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1.
Cell ; 186(23): 5041-5053.e19, 2023 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-37865089

RESUMO

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.


Assuntos
Proteoma , Espermatozoides , Animais , Masculino , Camundongos , Axonema/química , Microscopia Crioeletrônica/métodos , Flagelos/metabolismo , Microtúbulos/metabolismo , Sêmen , Espermatozoides/química , Proteoma/análise
2.
Nat Struct Mol Biol ; 30(3): 360-369, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36593309

RESUMO

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.


Assuntos
Axonema , Dineínas , Animais , Masculino , Humanos , Axonema/metabolismo , Dineínas/metabolismo , Tomografia com Microscopia Eletrônica , Sêmen/metabolismo , Espermatozoides , Microtúbulos/metabolismo , Flagelos/metabolismo , Mamíferos/metabolismo
3.
Nature ; 610(7932): 575-581, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36224386

RESUMO

RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies1,2. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development3. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain4. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.


Assuntos
DNA , Desoxirribonuclease I , RNA Guia de Cinetoplastídeos , Sistemas CRISPR-Cas , Desoxirribonuclease I/química , Desoxirribonuclease I/metabolismo , Desoxirribonuclease I/ultraestrutura , DNA/química , DNA/metabolismo , DNA/ultraestrutura , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , RNA Guia de Cinetoplastídeos/ultraestrutura , Microscopia Crioeletrônica , Proteínas Associadas a CRISPR/química
4.
PLoS One ; 17(8): e0271799, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35960737

RESUMO

Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.


Assuntos
Tomografia com Microscopia Eletrônica , Sinaptossomos , Animais , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Lasers , Camundongos , Microscopia Confocal , Sinapses
5.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34903670

RESUMO

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , RNA de Plantas/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA de Plantas , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Modelos Moleculares , Conformação Proteica , RNA de Plantas/genética , RNA Polimerase Dependente de RNA/genética , Transcrição Gênica
6.
Nat Neurosci ; 23(6): 741-753, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393895

RESUMO

During sleep and awake rest, the neocortex generates large-scale slow-wave (SW) activity. Here, we report that the claustrum coordinates neocortical SW generation. We established a transgenic mouse line that enabled the genetic interrogation of a subpopulation of claustral glutamatergic neurons. These neurons received inputs from and sent outputs to widespread neocortical areas. The claustral neuronal firings mostly correlated with cortical SW activity. In vitro optogenetic stimulation of the claustrum induced excitatory postsynaptic responses in most neocortical neurons, but elicited action potentials primarily in inhibitory interneurons. In vivo optogenetic stimulation induced a synchronized down-state featuring prolonged silencing of neural activity in all layers of many cortical areas, followed by a down-to-up state transition. In contrast, genetic ablation of claustral neurons attenuated SW activity in the frontal cortex. These results demonstrate a crucial role of claustral neurons in synchronizing inhibitory interneurons across wide cortical areas for the spatiotemporal coordination of SW activity.


Assuntos
Claustrum/fisiologia , Neocórtex/fisiologia , Sono de Ondas Lentas/fisiologia , Potenciais de Ação/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Interneurônios/fisiologia , Camundongos , Camundongos Transgênicos , Inibição Neural/fisiologia , Neurônios/fisiologia , Optogenética , Proteínas com Domínio T/genética
7.
Pediatr Diabetes ; 13(1): 26-32, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22060211

RESUMO

BACKGROUND: In Asians, mutations in the known maturity-onset diabetes of the young (MODY) genes have been identified in only <15% of patients. These results were obtained mostly through studies on adult patients. OBJECTIVE: To investigate the molecular basis of Japanese patients with pediatric-onset MODY-type diabetes. SUBJECTS: Eighty Japanese patients with pediatric-onset MODY-type diabetes. METHODS: Mitochondrial 3243A>G mutation was first tested by the polymerase chain reaction restriction fragment length polymorphism analysis for maternally inherited families. Then, all coding exons and exon-intron boundaries of the HNF1A, HNF1B, GCK, and HNF4A genes were amplified from genomic DNA and directly sequenced. Multiplex ligation-dependent probe amplification analysis was also performed to detect whole-exon deletions. RESULTS: After excluding one patient with a mitochondrial 3243A>G, mutations were identified in 38 (48.1%) patients; 18 had GCK mutations, 11 had HNF1A mutations, 3 had HNF4A mutations, and 6 had HNF1B mutations. In patients aged <8 yr, mutations were detected mostly in GCK at a higher frequency (63.6%). In patients >9 yr of age, mutations were identified less frequently (45.1%), with HNF1A mutations being the most frequent. A large fraction of mutation-negative patients showed elevated homeostasis model assessment (HOMA) insulin-resistance and normal HOMA-ß indices. Most of the HNF1B mutations were large deletions, and, interestingly, renal cysts were undetectable in two patients with whole-gene deletion of HNF1B. CONCLUSION: In Japanese patients with pediatric-onset MODY-type diabetes, mutations in known genes were identified at a much higher frequency than previously reported for adult Asians. A fraction of mutation-negative patients presented with insulin-resistance and normal insulin-secretory capacities resembling early-onset type 2 diabetes.


Assuntos
Povo Asiático/genética , Análise Mutacional de DNA , Diabetes Mellitus Tipo 2/genética , Adolescente , Idade de Início , Povo Asiático/estatística & dados numéricos , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/etnologia , Feminino , Frequência do Gene , Glucoquinase/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Humanos , Lactente , Masculino
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