RESUMEN
High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum, and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.
RESUMEN
The determination of the site occupancy of activators in phosphors is essential for precise synthesis, understanding the relationship between their luminescence properties and crystal structure, and tailoring their properties by modifying the host composition. Herein, one simple method was proposed to help determine the sites at which the doping of rare earth ions or transition metal ions occupies in the host lattice through site occupancy theory (SOT) for ions doped into the matrix lattice. SOT was established based on the fact that doping ions preferentially occupy the sites with the lowest bonding energy deviations. In order to provide detailed experimental evidence to prove the feasibility of SOT, several scheelite-type compounds were successfully synthesized using a high-temperature solid-phase method. When Eu3+ ions occupy a similar surrounding environment site, the photoluminescence spectra of the activators Eu3+ are similar. Therefore, by comparing the intensity ratio of photoluminescence spectra and the mechanism of all transitions of KEu(WO4)2, KY(WO4)2:Eu3+, Na5Eu(WO4)4, and Na5Y(WO4)4:Eu3+, it was proved that SOT can successfully confirm the site occupation when doped ions enter the matrix lattice. SOT was further applied to the sites occupied by Eu3+ ion-doped LiAl(MoO4)2 and LiLu(MoO4)2.
RESUMEN
Inflammation is a complex physiological process that enables the clearance of pathogens and repairing damaged tissues. Elevated serum copper concentration has been reported in cases of inflammation, but the role of copper in inflammatory responses remains unclear. This study used bovine macrophages to establish lipopolysaccharide (LPS)-induced inflammation model. There were five groups in the study: a group treated with LPS (100 ng/ml), a group treated with either copper chelator (tetrathiomolybdate, TTM) (20 µmol) or CuSO4 (25 µmol or 50 µmol) after LPS stimulation, and a control group. Copper concentrations increased in macrophages after the LPS treatment. TTM decreased mRNA expression of pro-inflammatory factors (IL-1ß, TNF-α, IL-6, iNOS, and COX-2), whereas copper supplement increased them. Compared to the control group, TLP4 and MyD88 protein levels were increased in the TTM and copper groups. However, TTM treatment decreased p-p65 and increased IкB-α while the copper supplement showed reversed results. In addition, the phagocytosis and migration of bovine macrophages decreased in the TTM treatment group while increased in the copper treatment groups. Results mentioned above indicated that copper could promote the LPS-induced inflammatory response in bovine macrophages, promote pro-inflammatory factors by activating the NF-кB pathway, and increase phagocytosis capacity and migration. Our study provides a possible targeted therapy for bovine inflammation.
RESUMEN
CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.
Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Bases de Datos Genéticas , Endodesoxirribonucleasas , Sistemas CRISPR-Cas/genética , Filogenia , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/clasificación , Proteínas Asociadas a CRISPR/genética , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/clasificación , Endodesoxirribonucleasas/genética , Enciclopedias como AsuntoRESUMEN
Alzheimer's disease (AD) is the most common neurodegenerative disorder, and is caused by multiple pathological factors, such as the overproduction of ß-amyloid (Aß) and the hyperphosphorylation of tau. However, there is limited knowledge of the mechanisms underlying AD pathogenesis and no effective biomarker for the early diagnosis of this disorder. Thus in this study, a quantitative phosphoproteomics analysis was performed to evaluate global protein phosphorylation in the hippocampus of Aß overexpressing APP/PS1 transgenic mice and tau overexpressing MAPT×P301S transgenic mice, two in vivo AD model systems. These animals, up to ten weeks old, do not exhibit cognitive dysfunctions and are widely used to simulate early-stage AD patients. The number of differentially phosphorylated proteins (DPPs) was greater for APP/PS1 transgenic mice than for MAPT×P301S transgenic mice. The function of the DPPs in APP/PS1 transgenic mice was mainly related to synapses, while the function of the DPPs in MAPT×P301S transgenic mice was mainly related to microtubules. In addition, an AD core network was established including seven phosphoproteins differentially expressed in both animal models, and the function of this core network was related to synapses and oxidative stress. The results of this study suggest that Aß and tau induce different protein phosphorylation profiles in the early stage of AD, leading to the dysfunctions in synapses and microtubule, respectively. And the detection of same DPPs in these animal models might be used for early AD diagnosis.
Asunto(s)
Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Ratones Transgénicos , Fosforilación , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismoRESUMEN
In this study, new magnetic nanoparticles (denotated as Fe3O4@mSiO2-PFIL-Ti4+) have been prepared by immobilizing titanium ions with phosphonate functionalized ionic liquid (PFIL) on the wall of core-shell structured mesoporous nanomaterials. The resulting nanoparticles possess large specific surface area, strong hydrophilicity and fast magnetic response. The composites can capture traces of phosphopeptides from the tryptic ß-casein digest (0.08 fmol), a digest mixture of ß-casein and BSA (1 : 10 000, molar ratio) as well as a blend of ß-casein digest and a great quantity of phosphorylated protein (ß-casein) and non-phosphorylated protein (BSA) (1 : 2000 : 2000, mass ratio), respectively, showing excellent sensitivity, selectivity and size exclusion ability. Additionally, Fe3O4@mSiO2-PFIL-Ti4+ shows excellent steadiness and can be reused at least 12 times. Moreover, this material was successfully applied to enrich endogenous phosphopeptides from complex bio-samples, including human saliva and serum.
RESUMEN
In this study, new graphene-based IMAC nanocomposites for phosphopeptide enrichment were prepared according to the guideline of our new design strategy. Superhydrophilic polyethyleneimine (PEI) was introduced, to which a phosphonate-functionalized ionic liquid (PFIL) was covalently bound, to form superhydrophilic and cationic surface layers with high densities of nitrogen atoms, phosphonate functional groups, and high-loading metal ions. Due to the combined features of superhydrophilicity, flexibility, highly dense metal binding sites, large surface area and excellent size-exclusion effect, the fabricated nanocomposite G@mSiO2@PEI-PFIL-Ti4+ exhibits superior detection sensitivity to enrich phosphopeptides (tryptic ß-casein digest, 0.1 fmol), and extraordinary enrichment specificity to enrich phosphopeptides from a digest mixture of ß-casein and bovine serum albumin (BSA) (molar ratio, 1 : 12 000). The excellent size-exclusion effect was also observed, and 27 endogenous phosphopeptides were identified in human saliva. All these results could be attributed to the unique superhydrophilic nanocomposite structure with a high density of a cationic linker modified with phosphonate functionality. Moreover, G@mSiO2@PEI-PFIL-Ti4+ adsorbents were used to extract phosphopeptides from the tryptic digests of hippocampal lysates for quantitative phosphoproteome analysis. The preliminary results indicate that 1649 phosphoproteins, 3286 phosphopeptides and 4075 phosphorylation sites were identified. A total of 13 Alzheimer's disease (AD)-related phosphopeptides within tau proteins were detected with a wide coverage from p-Thr111 to p-Ser404, in which the amounts of some phoshopeptides at certain sites in AD transgenic mice were found statistically higher than those in wild type littermates. Besides, phosphorylated neurofilament heavy chains, a potential biomarker for amyotrophic lateral sclerosis and traumatic brain injury, were also identified. Finally, the adsorbent was applied to human cerebrospinal fluid (CSF) and blood samples. 5 unique phosphopeptides of neuroendocrine specific VGF were identified in the CSF, while many phosphopeptides originated from the nervous system were found in the blood sample. All these results suggest that our new IMAC materials exhibit unbiased enrichment ability with superior detection sensitivity and specificity, allowing the global phosphoproteome analysis of complicated biological samples more convincible and indicating the potential use in disease diagnosis.
Asunto(s)
Enfermedad de Alzheimer , Grafito , Líquidos Iónicos , Nanocompuestos , Organofosfonatos , Animales , Caseínas/química , Hipocampo/química , Humanos , Indicadores y Reactivos , Iones , Ratones , Ratones Transgénicos , Nitrógeno , Fosfopéptidos/análisis , Fosfoproteínas/química , Fosforilación , Polietileneimina , Albúmina Sérica Bovina/química , Titanio/química , Proteínas tauRESUMEN
The human cerebral cortex has tremendous cellular diversity. How different cell types are organized in the human cortex and how cellular organization varies across species remain unclear. In this study, we performed spatially resolved single-cell profiling of 4000 genes using multiplexed error-robust fluorescence in situ hybridization (MERFISH), identified more than 100 transcriptionally distinct cell populations, and generated a molecularly defined and spatially resolved cell atlas of the human middle and superior temporal gyrus. We further explored cell-cell interactions arising from soma contact or proximity in a cell type-specific manner. Comparison of the human and mouse cortices showed conservation in the laminar organization of cells and differences in somatic interactions across species. Our data revealed human-specific cell-cell proximity patterns and a markedly increased enrichment for interactions between neurons and non-neuronal cells in the human cortex.
Asunto(s)
Corteza Cerebral , Perfilación de la Expresión Génica , Neuronas , Análisis de la Célula Individual , Animales , Comunicación Celular , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Humanos , Hibridación Fluorescente in Situ/métodos , Ratones , Neuronas/citología , Neuronas/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of potential candidate MPS-interacting proteins that span diverse functional categories. We examined representative proteins in several of these categories using super-resolution imaging, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, and observed periodic distributions characteristic of the MPS along the neurites for ~20 proteins. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide insights into the interactome of the MPS and suggest previously unknown functions of the MPS in neurons.
Asunto(s)
Proteómica , Espectrina , Actinas/metabolismo , Axones/metabolismo , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Espectrina/metabolismoRESUMEN
The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from â¼10,000 genes in individual cells with â¼80% detection efficiency and â¼4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing â¼1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Espacio Intracelular/diagnóstico por imagen , ARN Mensajero/análisis , División Celular/genética , Línea Celular Tumoral , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genes cdc/genética , Humanos , Hibridación Fluorescente in Situ/métodos , ARN Largo no Codificante/análisis , ARN Largo no Codificante/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual/métodos , Transcriptoma/genéticaRESUMEN
Actin, spectrin, and related molecules form a membrane-associated periodic skeleton (MPS) in neurons. The function of the MPS, however, remains poorly understood. Using super-resolution imaging, we observed that G protein-coupled receptors (GPCRs), cell adhesion molecules (CAMs), receptor tyrosine kinases (RTKs), and related signaling molecules were recruited to the MPS in response to extracellular stimuli, resulting in colocalization of these molecules and RTK transactivation by GPCRs and CAMs, giving rise to extracellular signal-regulated kinase (ERK) signaling. Disruption of the MPS prevented such molecular colocalizations and downstream ERK signaling. ERK signaling in turn caused calpain-dependent MPS degradation, providing a negative feedback that modulates signaling strength. These results reveal an important functional role of the MPS and establish it as a dynamically regulated platform for GPCR- and CAM-mediated RTK signaling.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Neuronas/enzimología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Espectrina/metabolismo , Animales , Antígeno CD56/metabolismo , Calpaína/metabolismo , Moléculas de Adhesión Celular/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Imagen Molecular , Cultivo Primario de Células , Transporte de Proteínas , Proteolisis , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows simultaneous imaging of numerous RNA species in their native cellular environment and hence spatially resolved single-cell transcriptomic measurements. However, the relatively modest brightness of signals from single RNA molecules can become limiting in a number of applications, such as increasing the imaging throughput, imaging shorter RNAs, and imaging samples with high degrees of background, such as some tissue samples. Here, we report a branched DNA (bDNA) amplification approach for MERFISH measurements. This approach produces a drastic signal increase in RNA FISH samples without increasing the fluorescent spot size for individual RNAs or increasing the variation in brightness from spot to spot, properties that are important for MERFISH imaging. Using this amplification approach in combination with MERFISH, we demonstrated RNA imaging and profiling with a near 100% detection efficiency. We further demonstrated that signal amplification improves MERFISH performance when fewer FISH probes are used for each RNA species, which should allow shorter RNAs to be imaged. We anticipate that the combination of bDNA amplification with MERFISH should facilitate many other applications and extend the range of biological questions that can be addressed by this technique in both cell culture and tissues.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Hibridación Fluorescente in Situ/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/análisis , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento , Humanos , Procesamiento de Imagen Asistido por Computador , Osteosarcoma/patología , ARN Neoplásico/análisis , Análisis de la Célula IndividualRESUMEN
To meet their purine demand, cells activate the de novo purine biosynthetic pathway and transiently cluster the pathway enzymes into metabolons called purinosomes. Recently, we have shown that purinosomes were spatially colocalized with mitochondria and microtubules, yet it remained unclear as to what drives these associations and whether a relationship between them exist. Here, we employed superresolution imaging methods to describe purinosome transit in the context of subcellular localization. Time-resolved imaging of purinosomes showed that these assemblies exhibit directed motion as they move along a microtubule toward mitochondria, where upon colocalization, a change in purinosome motion was observed. A majority of purinosomes colocalized with mitochondria were also deemed colocalized with microtubules. Nocodazole-dependent microtubule depolymerization resulted in a loss in the purinosome-mitochondria colocalization, suggesting that the association of purinosomes with mitochondria is facilitated by microtubule-directed transport, and thereby supporting our notion of an interdependency between these subcellular components in maximizing purine production through the de novo purine biosynthetic pathway.
Asunto(s)
Citosol/metabolismo , Metaboloma , Microtúbulos/metabolismo , Mitocondrias/metabolismo , Purinas/metabolismo , Vías Biosintéticas , Células HeLa , HumanosRESUMEN
Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. In the MPS, short actin filaments, capped by actin-capping proteins, form ring-like structures that wrap around the circumference of neurites, and these rings are periodically spaced along the neurite by spectrin tetramers, forming a quasi-1D lattice structure. This 1D MPS structure was initially observed in axons and exists extensively in axons, spanning nearly the entire axonal shaft of mature neurons. Such 1D MPS was also observed in dendrites, but the extent to which it exists and how it develops in dendrites remain unclear. It is also unclear whether other structural forms of the membrane skeleton are present in neurons. Here, we investigated the spatial organizations of spectrin, actin, and adducin, an actin-capping protein, in the dendrites and soma of cultured hippocampal neurons at different developmental stages, and compared results with those obtained in axons, using superresolution imaging. We observed that the 1D MPS exists in a substantial fraction of dendritic regions in relatively mature neurons, but this structure develops slower and forms with a lower propensity in dendrites than in axons. In addition, we observed that spectrin, actin, and adducin also form a 2D polygonal lattice structure, resembling the expanded erythrocyte membrane skeleton structure, in the somatodendritic compartment. This 2D lattice structure also develops substantially more slowly in the soma and dendrites than the development of the 1D MPS in axons. These results suggest membrane skeleton structures are differentially regulated across different subcompartments of neurons.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Dendritas/metabolismo , Hipocampo/metabolismo , Espectrina/metabolismo , Animales , Membrana Celular/ultraestructura , Citoesqueleto/ultraestructura , Dendritas/ultraestructura , Hipocampo/ultraestructura , Ratones , RatasRESUMEN
Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.
Asunto(s)
Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Nanoestructuras/ultraestructura , Orgánulos/ultraestructura , Compuestos de Boro/química , Carbocianinas/química , Membrana Celular/ultraestructura , Dendritas/ultraestructura , Retículo Endoplásmico/ultraestructura , Hipocampo/citología , Membrana Dobles de Lípidos , Lisosomas/ultraestructura , Microscopía Fluorescente/instrumentación , Mitocondrias/ultraestructura , Neuronas/ultraestructura , Seudópodos/ultraestructura , Procesos EstocásticosRESUMEN
Neuronal synapses are functional nodes in neural circuits. Their organization and activity define an individual's level of intelligence, emotional state and mental health. Changes in the structure and efficacy of synapses are the biological basis of learning and memory. However, investigation of the molecular architecture of synapses has been impeded by the lack of efficient techniques with sufficient resolution. Recent developments in state-of-the-art nano-imaging techniques have opened up a new window for dissecting the molecular organization of neuronal synapses with unprecedented resolution. Here, we review recent technological advances in nano-imaging techniques as well as their applications to the study of synapses, emphasizing super-resolution light microscopy and 3-dimensional electron tomography.
Asunto(s)
Encéfalo/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Neuronas/ultraestructura , Sinapsis/ultraestructura , Animales , Tomografía con Microscopio Electrónico/instrumentación , Humanos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodosRESUMEN
Cu2(OH)2CO3 and CuO hierarchical nanostructures with variable morphologies were synthesized by controlled heating hydrated nanoparticles. The growth of nanostructures started with nanoparticles which formed loose aggregates. The nanoparticles within aggregates reorganized to form urchin-like structures which consisted of dense nanorods. With the growth of nanorods, regular microspheres were formed. At the same time, the nanorods coalesced to form wedge-like structures. The various surface subunits were just the outer display of wedge-like structures under different conditions. CuO nanostructures were gained by pyrolysis of malachite precursors. The attractive electrostatic force was responsible for aggregation of nanoparticles. During growth of aggregates, nanorods acted as growing tips which adsorbed adjacent nanoparticles. The Brownian motion was responsible for reorientation of nanoparticles to achieve low-energy configuration. Adsorbed water played an important role during formation of malachite nanostructures. The effect of growing environments on nanostructures was investigated. XRD, SEM, TEM and BET and so on were used to characterize the products.