesting that TACC3/ch-TOG/clathrin bridges are the major class of bridge that is regulated by this kinase.