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Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.
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Reparación del ADN/genética , Conversión Génica , Recombinasa Rad51/metabolismo , Alelos , Animales , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromosomas de los Mamíferos/genética , Roturas del ADN de Doble Cadena , Embrión de Mamíferos , Femenino , Sitios Genéticos , Recombinación Homóloga/genética , Homocigoto , Humanos , Mutación INDEL/genética , Ratones Endogámicos C57BL , Mosaicismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple/genética , Ribonucleoproteínas/metabolismo , Cigoto/metabolismoRESUMEN
Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.
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Secuencia Conservada , Evolución Molecular , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Mamíferos , Neocórtex , Animales , Humanos , Ratones , Callithrix/genética , Cromatina/genética , Cromatina/metabolismo , Secuencia Conservada/genética , Metilación de ADN , Elementos Transponibles de ADN/genética , Epigenoma , Regulación de la Expresión Génica/genética , Macaca/genética , Mamíferos/genética , Corteza Motora/citología , Corteza Motora/metabolismo , Multiómica , Neocórtex/citología , Neocórtex/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Variación Genética/genéticaRESUMEN
In primates, high-acuity vision is mediated by the fovea, a small specialized central region of the retina. The fovea, unique to the anthropoid lineage among mammals, undergoes notable neuronal morphological changes during postnatal maturation. However, the extent of cellular similarity across anthropoid foveas and the molecular underpinnings of foveal maturation remain unclear. Here, we used high-throughput single-cell RNA sequencing to profile retinal cells of the common marmoset (Callithrix jacchus), an early divergent in anthropoid evolution from humans, apes, and macaques. We generated atlases of the marmoset fovea and peripheral retina for both neonates and adults. Our comparative analysis revealed that marmosets share almost all their foveal types with both humans and macaques, highlighting a conserved cellular structure among primate foveas. Furthermore, by tracing the developmental trajectory of cell types in the foveal and peripheral retina, we found distinct maturation paths for each. In-depth analysis of gene expression differences demonstrated that cone photoreceptors and Müller glia (MG), among others, show the greatest molecular divergence between these two regions. Utilizing single-cell ATAC-seq and gene-regulatory network inference, we uncovered distinct transcriptional regulations differentiating foveal cones from their peripheral counterparts. Further analysis of predicted ligand-receptor interactions suggested a potential role for MG in supporting the maturation of foveal cones. Together, these results provide valuable insights into foveal development, structure, and evolution.
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Callithrix , Retina , Humanos , Animales , Recién Nacido , Callithrix/anatomía & histología , Retina/metabolismo , Fóvea Central/fisiología , Células Fotorreceptoras Retinianas Conos , Macaca , MamíferosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Primates and rodents, which descended from a common ancestor around 90 million years ago1, exhibit profound differences in behaviour and cognitive capacity; the cellular basis for these differences is unknown. Here we use single-nucleus RNA sequencing to profile RNA expression in 188,776 individual interneurons across homologous brain regions from three primates (human, macaque and marmoset), a rodent (mouse) and a weasel (ferret). Homologous interneuron types-which were readily identified by their RNA-expression patterns-varied in abundance and RNA expression among ferrets, mice and primates, but varied less among primates. Only a modest fraction of the genes identified as 'markers' of specific interneuron subtypes in any one species had this property in another species. In the primate neocortex, dozens of genes showed spatial expression gradients among interneurons of the same type, which suggests that regional variation in cortical contexts shapes the RNA expression patterns of adult neocortical interneurons. We found that an interneuron type that was previously associated with the mouse hippocampus-the 'ivy cell', which has neurogliaform characteristics-has become abundant across the neocortex of humans, macaques and marmosets but not mice or ferrets. We also found a notable subcortical innovation: an abundant striatal interneuron type in primates that had no molecularly homologous counterpart in mice or ferrets. These interneurons expressed a unique combination of genes that encode transcription factors, receptors and neuropeptides and constituted around 30% of striatal interneurons in marmosets and humans.
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Interneuronas/citología , Primates , Animales , Callithrix , Corteza Cerebral/citología , Femenino , Hurones , Hipocampo/citología , Humanos , Interneuronas/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Macaca , Masculino , Ratones , Neostriado/citología , Proteínas del Tejido Nervioso/metabolismo , ARN/genética , Especificidad de la Especie , Factores de Transcripción/metabolismoRESUMEN
Despite tremendous effort, the molecular and cellular basis of cognitive deficits in schizophrenia remain poorly understood. Recent progress in elucidating the genetic architecture of schizophrenia has highlighted the association of multiple loci and rare variants that may impact susceptibility. One key example, given their potential etiopathogenic and therapeutic relevance, is a set of genes that encode proteins that regulate excitatory glutamatergic synapses in brain. A critical next step is to delineate specifically how such genetic variation impacts synaptic plasticity and to determine if and how the encoded proteins interact biochemically with one another to control cognitive function in a convergent manner. Towards this goal, here we study the roles of GPCR-kinase interacting protein 1 (GIT1), a synaptic scaffolding and signaling protein with damaging coding variants found in schizophrenia patients, as well as copy number variants found in patients with neurodevelopmental disorders. We generated conditional neural-selective GIT1 knockout mice and found that these mice have deficits in fear conditioning memory recall and spatial memory, as well as reduced cortical neuron dendritic spine density. Using global quantitative phospho-proteomics, we revealed that GIT1 deletion in brain perturbs specific networks of GIT1-interacting synaptic proteins. Importantly, several schizophrenia and neurodevelopmental disorder risk genes are present within these networks. We propose that GIT1 regulates the phosphorylation of a network of synaptic proteins and other critical regulators of neuroplasticity, and that perturbation of these networks may contribute specifically to cognitive deficits observed in schizophrenia and neurodevelopmental disorders.
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Proteínas de Ciclo Celular , Proteínas Activadoras de GTPasa , Esquizofrenia , Animales , Ratones , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Cognición , Proteínas Activadoras de GTPasa/genética , Ratones Noqueados , Fosforilación , Esquizofrenia/genética , Sinapsis/metabolismoRESUMEN
Intellectual disability is a common neurodevelopmental disorder characterized by impaired intellectual and adaptive functioning. Both environmental insults and genetic defects contribute to the etiology of intellectual disability. Copy number variations of SORBS2 have been linked to intellectual disability. However, the neurobiological function of SORBS2 in the brain is unknown. The SORBS2 gene encodes ArgBP2 (Arg/c-Abl kinase binding protein 2) protein in non-neuronal tissues and is alternatively spliced in the brain to encode nArgBP2 protein. We found nArgBP2 colocalized with F-actin at dendritic spines and growth cones in cultured hippocampal neurons. In the mouse brain, nArgBP2 was highly expressed in the cortex, amygdala, and hippocampus, and enriched in the outer one-third of the molecular layer in dentate gyrus. Genetic deletion of Sorbs2 in mice led to reduced dendritic complexity and decreased frequency of AMPAR-miniature spontaneous EPSCs in dentate gyrus granule cells. Behavioral characterization revealed that Sorbs2 deletion led to a reduced acoustic startle response, and defective long-term object recognition memory and contextual fear memory. Together, our findings demonstrate, for the first time, an important role for nArgBP2 in neuronal dendritic development and excitatory synaptic transmission, which may thus inform exploration of neurobiological basis of SORBS2 deficiency in intellectual disability. SIGNIFICANCE STATEMENT: Copy number variations of the SORBS2 gene are linked to intellectual disability, but the neurobiological mechanisms are unknown. We found that nArgBP2, the only neuronal isoform encoded by SORBS2, colocalizes with F-actin at neuronal dendritic growth cones and spines. nArgBP2 is highly expressed in the cortex, amygdala, and dentate gyrus in the mouse brain. Genetic deletion of Sorbs2 in mice leads to impaired dendritic complexity and reduced excitatory synaptic transmission in dentate gyrus granule cells, accompanied by behavioral deficits in acoustic startle response and long-term memory. This is the first study of Sorbs2 function in the brain, and our findings may facilitate the study of neurobiological mechanisms underlying SORBS2 deficiency in the development of intellectual disability.
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Encéfalo/crecimiento & desarrollo , Dendritas/patología , Memoria , Proteínas de Microfilamentos/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Conducta Animal , ADN/genética , Espinas Dendríticas/patología , Potenciales Postsinápticos Excitadores/fisiología , Conos de Crecimiento/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Memoria a Largo Plazo , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Proteínas de Unión al ARN , Reconocimiento en Psicología , Reflejo de Sobresalto/genéticaRESUMEN
Understanding cellular architectures and their connectivity is essential for interrogating system function and dysfunction. However, we lack technologies for mapping the multiscale details of individual cells and their connectivity in the human organ-scale system. We developed a platform that simultaneously extracts spatial, molecular, morphological, and connectivity information of individual cells from the same human brain. The platform includes three core elements: a vibrating microtome for ultraprecision slicing of large-scale tissues without losing cellular connectivity (MEGAtome), a polymer hydrogel-based tissue processing technology for multiplexed multiscale imaging of human organ-scale tissues (mELAST), and a computational pipeline for reconstructing three-dimensional connectivity across multiple brain slabs (UNSLICE). We applied this platform for analyzing human Alzheimer's disease pathology at multiple scales and demonstrating scalable neural connectivity mapping in the human brain.
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Enfermedad de Alzheimer , Encéfalo , Imagen Molecular , Humanos , Enfermedad de Alzheimer/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Imagen Molecular/métodos , Fenotipo , Hidrogeles/química , ConectomaRESUMEN
In primates, high-acuity vision is mediated by the fovea, a small specialized central region of the retina. The fovea, unique to the anthropoid lineage among mammals, undergoes notable neuronal morphological changes during postnatal maturation. However, the extent of cellular similarity across anthropoid foveas and the molecular underpinnings of foveal maturation remain unclear. Here, we used high throughput single cell RNA sequencing to profile retinal cells of the common marmoset ( Callithrix jacchus ), an early divergent in anthropoid evolution from humans, apes, and macaques. We generated atlases of the marmoset fovea and peripheral retina for both neonates and adults. Our comparative analysis revealed that marmosets share almost all its foveal types with both humans and macaques, highlighting a conserved cellular structure among primate foveas. Furthermore, by tracing the developmental trajectory of cell types in the foveal and peripheral retina, we found distinct maturation paths for each. In-depth analysis of gene expression differences demonstrated that cone photoreceptors and Müller glia, among others, show the greatest molecular divergence between these two regions. Utilizing single-cell ATAC-seq and gene-regulatory network inference, we uncovered distinct transcriptional regulations differentiating foveal cones from their peripheral counterparts. Further analysis of predicted ligand-receptor interactions suggested a potential role for Müller glia in supporting the maturation of foveal cones. Together, these results provide valuable insights into foveal development, structure, and evolution. Significance statement: The sharpness of our eyesight hinges on a tiny retinal region known as the fovea. The fovea is pivotal for primate vision and is susceptible to diseases like age-related macular degeneration. We studied the fovea in the marmoset-a primate with ancient evolutionary ties. Our data illustrated the cellular and molecular composition of its fovea across different developmental ages. Our findings highlighted a profound cellular consistency among marmosets, humans, and macaques, emphasizing the value of marmosets in visual research and the study of visual diseases.
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Deletion of the obsessive-compulsive disorder (OCD)-associated gene SAP90/PSD-95-associated protein 3 (Sapap3), which encodes a postsynaptic anchoring protein at corticostriatal synapses, causes OCD-like motor behaviors in mice. While corticostriatal synaptic dysfunction is central to this phenotype, the striatum efficiently adapts to pathological changes, often in ways that expand upon the original circuit impairment. Here, we show that SAPAP3 deletion causes non-synaptic and pathway-specific alterations in dorsolateral striatum circuit function. While somatic excitability was elevated in striatal projection neurons (SPNs), dendritic excitability was exclusively enhanced in direct pathway SPNs. Layered on top of this, cholinergic modulation was altered in opposing ways: striatal cholinergic interneuron density and evoked acetylcholine release were elevated, while basal muscarinic modulation of SPNs was reduced. These data describe how SAPAP3 deletion alters the striatal landscape upon which impaired corticostriatal inputs will act, offering a basis for how pathological synaptic integration and unbalanced striatal output underlying OCD-like behaviors may be shaped.
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Proteínas del Tejido Nervioso , Trastorno Obsesivo Compulsivo , Ratones , Animales , Proteínas del Tejido Nervioso/metabolismo , Cuerpo Estriado/metabolismo , Neostriado/metabolismo , Trastorno Obsesivo Compulsivo/genética , Colinérgicos/metabolismoRESUMEN
Chimerism happens rarely among most mammals but is common in marmosets and tamarins, a result of fraternal twin or triplet birth patterns in which in utero connected circulatory systems (through which stem cells transit) lead to persistent blood chimerism (12-80%) throughout life. The presence of Y-chromosome DNA sequences in other organs of female marmosets has long suggested that chimerism might also affect these organs. However, a longstanding question is whether this chimerism is driven by blood-derived cells or involves contributions from other cell types. To address this question, we analyzed single-cell RNA-seq data from blood, liver, kidney and multiple brain regions across a number of marmosets, using transcribed single nucleotide polymorphisms (SNPs) to identify cells with the sibling's genome in various cell types within these tissues. Sibling-derived chimerism in all tissues arose entirely from cells of hematopoietic origin (i.e., myeloid and lymphoid lineages). In brain tissue this was reflected as sibling-derived chimerism among microglia (20-52%) and macrophages (18-64%) but not among other resident cell types (i.e., neurons, glia or ependymal cells). The percentage of microglia that were sibling-derived showed significant variation across brain regions, even within individual animals, likely reflecting distinct responses by siblings' microglia to local recruitment or proliferation cues or, potentially, distinct clonal expansion histories in different brain areas. In the animals and tissues we analyzed, microglial gene expression profiles bore a much stronger relationship to local/host context than to sibling genetic differences. Naturally occurring marmoset chimerism will provide new ways to understand the effects of genes, mutations and brain contexts on microglial biology and to distinguish between effects of microglia and other cell types on brain phenotypes.
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Sequence divergence of cis- regulatory elements drives species-specific traits, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains to be elucidated. We investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset, and mouse with single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome, and chromosomal conformation profiles from a total of over 180,000 cells. For each modality, we determined species-specific, divergent, and conserved gene expression and epigenetic features at multiple levels. We find that cell type-specific gene expression evolves more rapidly than broadly expressed genes and that epigenetic status at distal candidate cis -regulatory elements (cCREs) evolves faster than promoters. Strikingly, transposable elements (TEs) contribute to nearly 80% of the human-specific cCREs in cortical cells. Through machine learning, we develop sequence-based predictors of cCREs in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Lastly, we show that epigenetic conservation combined with sequence similarity helps uncover functional cis -regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.
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The mammalian brain is composed of many brain structures, each with its own ontogenetic and developmental history. We used single-nucleus RNA sequencing to sample over 2.4 million brain cells across 18 locations in the common marmoset, a New World monkey primed for genetic engineering, and examined gene expression patterns of cell types within and across brain structures. The adult transcriptomic identity of most neuronal types is shaped more by developmental origin than by neurotransmitter signaling repertoire. Quantitative mapping of GABAergic types with single-molecule FISH (smFISH) reveals that interneurons in the striatum and neocortex follow distinct spatial principles, and that lateral prefrontal and other higher-order cortical association areas are distinguished by high proportions of VIP+ neurons. We use cell type-specific enhancers to drive AAV-GFP and reconstruct the morphologies of molecularly resolved interneuron types in neocortex and striatum. Our analyses highlight how lineage, local context, and functional class contribute to the transcriptional identity and biodistribution of primate brain cell types.
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Callithrix , Neocórtex , Animales , Neocórtex/fisiología , Neuronas/fisiología , Distribución TisularRESUMEN
Adhesion and detachment are coordinated critical steps during cell migration. Conceptually, efficient migration requires both effective stabilization of membrane protrusions at the leading edge via nascent adhesions and their successful persistence during retraction of the trailing side via disruption of focal adhesions. As nascent adhesions are much smaller in size than focal adhesions, they are expected to exhibit a stronger adhesivity in order to achieve the coordination between cell front and back. Here, we show that Nudel knockdown by interference RNA (RNAi) resulted in cell edge shrinkage due to poor adhesions of membrane protrusions. Nudel bound to paxillin, a scaffold protein of focal contacts, and colocalized with it in areas of active membrane protrusions, presumably at nascent adhesions. The Nudel-paxillin interaction was disrupted by focal adhesion kinase (FAK) in a paxillin-binding-dependent manner. Forced localization of Nudel in all focal contacts by fusing it to paxillin markedly strengthened their adhesivity, whereas overexpression of structurally activated FAK or any paxillin-binding FAK mutant lacking the N-terminal autoinhibitory domain caused cell edge shrinkage. These results suggest a novel mechanism for selective reinforcement of nascent adhesions via interplays of Nudel and FAK with paxillin to facilitate cell migration.
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Proteínas Portadoras/metabolismo , Adhesión Celular , Quinasa 1 de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Paxillin/metabolismo , Proteínas Portadoras/genética , Línea Celular , Movimiento Celular , Células Epiteliales/metabolismo , Quinasa 1 de Adhesión Focal/genética , Células HeLa , Humanos , Riñón/citología , Microscopía Confocal/métodos , Mutación , Paxillin/genética , Interferencia de ARN , Factores de Tiempo , Vejiga Urinaria/citologíaRESUMEN
Fragile X syndrome (FXS) is a leading monogenic cause of intellectual disability and autism spectrum disorders, spurring decades of intense research and a multitude of mouse models. So far, these models do not recapitulate the genetic underpinning of classical FXS-CGG repeat-induced methylation of the Fmr1 locus-and their findings have failed to translate into the clinic. We sought to answer whether this disparity was because of low repeat length and generated a novel mouse line with 341 repeats, Fmr1hs341 , which is the largest allele in mice reported to date. This repeat length is significantly longer than the 200 repeats generally required for methylation of the repeat tract and promoter region in FXS patients, which leads to silencing of the FMR1 gene. Bisulfite sequencing fails to detect the robust methylation expected of FXS in Fmr1hs341 mice. Quantitative real-time PCR and Western blotting results also do not resemble FXS and instead produce a biochemical profile consistent with the fragile X-associated premutation disorders. These findings suggest that repeat length is unlikely to be the core determinant preventing methylation in mice, and other organisms phylogenetically closer to humans may be required to effectively model FXS.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Animales , Metilación de ADN , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Humanos , Ratones , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Genetic intervention is increasingly being explored as a therapeutic option for debilitating disorders of the central nervous system. The safety and efficacy of gene therapies rely upon expressing a transgene in affected cells while minimizing off-target expression. Here we show organ-specific targeting of adeno-associated virus (AAV) capsids after intravenous delivery, which we achieved by employing a Cre-transgenic-based screening platform and sequential engineering of AAV-PHP.eB between the surface-exposed AA452 and AA460 of VP3. From this selection, we identified capsid variants that were enriched in the brain and targeted away from the liver in C57BL/6J mice. This tropism extends to marmoset (Callithrix jacchus), enabling robust, non-invasive gene delivery to the marmoset brain after intravenous administration. Notably, the capsids identified result in distinct transgene expression profiles within the brain, with one exhibiting high specificity to neurons. The ability to cross the blood-brain barrier with neuronal specificity in rodents and non-human primates enables new avenues for basic research and therapeutic possibilities unattainable with naturally occurring serotypes.
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Cápside , Dependovirus , Administración Intravenosa , Animales , Encéfalo/metabolismo , Callithrix/genética , Dependovirus/genética , Vectores Genéticos , Hígado , Ratones , Ratones Endogámicos C57BL , Transducción Genética , TransgenesRESUMEN
Axonal transport is critical for neuronal function and survival. Cytoplasmic dynein and its accessory complex dynactin form a microtubule minus end-directed motor in charge of retrograde transport. In this study, we show that Nudel, a dynein regulator, was highly expressed in dorsal root ganglion (DRG) neurons. Microinjection of anti-Nudel antibody into cultured DRG neurons abolished retrograde transport of membranous organelles in the axon and led to dispersions of Golgi cisternae in the soma. As a result, lysosomes, which are normally enriched in the soma, moved persistently into and thus accumulated in axons. Endo-lysosome formation was also markedly delayed. As anterograde motility of mitochondria was not inhibited, the antibody apparently did not abolish retrograde transport by destructing axonal microtubule tracks. Similar results were obtained by microinjecting N-terminal Nudel, anti-dynein antibody or a p150(Glued) mutant capable of abrogating the dynein-dynactin association. These results indicate a critical role of Nudel in dynein-mediated axonal transport. Moreover, the effects of dynein on endolysosome formation and regional sequestration of lysosomes may contribute to defects in the endocytic pathway seen in neurons of patients or animals with malfunction of dynein.
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Transporte Axonal , Axones/metabolismo , Proteínas Portadoras/fisiología , Dineínas/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/farmacología , Transporte Axonal/efectos de los fármacos , Axones/fisiología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ratones , Microinyecciones , Microscopía Confocal , Microscopía Fluorescente , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Synthetic tissue-hydrogel methods have enabled superresolution investigation of biological systems using diffraction-limited microscopy. However, chemical modification by fixatives can cause loss of antigenicity, limiting molecular interrogation of the tissue gel. Here, we present epitope-preserving magnified analysis of proteome (eMAP) that uses purely physical tissue-gel hybridization to minimize the loss of antigenicity while allowing permanent anchoring of biomolecules. We achieved success rates of 96% and 94% with synaptic antibodies for mouse and marmoset brains, respectively. Maximal preservation of antigenicity allows imaging of nanoscopic architectures in 1000-fold expanded tissues without additional signal amplification. eMAP-processed tissue gel can endure repeated staining and destaining without epitope loss or structural damage, enabling highly multiplexed proteomic analysis. We demonstrated the utility of eMAP as a nanoscopic proteomic interrogation tool by investigating molecular heterogeneity in inhibitory synapses in the mouse brain neocortex and characterizing the spatial distributions of synaptic proteins within synapses in mouse and marmoset brains.
RESUMEN
The microtubule-based motor cytoplasmic dynein/dynactin is a force generator at the kinetochore. It also transports proteins away from kinetochores to spindle poles. Regulation of such diverse functions, however, is poorly understood. We have previously shown that Nudel is critical for dynein-mediated protein transport, whereas mitosin, a kinetochore protein that binds Nudel, is involved in retention of kinetochore dynein/dynactin against microtubule-dependent stripping. Here we demonstrate that Nudel is required for robust localization of dynein/dynactin at the kinetochore. It localizes to kinetochores after nuclear envelope breakdown, depending mostly ( approximately 78%) on mitosin and slightly on dynein/dynactin. Depletion of Nudel by RNA interference (RNAi) or overexpression of its mutant incapable of binding either Lis1 or dynein heavy chain abolishes the kinetochore protein transport and mitotic progression. Similar to mitosin RNAi, Nudel RNAi also leads to increased stripping of kinetochore dynein/dynactin in the presence of microtubules. Taking together, our results suggest a dual role of kinetochore Nudel: it activates dynein-mediated protein transport and, when interacting with both mitosin and dynein, stabilizes kinetochore dynein/dynactin against microtubule-dependent stripping to facilitate the force generation function of the motor.