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1.
Cell ; 187(21): 6088-6103.e18, 2024 Oct 17.
Article in English | MEDLINE | ID: mdl-39214079

ABSTRACT

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression.


Subject(s)
Cytosine , Epigenesis, Genetic , RNA Polymerase III , Zygote , Animals , Cytosine/metabolism , Cytosine/analogs & derivatives , Mice , Zygote/metabolism , RNA Polymerase III/metabolism , RNA Polymerase III/genetics , RNA, Transfer/metabolism , RNA, Transfer/genetics , Xenopus laevis/metabolism , Xenopus laevis/embryology , Xenopus laevis/genetics , Xenopus/metabolism , Xenopus/embryology , Xenopus/genetics , Female , Cellular Reprogramming , Gene Expression Regulation, Developmental , Oocytes/metabolism
2.
Cell ; 187(8): 1889-1906.e24, 2024 Apr 11.
Article in English | MEDLINE | ID: mdl-38503281

ABSTRACT

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.


Subject(s)
Cell Nucleolus , Nuclear Proteins , Proton-Motive Force , Cell Nucleolus/chemistry , Cell Nucleus/chemistry , Nuclear Proteins/chemistry , RNA/metabolism , Phase Separation , Intrinsically Disordered Proteins/chemistry , Animals , Xenopus laevis , Oocytes/chemistry , Oocytes/cytology
3.
Cell ; 178(6): 1421-1436.e24, 2019 09 05.
Article in English | MEDLINE | ID: mdl-31491386

ABSTRACT

The developmental disorder Floating-Harbor syndrome (FHS) is caused by heterozygous truncating mutations in SRCAP, a gene encoding a chromatin remodeler mediating incorporation of histone variant H2A.Z. Here, we demonstrate that FHS-associated mutations result in loss of SRCAP nuclear localization, alter neural crest gene programs in human in vitro models and Xenopus embryos, and cause craniofacial defects. These defects are mediated by one of two H2A.Z subtypes, H2A.Z.2, whose knockdown mimics and whose overexpression rescues the FHS phenotype. Selective rescue by H2A.Z.2 is conferred by one of the three amino acid differences between the H2A.Z subtypes, S38/T38. We further show that H2A.Z.1 and H2A.Z.2 genomic occupancy patterns are qualitatively similar, but quantitatively distinct, and H2A.Z.2 incorporation at AT-rich enhancers and expression of their associated genes are both sensitized to SRCAP truncations. Altogether, our results illuminate the mechanism underlying a human syndrome and uncover selective functions of H2A.Z subtypes during development.


Subject(s)
Abnormalities, Multiple/genetics , Chromatin Assembly and Disassembly , Chromatin/metabolism , Craniofacial Abnormalities/genetics , Growth Disorders/genetics , Heart Septal Defects, Ventricular/genetics , Histones/genetics , Adenosine Triphosphatases/genetics , Amino Acid Substitution , Animals , Embryonic Stem Cells , HEK293 Cells , Humans , Mutation , Xenopus laevis
4.
Cell ; 176(6): 1490-1501.e12, 2019 03 07.
Article in English | MEDLINE | ID: mdl-30765112

ABSTRACT

Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.


Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Animals , Catalytic Domain , Chromatin/metabolism , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/ultrastructure , Histones/chemistry , Histones/genetics , Humans , Methylation , Models, Molecular , Nucleosomes/metabolism , Protein Processing, Post-Translational , Receptor Cross-Talk , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitination , Xenopus laevis
5.
Cell ; 176(1-2): 167-181.e21, 2019 01 10.
Article in English | MEDLINE | ID: mdl-30595447

ABSTRACT

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.


Subject(s)
DNA Helicases/metabolism , DNA Helicases/physiology , DNA Repair/physiology , Animals , Cell Cycle Proteins/metabolism , DNA/metabolism , DNA Replication , DNA, Single-Stranded , DNA-Binding Proteins/physiology , Female , Male , Proteolysis , Single Molecule Imaging/methods , Xenopus laevis/metabolism
6.
Cell ; 176(4): 805-815.e8, 2019 02 07.
Article in English | MEDLINE | ID: mdl-30639102

ABSTRACT

Early embryogenesis is accompanied by reductive cell divisions requiring that subcellular structures adapt to a range of cell sizes. The interphase nucleus and mitotic spindle scale with cell size through both physical and biochemical mechanisms, but control systems that coordinately scale intracellular structures are unknown. We show that the nuclear transport receptor importin α is modified by palmitoylation, which targets it to the plasma membrane and modulates its binding to nuclear localization signal (NLS)-containing proteins that regulate nuclear and spindle size in Xenopus egg extracts. Reconstitution of importin α targeting to the outer boundary of extract droplets mimicking cell-like compartments recapitulated scaling relationships observed during embryogenesis, which were altered by inhibitors that shift levels of importin α palmitoylation. Modulation of importin α palmitoylation in human cells similarly affected nuclear and spindle size. These experiments identify importin α as a conserved surface area-to-volume sensor that scales intracellular structures to cell size.


Subject(s)
Cell Division/physiology , alpha Karyopherins/metabolism , alpha Karyopherins/physiology , Active Transport, Cell Nucleus , Animals , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cell Size , Cytoplasm/metabolism , Lipoylation , Membrane Proteins/metabolism , Nuclear Proteins/metabolism , Ovum/cytology , Spindle Apparatus/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/metabolism
7.
Cell ; 176(1-2): 56-72.e15, 2019 01 10.
Article in English | MEDLINE | ID: mdl-30612743

ABSTRACT

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.


Subject(s)
Endosomes/physiology , Protein Biosynthesis/physiology , rab GTP-Binding Proteins/metabolism , Animals , Axons/metabolism , Endosomes/metabolism , Mitochondria/genetics , Mitochondria/metabolism , RNA/metabolism , RNA, Messenger/metabolism , RNA, Messenger/physiology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/physiology , Ribosomes/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/physiology , rab7 GTP-Binding Proteins
8.
Cell ; 173(3): 720-734.e15, 2018 04 19.
Article in English | MEDLINE | ID: mdl-29677515

ABSTRACT

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.


Subject(s)
Arginine/chemistry , Molecular Chaperones/chemistry , RNA-Binding Protein FUS/chemistry , Amyotrophic Lateral Sclerosis/metabolism , Animals , Cations , DNA Methylation , Frontotemporal Dementia/metabolism , Frontotemporal Lobar Degeneration/metabolism , Humans , Microscopy, Atomic Force , Microscopy, Fluorescence , Protein Binding , Protein Domains , Protein Processing, Post-Translational , Protein Structure, Secondary , RNA-Binding Protein FUS/metabolism , Tyrosine/chemistry , Xenopus laevis
9.
Cell ; 174(2): 312-324.e16, 2018 07 12.
Article in English | MEDLINE | ID: mdl-29804838

ABSTRACT

The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.


Subject(s)
Hedgehog Proteins/metabolism , Smoothened Receptor/chemistry , Xenopus Proteins/chemistry , Allosteric Regulation , Animals , Binding Sites , Cell Line , Cholesterol/chemistry , Cholesterol/metabolism , Crystallography, X-Ray , Flow Cytometry , Hedgehog Proteins/genetics , Humans , Mice , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Structure, Tertiary , Signal Transduction , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Smoothened Receptor/antagonists & inhibitors , Smoothened Receptor/metabolism , Veratrum Alkaloids/chemistry , Veratrum Alkaloids/metabolism , Xenopus Proteins/antagonists & inhibitors , Xenopus Proteins/metabolism , Xenopus laevis/metabolism
10.
Cell ; 169(1): 85-95.e8, 2017 03 23.
Article in English | MEDLINE | ID: mdl-28340353

ABSTRACT

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , ATP-Binding Cassette Transporters/chemistry , Adenosine Triphosphate/metabolism , Animals , Cattle , Cryoelectron Microscopy , Humans , Hydrolysis , Models, Molecular , Protein Domains , Xenopus laevis , Zebrafish , Zebrafish Proteins/chemistry
11.
Cell ; 169(6): 1042-1050.e9, 2017 Jun 01.
Article in English | MEDLINE | ID: mdl-28575668

ABSTRACT

KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (IKs) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP2-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP2. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an electrophysiological assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP2, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS.


Subject(s)
Calmodulin/chemistry , KCNQ1 Potassium Channel/chemistry , Long QT Syndrome/metabolism , Amino Acid Sequence , Animals , Calmodulin/metabolism , Cryoelectron Microscopy , Humans , KCNQ1 Potassium Channel/genetics , KCNQ1 Potassium Channel/metabolism , Models, Molecular , Mutation , Sequence Alignment , Xenopus laevis
12.
Mol Cell ; 84(13): 2490-2510.e9, 2024 Jul 11.
Article in English | MEDLINE | ID: mdl-38996459

ABSTRACT

The formation of dynamic protein filaments contributes to various biological functions by clustering individual molecules together and enhancing their binding to ligands. We report such a propensity for the BTB domains of certain proteins from the ZBTB family, a large eukaryotic transcription factor family implicated in differentiation and cancer. Working with Xenopus laevis and human proteins, we solved the crystal structures of filaments formed by dimers of the BTB domains of ZBTB8A and ZBTB18 and demonstrated concentration-dependent higher-order assemblies of these dimers in solution. In cells, the BTB-domain filamentation supports clustering of full-length human ZBTB8A and ZBTB18 into dynamic nuclear foci and contributes to the ZBTB18-mediated repression of a reporter gene. The BTB domains of up to 21 human ZBTB family members and two related proteins, NACC1 and NACC2, are predicted to behave in a similar manner. Our results suggest that filamentation is a more common feature of transcription factors than is currently appreciated.


Subject(s)
BTB-POZ Domain , Transcription Factors , Xenopus Proteins , Animals , Humans , Cell Nucleus/metabolism , Cell Nucleus/genetics , Crystallography, X-Ray , HEK293 Cells , Models, Molecular , Protein Binding , Protein Multimerization , Repressor Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/chemistry , Transcription Factors/metabolism , Transcription Factors/genetics , Xenopus laevis , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus Proteins/chemistry
13.
Mol Cell ; 84(16): 3026-3043.e11, 2024 Aug 22.
Article in English | MEDLINE | ID: mdl-39178838

ABSTRACT

Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.


Subject(s)
BRCA2 Protein , DNA Damage , DNA Repair , DNA Replication , DNA, Single-Stranded , Rad51 Recombinase , Xenopus laevis , Humans , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Animals , DNA, Single-Stranded/metabolism , DNA, Single-Stranded/genetics , Cryoelectron Microscopy , DNA Polymerase theta , DNA Methylation , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , MRE11 Homologue Protein/metabolism , MRE11 Homologue Protein/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics
14.
Annu Rev Biochem ; 85: 659-83, 2016 Jun 02.
Article in English | MEDLINE | ID: mdl-27145846

ABSTRACT

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ∼200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes.


Subject(s)
Centrosome/metabolism , Kinetochores/metabolism , Metaphase , Microtubules/metabolism , Spindle Apparatus/metabolism , Animals , Centrosome/ultrastructure , Chromosome Segregation , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Gene Expression Regulation , Humans , Kinesins/genetics , Kinesins/metabolism , Kinetochores/ultrastructure , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/ultrastructure , Signal Transduction , Spindle Apparatus/ultrastructure , Tubulin/genetics , Tubulin/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/genetics , Xenopus laevis/metabolism , Zygote/cytology , Zygote/metabolism
15.
Cell ; 165(3): 704-14, 2016 Apr 21.
Article in English | MEDLINE | ID: mdl-27062927

ABSTRACT

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that mediate synaptic transmission and underpin learning and memory. NMDAR dysfunction is directly implicated in diseases ranging from seizure to ischemia. Despite its fundamental importance, little is known about how the NMDAR transitions between inactive and active states and how small molecules inhibit or activate ion channel gating. Here, we report electron cryo-microscopy structures of the GluN1-GluN2B NMDA receptor in an ensemble of competitive antagonist-bound states, an agonist-bound form, and a state bound with agonists and the allosteric inhibitor Ro25-6981. Together with double electron-electron resonance experiments, we show how competitive antagonists rupture the ligand binding domain (LBD) gating "ring," how agonists retain the ring in a dimer-of-dimers configuration, and how allosteric inhibitors, acting within the amino terminal domain, further stabilize the LBD layer. These studies illuminate how the LBD gating ring is fundamental to signal transduction and gating in NMDARs.


Subject(s)
Receptors, N-Methyl-D-Aspartate/chemistry , Xenopus Proteins/chemistry , Animals , Cryoelectron Microscopy , Electron Spin Resonance Spectroscopy , Models, Molecular , Protein Domains , Protein Subunits/chemistry , Receptors, N-Methyl-D-Aspartate/agonists , Xenopus laevis
16.
Cell ; 164(3): 420-32, 2016 Jan 28.
Article in English | MEDLINE | ID: mdl-26824655

ABSTRACT

Reproduction is heavily influenced by nutrition and metabolic state. Many common reproductive disorders in humans are associated with diabetes and metabolic syndrome. We characterized the metabolic mechanisms that support oogenesis and found that mitochondria in mature Drosophila oocytes enter a low-activity state of respiratory quiescence by remodeling the electron transport chain (ETC). This shift in mitochondrial function leads to extensive glycogen accumulation late in oogenesis and is required for the developmental competence of the oocyte. Decreased insulin signaling initiates ETC remodeling and mitochondrial respiratory quiescence through glycogen synthase kinase 3 (GSK3). Intriguingly, we observed similar ETC remodeling and glycogen uptake in maturing Xenopus oocytes, suggesting that these processes are evolutionarily conserved aspects of oocyte development. Our studies reveal an important link between metabolism and oocyte maturation.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Electron Transport Chain Complex Proteins/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen/metabolism , Oogenesis , Xenopus laevis/embryology , Animals , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/metabolism , Embryonic Development , Female , Forkhead Transcription Factors/metabolism , Mitochondria/metabolism , Oncogene Protein v-akt/metabolism , Oocytes/cytology , Oocytes/metabolism , Xenopus laevis/metabolism
17.
Cell ; 166(5): 1176-1187.e14, 2016 Aug 25.
Article in English | MEDLINE | ID: mdl-27545348

ABSTRACT

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.


Subject(s)
Cholesterol/metabolism , Hedgehog Proteins/metabolism , Smoothened Receptor/agonists , Animals , Cholesterol/chemistry , Crystallography, X-Ray , Mice , NIH 3T3 Cells , Oxysterols/chemistry , Oxysterols/metabolism , Protein Binding , Protein Conformation , Signal Transduction , Smoothened Receptor/chemistry , Smoothened Receptor/metabolism , Xenopus laevis
18.
Cell ; 165(7): 1686-1697, 2016 Jun 16.
Article in English | MEDLINE | ID: mdl-27212236

ABSTRACT

The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP.


Subject(s)
Cell Nucleolus/chemistry , Animals , Caenorhabditis elegans , Cells, Cultured , Chromosomal Proteins, Non-Histone/analysis , Intestines/chemistry , Intestines/cytology , Mammals , Nuclear Proteins/analysis , Nucleophosmin , Oocytes/chemistry , Oocytes/cytology , RNA Processing, Post-Transcriptional , Ribonucleoproteins/metabolism , Xenopus laevis
19.
Cell ; 167(2): 498-511.e14, 2016 Oct 06.
Article in English | MEDLINE | ID: mdl-27693351

ABSTRACT

During eukaryotic DNA interstrand cross-link (ICL) repair, cross-links are resolved ("unhooked") by nucleolytic incisions surrounding the lesion. In vertebrates, ICL repair is triggered when replication forks collide with the lesion, leading to FANCI-FANCD2-dependent unhooking and formation of a double-strand break (DSB) intermediate. Using Xenopus egg extracts, we describe here a replication-coupled ICL repair pathway that does not require incisions or FANCI-FANCD2. Instead, the ICL is unhooked when one of the two N-glycosyl bonds forming the cross-link is cleaved by the DNA glycosylase NEIL3. Cleavage by NEIL3 is the primary unhooking mechanism for psoralen and abasic site ICLs. When N-glycosyl bond cleavage is prevented, unhooking occurs via FANCI-FANCD2-dependent incisions. In summary, we identify an incision-independent unhooking mechanism that avoids DSB formation and represents the preferred pathway of ICL repair in a vertebrate cell-free system.


Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , DNA Replication , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group Proteins/metabolism , N-Glycosyl Hydrolases/metabolism , Animals , Cell-Free System/chemistry , Cross-Linking Reagents/chemistry , DNA/biosynthesis , DNA/chemistry , Fanconi Anemia Complementation Group D2 Protein/chemistry , Fanconi Anemia Complementation Group Proteins/chemistry , Ficusin/chemistry , N-Glycosyl Hydrolases/chemistry , Xenopus laevis
20.
Cell ; 166(3): 637-650, 2016 Jul 28.
Article in English | MEDLINE | ID: mdl-27471966

ABSTRACT

Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells.


Subject(s)
Amyloid/metabolism , Organelle Biogenesis , T-Box Domain Proteins/metabolism , Xenopus Proteins/metabolism , Animals , Benzothiazoles , Female , Fluorescent Dyes , Mitochondria/metabolism , Oocytes/cytology , Organelles/metabolism , Prions/chemistry , Protein Domains , Protein Transport , RNA, Messenger/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sf9 Cells , T-Box Domain Proteins/chemistry , T-Box Domain Proteins/genetics , Thiazoles , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus laevis , Zebrafish
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