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1.
Nat Commun ; 12(1): 2005, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790271

RESUMEN

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra. We develop the theory underlying this method and validate our approach using numerical simulations. To apply our system, we develop rsAKARev, a photochromic biosensor for cAMP-dependent protein kinase (PKA), and combine it with the spectrally-identical biosensor EKARev, a reporter for extracellular signal-regulated kinase (ERK) activity, to deliver simultaneous readout of both activities in the same cell. We further perform multiplexed PKA, ERK, and calcium measurements by including a third, spectrally-shifted biosensor. Our work demonstrates that exploiting donor photochromism in FRET can be a powerful approach to simultaneously read out multiple associations within living cells.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Algoritmos , Animales , Técnicas Biosensibles/métodos , Células COS , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Imagen de Lapso de Tiempo/métodos
2.
Int J Nanomedicine ; 16: 2879-2896, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33883896

RESUMEN

Background: Effective treatment strategy for cervical carcinoma is subject to the limitation of its anatomical location and histological characteristics. Comprehensive imaging before cervical carcinoma treatment is of great significance for the patients. Current imaging methods cannot meet the requirements of high resolution, deep imaging depth and non-invasive imaging at the same time. Fortunately, Photoacoustic imaging (PAI) is a novel imaging method that combines rich optical contrast, high ultrasonic spatial resolution, and deep penetration depth in a single modality. Moreover, PAI-guided photothermal therapy (PTT) by aid of targeting nanoparticles is an emerging and effective cancer treatment in recent years. Methods: Here, strong near-infrared region (NIR) absorption-conjugated polymer PIIGDTS (PD) nanoparticles with folic acid (FA) modification (namely, PD-FA) that targeted at Hela cell were specifically designed as cervical tumor imaging contrast agents and photothermal agents. Results: The obtained PD-FA nanoparticles exhibited admirable photoacoustic contrast-enhancing ability and desirable PTT behavior with the photothermal conversion efficiency as high as 62.6% in vitro. Furthermore, the PAI performance and PTT efficiency were tested in HeLa tumor-bearing nude mice after injection of PD-FA nanoparticles. In vivo multi-scale, PAI provided B-san and 3D dimension imaging for intuitive and comprehensive information of Hela tumor. Moreover, the Hela tumor can be completely eliminated within 18 days after PTT, with no toxicity and side effects. Conclusion: In summary, PD-FA injection combined with PAI and PTT systems provides a novel powerful tool for early diagnosis and precise treatment of cervical cancer.


Asunto(s)
Rayos Láser , Nanopartículas/química , Técnicas Fotoacústicas/métodos , Neoplasias del Cuello Uterino/diagnóstico por imagen , Neoplasias del Cuello Uterino/terapia , Animales , Células COS , Chlorocebus aethiops , Femenino , Ácido Fólico/química , Células HeLa , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/administración & dosificación , Nanopartículas/ultraestructura , Especificidad de Órganos , Fantasmas de Imagen , Polímeros/química
3.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33807610

RESUMEN

Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genes Dominantes/genética , Mutación/genética , Retinitis Pigmentosa/genética , Alelos , Animales , Secuencia de Bases , Células COS , Línea Celular , Chlorocebus aethiops , Edición Génica/métodos , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Receptores Nucleares Huérfanos/genética , Retina/fisiología
4.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-33800200

RESUMEN

The identification of soluble fibroblast growth factor (FGF) receptors in blood and the extracellular matrix has led to the prediction that these proteins modulate the diverse biological activities of the FGF family of ligands in vivo. A recent structural characterization of the soluble FGF receptors revealed that they are primarily generated by proteolytic cleavage of the FGFR-1 ectodomain. Efforts to examine their biological properties are now focused on understanding the functional consequences of FGFR-1 ectodomain shedding and how the shedding event is regulated. We have purified an FGFR-1 ectodomain that is constitutively cleaved from the full-length FGFR-1(IIIc) receptor and released into conditioned media. This shed receptor binds FGF-2; inhibits FGF-2-induced cellular proliferation; and competes with high affinity, cell surface FGF receptors for ligand binding. FGFR-1 ectodomain shedding downregulates the number of high affinity receptors from the cell surface. The shedding mechanism is regulated by ligand binding and by activators of PKC, and the two signaling pathways appear to be independent of each other. Deletions and substitutions at the proposed cleavage site of FGFR-1 do not prevent ectodomain shedding. Broad spectrum inhibitors of matrix metalloproteases decrease FGFR-1 ectodomain shedding, suggesting that the enzyme responsible for constitutive, ligand-activated, and protein kinase C-activated shedding is a matrix metalloprotease. In summary, shedding of the FGFR-1 ectodomain is a highly regulated event, sharing many features with a common system that governs the release of diverse membrane proteins from the cell surface. Most importantly, the FGFR ectodomains are biologically active after shedding and are capable of functioning as inhibitors of FGF-2.


Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Factor 2 de Crecimiento de Fibroblastos/genética , Humanos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Solubilidad
5.
Science ; 371(6533)2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33649166

RESUMEN

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Neoplasias/terapia , Proteína p53 Supresora de Tumor/inmunología , Alelos , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/uso terapéutico , Arginina/genética , Células COS , Chlorocebus aethiops , Femenino , Células HEK293 , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Histidina/genética , Humanos , Inmunización Pasiva , Células Jurkat , Activación de Linfocitos , Ratones Endogámicos NOD , Mutación , Linfocitos T/inmunología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Nat Commun ; 12(1): 1809, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753744

RESUMEN

Dynamic membraneless compartments formed by protein condensates have multifunctional roles in cellular biology. Tools that inducibly trigger condensate formation have been useful for exploring their cellular function, however, there are few tools that provide inducible control over condensate disruption. To address this need we developed DisCo (Disassembly of Condensates), which relies on the use of chemical dimerizers to inducibly recruit a ligand to the condensate-forming protein, triggering condensate dissociation. We demonstrate use of DisCo to disrupt condensates of FUS, associated with amyotrophic lateral sclerosis, and to prevent formation of polyglutamine-containing huntingtin condensates, associated with Huntington's disease. In addition, we combined DisCo with a tool to induce condensates with light, CRY2olig, achieving bidirectional control of condensate formation and disassembly using orthogonal inputs of light and rapamycin. Our results demonstrate a method to manipulate condensate states that will have broad utility, enabling better understanding of the biological role of condensates in health and disease.


Asunto(s)
Proteínas Fluorescentes Verdes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Multimerización de Proteína , Proteínas/química , Animales , Células COS , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Fluorescente/métodos , Proteínas/genética , Proteínas/metabolismo , Proteína FUS de Unión a ARN/química , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo
7.
J Vis Exp ; (168)2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33645581

RESUMEN

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4',5'-dimethylthiazol-2'-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.


Asunto(s)
Proliferación Celular , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , Técnicas de Síntesis en Fase Sólida/métodos , Compuestos de Espiro/síntesis química , Compuestos de Espiro/farmacología , Animales , Células COS , Chlorocebus aethiops
8.
Nat Commun ; 12(1): 1444, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33664271

RESUMEN

TRPV4 is a cell surface-expressed calcium-permeable cation channel that mediates cell-specific effects on cellular morphology and function. Dominant missense mutations of TRPV4 cause distinct, tissue-specific diseases, but the pathogenic mechanisms are unknown. Mutations causing peripheral neuropathy localize to the intracellular N-terminal domain whereas skeletal dysplasia mutations are in multiple domains. Using an unbiased screen, we identified the cytoskeletal remodeling GTPase RhoA as a TRPV4 interactor. TRPV4-RhoA binding occurs via the TRPV4 N-terminal domain, resulting in suppression of TRPV4 channel activity, inhibition of RhoA activation, and extension of neurites in vitro. Neuropathy but not skeletal dysplasia mutations disrupt TRPV4-RhoA binding and cytoskeletal outgrowth. However, inhibition of RhoA restores neurite length in vitro and in a fly model of TRPV4 neuropathy. Together these results identify RhoA as a critical mediator of TRPV4-induced cell structure changes and suggest that disruption of TRPV4-RhoA binding may contribute to tissue-specific toxicity of TRPV4 neuropathy mutations.


Asunto(s)
Neuritas/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Células COS , Calcio/metabolismo , Línea Celular , Chlorocebus aethiops , Drosophila , Células HEK293 , Humanos
9.
Eur J Med Chem ; 216: 113285, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33662676

RESUMEN

The development of resistance poses a serious problem in the therapy of cancer due to the necessity of a multiple-drug and unlimited treatment of affected patients. In chronic myeloid leukemia (CML), the introduction of imatinib has revolutionized the therapy. The persistence of an untreatable cancer stem cell pool and other resistance-causing factors, however, also impede the cure of this malignancy. New therapeutic approaches are therefore essential to overcome current treatment drawbacks. In this regard, an intervention in the STAT5 signaling pathway can significantly improve drug response, as this central signaling node induces the formation of highly resistant CML cells. In the present study, we continued the design of efficient chemosensitizers derived from the partial PPARγ agonist telmisartan. The developed 2-carbonitriles or 2-carboxymethyl esters showed improved potency in sensitizing K562-resistant cells to imatinib treatment, even at concentrations, which are considered patient-relevant. At 5 µM, for instance, 2d sensitized the cells in such a manner that the resistance was fully overcome and the recovered efficacy of imatinib resulted in >76% cell death. Importantly, all compounds were non-cytotoxic per se. A transactivation experiment showed that only the carbonitriles are partial agonists of PPARγ, which does not seem to be involved in the mode of action. Yet, immunoassays revealed a suppression of the STAT5 phosphorylation status by co-application of the most active derivatives with imatinib. This mechanism consequently resulted in reduced cell proliferation and induction of cell death in resistant CML cells.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Nitrilos/química , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Diseño de Fármacos , Regulación de la Expresión Génica , Humanos , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Nitrilos/farmacología , PPAR gamma/genética , PPAR gamma/metabolismo , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Telmisartán/química , Telmisartán/farmacología , Activación Transcripcional/efectos de los fármacos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
10.
Nat Commun ; 12(1): 1773, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741995

RESUMEN

The exploration of artificial luminogens with bright emission has been fully developed with the advancement of synthetic chemistry. However, many of them face problems like weakened emission in the aggregated state as well as poor renewability and sustainability. Therefore, the development of renewable and sustainable luminogens with anti-quenching function in the solid state, as well as to unveil the key factors that influence their luminescence behavior become highly significant. Herein, a new class of natural rosin-derived luminogens with aggregation-induced emission property (AIEgens) have been facilely obtained with good biocompatibility and targeted organelle imaging capability as well as photochromic behavior in the solid state. Mechanistic study indicates that the introduction of the alicyclic moiety helps suppress the excited-state molecular motion to enhance the solid-state emission. The current work fundamentally elucidates the role of alicyclic moiety in luminogen design and practically demonstrates a new source to large-scalely obtain biocompatible AIEgens.


Asunto(s)
Materiales Biocompatibles/química , Colorantes Fluorescentes/química , Luminiscencia , Resinas de Plantas/química , Animales , Materiales Biocompatibles/farmacología , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Escherichia coli/efectos de los fármacos , Colorantes Fluorescentes/farmacología , Microscopía Confocal , Estructura Molecular , Movimiento (Física) , Imagen Óptica/métodos , Orgánulos/química , Orgánulos/metabolismo , Resinas de Plantas/farmacología , Relación Estructura-Actividad
11.
Nat Commun ; 12(1): 856, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558528

RESUMEN

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein-protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.


Asunto(s)
Proteínas Luminiscentes/metabolismo , Acetilcolina/metabolismo , Animales , Células COS , Calcio/metabolismo , Chlorocebus aethiops , Fluorescencia , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/química , Modelos Moleculares
12.
Nat Commun ; 12(1): 1252, 2021 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-33623047

RESUMEN

Upon starvation, cells rewire their metabolism, switching from glucose-based metabolism to mitochondrial oxidation of fatty acids, which require the transfer of FAs from lipid droplets (LDs) to mitochondria at mitochondria-LD membrane contact sites (MCSs). However, factors responsible for FA transfer at these MCSs remain uncharacterized. Here, we demonstrate that vacuolar protein sorting-associated protein 13D (VPS13D), loss-of-function mutations of which cause spastic ataxia, coordinates FA trafficking in conjunction with the endosomal sorting complex required for transport (ESCRT) protein tumor susceptibility 101 (TSG101). The VPS13 adaptor-binding domain of VPS13D and TSG101 directly remodels LD membranes in a cooperative manner. The lipid transfer domain of human VPS13D binds glycerophospholipids and FAs in vitro. Depletion of VPS13D, TSG101, or ESCRT-III proteins inhibits FA trafficking from LDs to mitochondria. Our findings suggest that VPS13D mediates the ESCRT-dependent remodeling of LD membranes to facilitate FA transfer at mitochondria-LD contacts.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Ácidos Grasos/metabolismo , Gotas Lipídicas/metabolismo , Mitocondrias/metabolismo , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Fluorescencia , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas/química
13.
Nucleic Acids Res ; 49(5): 2435-2449, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33577685

RESUMEN

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.


Asunto(s)
Nucleótidos/química , Interferencia de ARN , ARN Interferente Pequeño/química , Animales , Proteínas Argonauta/química , Células COS , Células Cultivadas , Chlorocebus aethiops , Polimerasa del ADN Mitocondrial/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Ratones , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/química , Prealbúmina/genética , Nucleótidos de Pirimidina/síntesis química , Nucleótidos de Pirimidina/química , Uridina/análogos & derivados
14.
Nat Commun ; 12(1): 1305, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33637723

RESUMEN

Imaging the spatial distribution of biomolecules is at the core of modern biology. The development of fluorescence techniques has enabled researchers to investigate subcellular structures with nanometer precision. However, multiplexed imaging, i.e. observing complex biological networks and interactions, is mainly limited by the fundamental 'spectral crowding' of fluorescent materials. Raman spectroscopy-based methods, on the other hand, have a much greater spectral resolution, but often lack the required sensitivity for practical imaging of biomarkers. Addressing the pressing need for new Raman probes, herein we present a series of Raman-active  nanoparticles (Rdots) that exhibit the combined advantages of ultra-brightness and compact sizes (~20 nm). When coupled with the emerging stimulated Raman scattering (SRS) microscopy, these Rdots are brighter than previously reported Raman-active organic probes by two to three orders of magnitude. We further obtain evidence supporting for SRS imaging of Rdots at single particle level. The compact size and ultra-brightness of Rdots allows immunostaining of specific protein targets (including cytoskeleton and low-abundant surface proteins) in mammalian cells and tissue slices with high imaging contrast. These Rdots thus offer a promising tool for a large range of studies on complex biological networks.


Asunto(s)
Imagen Óptica/métodos , Espectrometría Raman/métodos , Animales , Biomarcadores , Células COS , Chlorocebus aethiops , Citoesqueleto , Células HeLa , Humanos , Microscopía/métodos , Nanopartículas , Imagen Óptica/instrumentación
15.
Nat Commun ; 12(1): 517, 2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33483489

RESUMEN

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.


Asunto(s)
Fluorescencia , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Imagen Individual de Molécula/métodos , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Microtúbulos/metabolismo , Fotometría/métodos
16.
Nat Commun ; 12(1): 263, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431828

RESUMEN

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. While SVs are mobile within the clusters, the clusters have no boundaries consistent with a liquid phase. We previously found that purified synapsin, a peripheral SV protein, can assemble into liquid condensates and trap liposomes into them. How this finding relates to the physiological formation of SV clusters in living cells remains unclear. Here, we report that synapsin alone, when expressed in fibroblasts, has a diffuse cytosolic distribution. However, when expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. This minimal reconstitution system can be a powerful model to gain mechanistic insight into the assembly of structures which are of fundamental importance in synaptic transmission.


Asunto(s)
Neuronas/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , Animales , Células COS , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citosol/metabolismo , Endocitosis , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Ratones , Electricidad Estática , Vesículas Sinápticas/ultraestructura
17.
Mol Cell ; 81(5): 905-921.e5, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33497605

RESUMEN

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation. We investigated this matter in five mammalian aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and ADGRL1) and demonstrate that intact aGPCR heterodimers exist at the cell surface, that the core TA region becomes unmasked in the cleaved GAIN domain, and that intra-GAIN domain movements regulate the level of tethered agonist exposure, thereby likely controlling aGPCR activity. Collectively, these findings delineate a unifying mechanism for TA-dependent signaling of intact aGPCRs.


Asunto(s)
Antígenos CD/química , Proteínas del Tejido Nervioso/química , Péptidos/química , Receptores Acoplados a Proteínas G/química , Receptores de Péptidos/química , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteolisis , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal
18.
Int J Mol Sci ; 22(1)2020 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-33375396

RESUMEN

ABCA4 is an ATP-binding cassette (ABC) transporter expressed in photoreceptors, where it transports its substrate, N-retinylidene-phosphatidylethanolamine (N-Ret-PE), across outer segment membranes to facilitate the clearance of retinal from photoreceptors. Mutations in ABCA4 cause Stargardt macular degeneration (STGD1), an autosomal recessive disorder characterized by a loss of central vision and the accumulation of bisretinoid compounds. The purpose of this study was to determine the molecular properties of ABCA4 variants harboring disease-causing missense mutations in the transmembrane domains. Thirty-eight variants expressed in culture cells were analyzed for expression, ATPase activities, and substrate binding. On the basis of these properties, the variants were divided into three classes: Class 1 (severe variants) exhibited significantly reduced ABCA4 expression and basal ATPase activity that was not stimulated by its substrate N-Ret-PE; Class 2 (moderate variants) showed a partial reduction in expression and basal ATPase activity that was modestly stimulated by N-Ret-PE; and Class 3 (mild variants) displayed expression and functional properties comparable to normal ABCA4. The p.R653C variant displayed normal expression and basal ATPase activity, but lacked substrate binding and ATPase activation, suggesting that arginine 653 contributes to N-Ret-PE binding. Our classification provides a basis for better understanding genotype-phenotype correlations and evaluating therapeutic treatments for STGD1.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico Activo/genética , Enfermedad de Stargardt/genética , Enfermedad de Stargardt/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Animales , Células COS , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Expresión Génica , Estudios de Asociación Genética , Células HEK293 , Humanos , Modelos Moleculares , Mutación Missense , Fosfatidiletanolaminas/metabolismo , Unión Proteica , Dominios Proteicos , Enfermedades de la Retina/congénito , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Retinoides/metabolismo , Enfermedad de Stargardt/enzimología
19.
Viruses ; 12(12)2020 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-33371460

RESUMEN

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas.


Asunto(s)
Virus de la Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Protección Cruzada/inmunología , Vacunas Atenuadas/inmunología , Vacunas Virales/inmunología , Fiebre Porcina Africana/prevención & control , Virus de la Fiebre Porcina Africana/genética , Animales , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Células COS , Línea Celular , Chlorocebus aethiops , Genotipo , Inmunización , Inmunoglobulina G/inmunología , Porcinos , Proteínas Virales/inmunología
20.
Nutrients ; 13(1)2020 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-33375084

RESUMEN

Congenital sucrase-isomaltase deficiency (CSID) is a rare metabolic intestinal disorder with reduced or absent activity levels of sucrase-isomaltase (SI). Interestingly, the main symptoms of CSID overlap with those in irritable bowel syndrome (IBS), a common functional gastrointestinal disorder with unknown etiology. Recent advances in genetic screening of IBS patients have revealed rare SI gene variants that are associated with IBS. Here, we investigated the biochemical, cellular and functional phenotypes of several of these variants. The data demonstrate that the SI mutants can be categorized into three groups including immature, mature but slowly transported, and finally mature and properly transported but with reduced enzymatic activity. We also identified SI mutant phenotypes that are deficient but generally not as severe as those characterized in CSID patients. The variable effects on the trafficking and function of the mutations analyzed in this study support the view that both CSID and IBS are heterogeneous disorders, the severity of which is likely related to the biochemical phenotypes of the SI mutants as well as the environment and diet of patients. Our study underlines the necessity to screen for SI mutations in IBS patients and to consider enzyme replacement therapy as an appropriate therapy as in CSID.


Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/genética , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/metabolismo , Mutación , Transporte de Proteínas , Complejo Sacarasa-Isomaltasa/deficiencia , Animales , Células COS , Chlorocebus aethiops , Oligo-1,6-Glucosidasa/genética , Oligo-1,6-Glucosidasa/metabolismo , Fenotipo , Complejo Sacarasa-Isomaltasa/genética , Complejo Sacarasa-Isomaltasa/metabolismo
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