RESUMO
The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.
Assuntos
Proteína Centromérica A , Centrômero , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/química , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Ligação Proteica , Ciclo Celular/genética , Modelos Moleculares , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Nucleossomos/metabolismo , Nucleossomos/química , Proteínas Adaptadoras de Transdução de SinalRESUMO
During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at ~1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.
RESUMO
SPOUT1/CENP-32 encodes a putative SPOUT RNA methyltransferase previously identified as a mitotic chromosome associated protein. SPOUT1/CENP-32 depletion leads to centrosome detachment from the spindle poles and chromosome misalignment. Aided by gene matching platforms, we identified 24 individuals with neurodevelopmental delays from 18 families with bi-allelic variants in SPOUT1/CENP-32 detected by exome/genome sequencing. Zebrafish spout1/cenp-32 mutants showed reduction in larval head size with concomitant apoptosis likely associated with altered cell cycle progression. In vivo complementation assays in zebrafish indicated that SPOUT1/CENP-32 missense variants identified in humans are pathogenic. Crystal structure analysis of SPOUT1/CENP-32 revealed that most disease-associated missense variants mapped to the catalytic domain. Additionally, SPOUT1/CENP-32 recurrent missense variants had reduced methyltransferase activity in vitro and compromised centrosome tethering to the spindle poles in human cells. Thus, SPOUT1/CENP-32 pathogenic variants cause an autosomal recessive neurodevelopmental disorder: SpADMiSS ( SPOUT1 Associated Development delay Microcephaly Seizures Short stature) underpinned by mitotic spindle organization defects and consequent chromosome segregation errors.
RESUMO
Background: Unicorn™ software on Äkta liquid chromatography instruments outputs chromatography profiles of purified biological macromolecules. While the plots generated by the instrument software are very helpful to inspect basic chromatogram properties, they lack a range of useful annotation, customization and export options. Methods: We use the R Shiny framework to build an interactive app that facilitates the interpretation of chromatograms and the generation of figures for publications. Results: The app allows users to fit a baseline, to highlight selected fractions and elution volumes inside or under the plot (e.g. those used for downstream biochemical/biophysical/structural analysis) and to zoom into the plot. The app is freely available at https://ChromatoShiny.bio.ed.ac.uk. Conclusions: It requires no programming experience, so we anticipate that it will enable chromatography users to create informative, annotated chromatogram plots quickly and simply.
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Increased nuclear size correlates with lower survival rates and higher grades for prostate cancer. The short-chain dehydrogenase/reductase (SDR) family member DHRS7 was suggested as a biomarker for use in prostate cancer grading because it is largely lost in higher-grade tumors. Here, we found that reduction in DHRS7 from the LNCaP prostate cancer cell line with normally high levels of DHRS7 increases nuclear size, potentially explaining the nuclear size increase observed in higher-grade prostate tumors where it is lost. An exogenous expression of DHRS7 in the PC3 prostate cancer cell line with normally low DHRS7 levels correspondingly decreases nuclear size. We separately tested 80 compounds from the Microsource Spectrum library for their ability to restore normal smaller nuclear size to PC3 cells, finding that estradiol propionate had the same effect as the re-expression of DHRS7 in PC3 cells. However, the drug had no effect on LNCaP cells or PC3 cells re-expressing DHRS7. We speculate that separately reported beneficial effects of estrogens in androgen-independent prostate cancer may only occur with the loss of DHRS7/ increased nuclear size, and thus propose DHRS7 levels and nuclear size as potential biomarkers for the likely effectiveness of estrogen-based treatments.
Assuntos
Estradiol , Neoplasias da Próstata , Masculino , Humanos , Estradiol/farmacologia , Propionatos , Neoplasias da Próstata/tratamento farmacológico , Próstata , Estrogênios , OxirredutasesRESUMO
Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded â¼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only â¼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.
Assuntos
Proteínas do Citoesqueleto , Cinetocoros , Microtúbulos , Mitose , Aurora Quinases/metabolismo , Proteína Quinase CDC2/metabolismo , Segregação de Cromossomos , Ciclina B1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , FosforilaçãoRESUMO
Centromere association of the chromosomal passenger complex (CPC; Borealin-Survivin-INCENP-Aurora B) and Sgo1 is crucial for chromosome biorientation, a process essential for error-free chromosome segregation. Phosphorylated histone H3 Thr3 (H3T3ph; directly recognized by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognized via Sgo1), together with CPC's intrinsic nucleosome-binding ability, facilitate CPC centromere recruitment. However, the molecular basis for CPC-Sgo1 binding and how their physical interaction influences CPC centromere localization are lacking. Here, using an integrative structure-function approach, we show that the "histone H3-like" Sgo1 N-terminal tail-Survivin BIR domain interaction acts as a hotspot essential for CPC-Sgo1 assembly, while downstream Sgo1 residues and Borealin contribute for high-affinity binding. Disrupting Sgo1-Survivin interaction abolished CPC-Sgo1 assembly and perturbed CPC centromere localization and function. Our findings reveal that Sgo1 and H3T3ph use the same surface on Survivin to bind CPC. Hence, it is likely that these interactions take place in a spatiotemporally restricted manner, providing a rationale for the Sgo1-mediated "kinetochore-proximal" CPC centromere pool.
Assuntos
Proteínas de Ciclo Celular , Centrômero , Histonas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Histonas/genética , Histonas/metabolismo , Cinetocoros/metabolismo , Fosforilação , Survivina/genética , Survivina/metabolismoRESUMO
Aneuploidy, the gain or loss of chromosomes in a cell, is a hallmark of cancer. Although our understanding of the contribution of aneuploidy to cancer initiation and progression is incomplete, significant progress has been made in uncovering the cellular consequences of aneuploidy and how aneuploid cancer cells self-adapt to promote tumorigenesis. Aneuploidy is physiologically associated with significant cellular stress but, paradoxically, it favors tumor progression. Although more common in solid tumors, different forms of aneuploidy represent the initiating oncogenic lesion in patients with B cell acute lymphoblastic leukemia (B-ALL), making B-ALL an excellent model for studying the role of aneuploidy in tumorigenesis. We review the molecular mechanisms underlying aneuploidy and discuss its contributions to B-ALL initiation and progression.
Assuntos
Aneuploidia , Carcinogênese/genética , Segregação de Cromossomos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adenosina Trifosfatases/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Humanos , Complexos Multiproteicos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.
Assuntos
Cromossomos Artificiais Humanos/metabolismo , Rearranjo Gênico/fisiologia , Linhagem Celular Tumoral , Centrômero/metabolismo , Proteína B de Centrômero/genética , Instabilidade Cromossômica , Epigênese Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , HumanosRESUMO
The endosomal sorting complex required for transport (ESCRT)-III proteins are known to assemble into filaments that mediate membrane remodeling and fission in various biological processes, including the formation of endosomal multivesicular bodies, viral budding, cytokinesis, plasma membrane repair, nuclear pore quality control, nuclear envelope reformation, and neuron pruning. The study of the regulation and function of ESCRT-III proteins is therefore crucial to understand these events and requires a combination of in vivo and in vitro experimental techniques. Here we describe two protocols for the purification of human and Drosophila ESCRT-III proteins from bacteria and their use in in vitro phosphorylation assays and atomic force microscopy experiments on membrane lipid bilayers. These protocols can also be applied for the purification of other proteins that are insoluble when expressed in bacteria.
Assuntos
Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica/métodos , Proteínas Recombinantes/metabolismo , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Clonagem Molecular/métodos , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/isolamento & purificação , Vetores Genéticos/genética , Fosforilação , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Transformação BacterianaRESUMO
The centromere, a chromosomal locus that acts as a microtubule attachment site, is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during the cell cycle requires HJURP-mediated CENP-A deposition, a process regulated by the Mis18 complex (Mis18α/Mis18ß/Mis18BP1). Spatial and temporal regulation of Mis18 complex assembly is crucial for its centromere association and function. Here, we provide the molecular basis for the assembly and regulation of the Mis18 complex. We show that the N-terminal region of Mis18BP1 spanning amino acid residues 20-130 directly interacts with Mis18α/ß to form the Mis18 complex. Within Mis18α/ß, the Mis18α MeDiY domain can directly interact with Mis18BP1. Mis18α/ß forms a hetero-hexamer with 4 Mis18α and 2 Mis18ß. However, only two copies of Mis18BP1 interact with Mis18α/ß to form a hetero-octameric assembly, highlighting the role of Mis18 oligomerization in limiting the number of Mis18BP1 within the Mis18 complex. Furthermore, we demonstrate the involvement of consensus Cdk1 phosphorylation sites on Mis18 complex assembly and thus provide a rationale for cell cycle-regulated timing of Mis18 assembly and CENP-A deposition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/farmacocinética , Proteína Centromérica A/metabolismo , Regulação da Expressão Gênica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína Quinase CDC2/genética , Ciclo Celular/genética , Centrômero/genética , Centrômero/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Nucleossomos , Fosforilação , Ligação ProteicaRESUMO
Centromeres consist of specialized centrochromatin containing CENP-A nucleosomes intermingled with H3 nucleosomes carrying transcription-associated modifications. We have designed a novel synthetic biology 'in situ epistasis' analysis in which H3 dimethylated on lysine 4 (H3K4me2) demethylase LSD2 plus synthetic modules with competing activities are simultaneously targeted to a synthetic alphoidtetO HAC centromere. This allows us to uncouple transcription from histone modifications at the centromere. Here, we report that H3K4me2 loss decreases centromeric transcription, CENP-A assembly and stability and causes spreading of H3K9me3 across the HAC, ultimately inactivating the centromere. Surprisingly, CENP-28/Eaf6-induced transcription of the alphoidtetO array associated with H4K12 acetylation does not rescue the phenotype, whereas p65-induced transcription associated with H3K9 acetylation does rescue. Thus mitotic transcription plus histone modifications including H3K9ac constitute the 'epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres.
Assuntos
Epigênese Genética , Código das Histonas , Cinetocoros/metabolismo , Transcrição Gênica , Acetilação , Linhagem Celular Tumoral , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Células HeLa , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
The chromosomal passenger complex (CPC)-composed of Aurora B kinase, Borealin, Survivin and INCENP-surveys the fidelity of genome segregation throughout cell division. The CPC has been proposed to prevent polyploidy by controlling the final separation (known as abscission) of the two daughter cells via regulation of the ESCRT-III CHMP4C component. The molecular details are, however, still unclear. Using atomic force microscopy, we show that CHMP4C binds to and remodels membranes in vitro Borealin prevents the association of CHMP4C with membranes, whereas Aurora B interferes with CHMP4C's membrane remodelling activity. Moreover, we show that CHMP4C phosphorylation is not required for its assembly into spiral filaments at the abscission site and that two distinctly localized pools of phosphorylated CHMP4C exist during cytokinesis. We also characterized the CHMP4C interactome in telophase cells and show that the centralspindlin complex associates preferentially with unphosphorylated CHMP4C in cytokinesis. Our findings indicate that gradual dephosphorylation of CHMP4C triggers a 'relay' mechanism between the CPC and centralspindlin that regulates the timely distribution and activation of CHMP4C for the execution of abscission.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Aurora Quinase B/metabolismo , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Células HeLa , Humanos , Microscopia de Força Atômica , Fosforilação , Ligação Proteica , Mapas de Interação de Proteínas , TelófaseRESUMO
The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis.
RESUMO
In mature neurons, the number of synapses is determined by a neuronal activity-dependent dynamic equilibrium between positive and negative regulatory factors. We hypothesized that neuronal pentraxin (NP1), a proapoptotic protein induced by low neuronal activity, could be a negative regulator of synapse density because it is found in dystrophic neurites in Alzheimer's disease-affected brains. Here, we report that knockdown of NP1 increases the number of excitatory synapses and neuronal excitability in cultured rat cortical neurons and enhances excitatory drive and long-term potentiation in the hippocampus of behaving mice. Moreover, we found that NP1 regulates the surface expression of the Kv7.2 subunit of the Kv7 family of potassium channels that control neuronal excitability. Furthermore, pharmacological activation of Kv7 channels prevents, whereas inhibition mimics, the increase in synaptic proteins evoked by the knockdown of NP1. These results indicate that NP1 negatively regulates excitatory synapse number by modulating neuronal excitability and show that NP1 restricts excitatory synaptic plasticity.
Assuntos
Proteína C-Reativa/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Proteína C-Reativa/genética , Células Cultivadas , Córtex Cerebral/citologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Antagonistas de Receptores de GABA-A/farmacologia , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/genética , Vigília/efeitos dos fármacosRESUMO
The ability of kinetochores (KTs) to maintain stable attachments to dynamic microtubule structures ('straight' during microtubule polymerization and 'curved' during microtubule depolymerization) is an essential requirement for accurate chromosome segregation. Here we show that the kinetochore-associated Ska complex interacts with tubulin monomers via the carboxy-terminal winged-helix domain of Ska1, providing the structural basis for the ability to bind both straight and curved microtubule structures. This contrasts with the Ndc80 complex, which binds straight microtubules by recognizing the dimeric interface of tubulin. The Ska1 microtubule-binding domain interacts with tubulins using multiple contact sites that allow the Ska complex to bind microtubules in multiple modes. Disrupting either the flexibility or the tubulin contact sites of the Ska1 microtubule-binding domain perturbs normal mitotic progression, explaining the critical role of the Ska complex in maintaining a firm grip on dynamic microtubules.