Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 151
Filtrar
1.
Mol Microbiol ; 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34514656

RESUMO

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.

3.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
4.
Cell Death Differ ; 28(10): 2957-2969, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34285384

RESUMO

SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.

5.
Oncogene ; 40(25): 4352-4367, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34103685

RESUMO

Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

6.
J Phys Chem B ; 125(22): 5716-5721, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34042461

RESUMO

Understanding the function of protein complexes requires information on their molecular organization, specifically, their oligomerization level. Optical super-resolution microscopy can localize single protein complexes in cells with high precision, however, the quantification of their oligomerization level, remains a challenge. Here, we present a Quantitative Algorithm for Fluorescent Kinetics Analysis (QAFKA), that serves as a fully automated workflow for quantitative analysis of single-molecule localization microscopy (SMLM) data by extracting fluorophore "blinking" events. QAFKA includes an automated localization algorithm, the extraction of emission features per localization cluster, and a deep neural network-based estimator that reports the ratios of cluster types within the population. We demonstrate molecular quantification of protein monomers and dimers on simulated and experimental SMLM data. We further demonstrate that QAFKA accurately reports quantitative information on the monomer/dimer equilibrium of membrane receptors in single immobilized cells, opening the door to single-cell single-protein analysis.


Assuntos
Corantes Fluorescentes , Imagem Individual de Molécula , Algoritmos , Cinética , Microscopia de Fluorescência
7.
Nat Commun ; 12(1): 2276, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859193

RESUMO

Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm and innovations fuelled by DL technology, the need to access powerful and compatible resources to train DL networks leads to an accessibility barrier that novice users often find difficult to overcome. Here, we present ZeroCostDL4Mic, an entry-level platform simplifying DL access by leveraging the free, cloud-based computational resources of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise to train and apply key DL networks to perform tasks including segmentation (using U-Net and StarDist), object detection (using YOLOv2), denoising (using CARE and Noise2Void), super-resolution microscopy (using Deep-STORM), and image-to-image translation (using Label-free prediction - fnet, pix2pix and CycleGAN). Importantly, we provide suitable quantitative tools for each network to evaluate model performance, allowing model optimisation. We demonstrate the application of the platform to study multiple biological processes.


Assuntos
Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Animais , Linhagem Celular Tumoral , Computação em Nuvem , Conjuntos de Dados como Assunto , Humanos , Cultura Primária de Células , Ratos , Software
8.
ACS Chem Biol ; 16(3): 447-451, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33596038

RESUMO

Bacteria produce a plethora of specialized metabolites (SM), with the ecological function of most of them not known. A major group of SM are peptides derived from nonribosomal peptide synthetases (NRPS). In entomopathogenic bacteria of the genus Xenorhabdus, PAX (peptide-antimicrobial-Xenorhabdus) were described as NRPS-derived lipopeptides, which show antimicrobial activities against bacteria and fungi. We analyzed the production of PAX in Xenorhabdus doucetiae and found the majority bound to the cells. We derivatized PAX with fluorophores and show binding to cells when added externally using super-resolution microscopy. Externally added PAX in X. doucetiae and E. coli as well as inducible PAX production in X. doucetiae showed a protective effect against various antimicrobial peptides (AMPs) from insects, where they are used as a defense mechanism against pathogens. Because AMPs are often positively charged, our results suggest a PAX-induced repulsive force due to positive charge at the bacterial cell wall.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Produtos Biológicos/química , Insetos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Mecanismos de Defesa , Escherichia coli/química , Corantes Fluorescentes/química , Lipopeptídeos/química , Peptídeo Sintases/metabolismo , Xenorhabdus/química
9.
Angew Chem Int Ed Engl ; 60(12): 6310-6313, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33301653

RESUMO

Super-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI.

10.
J Leukoc Biol ; 109(2): 363-371, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32401398

RESUMO

TNFR1 is a crucial regulator of NF-ĸB-mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNFα- and TNFR1-controlled NF-ĸB signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNFα might also affect TNFR2-mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single-molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1-mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand-independent preligand assembly domain (PLAD)-mediated TNFR1 dimerization as well as TNFα-induced TNFR1 oligomerization. In addition, zafirlukast-mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF-ĸB signaling in reconstituted TNFR1-mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD-mediated, ligand-independent TNFR1 dimerization for NF-ĸB activation, highlight the PLAD as central regulator of TNFα-induced TNFR1 oligomerization, and demonstrate that TNFR1-mEos2 MEFs can be used to investigate TNFR1-antagonizing compounds employing single-molecule quantification and functional NF-ĸB assays at physiologic conditions.


Assuntos
NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Transdução de Sinais , Imagem Individual de Molécula , Compostos de Tosil/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Citocinas/biossíntese , Células HeLa , Humanos , Camundongos , Multimerização Proteica/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
11.
Chem Commun (Camb) ; 56(69): 10026-10029, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32728684

RESUMO

Conjugate Sn(iv)(pyropheophorbide a)dichloride-(peptide nucleic acid) catalyzes reduction of azobenzene derivatives in the presence of complementary nucleic acid (NA) upon irridiation with red light (660 nm). This is the first red light-induced NA-templated photoreduction. It is highly sensitive to single mismatches in the NA-template and can detect down to 5 nM NAs.


Assuntos
Luz , Ácidos Nucleicos/análise , Compostos Azo/química , Sequência de Bases , Catálise , Clorofila/análogos & derivados , Clorofila/química , Complexos de Coordenação/química , Limite de Detecção , Ácidos Nucleicos/química , Oxirredução , Ácidos Nucleicos Peptídicos/química , Estanho/química
12.
Nat Chem Biol ; 16(9): 946-954, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32541966

RESUMO

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with ß-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.


Assuntos
Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Imagem Individual de Molécula/métodos , Animais , Células CHO , Cricetulus , Ala(2)-MePhe(4)-Gly(5)-Encefalina/química , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Transferência Ressonante de Energia de Fluorescência , Morfina/química , Morfina/farmacologia , Mutação , Naloxona/química , Naloxona/farmacologia , Naltrexona/análogos & derivados , Naltrexona/química , Naltrexona/farmacologia , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/farmacologia , Fosforilação , Multimerização Proteica , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/genética , beta-Arrestinas/metabolismo
13.
Leukemia ; 34(8): 2087-2101, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32439895

RESUMO

Therapy resistance in leukemia may be due to cancer cell-intrinsic and/or -extrinsic mechanisms. Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid leukemia (CML), lead to resistance to tyrosine kinase inhibitors (TKI), and some are associated with clinically more aggressive disease and worse outcome. Using the retroviral transduction/transplantation model of CML and human cell lines we faithfully recapitulate accelerated disease course in TKI resistance. We show in various models, that murine and human imatinib-resistant leukemia cells positive for the oncogene BCR-ABL1T315I differ from BCR-ABL1 native (BCR-ABL1) cells with regards to niche location and specific niche interactions. We implicate a pathway via integrin ß3, integrin-linked kinase (ILK) and its role in deposition of the extracellular matrix (ECM) protein fibronectin as causative of these differences. We demonstrate a trend towards a reduced BCR-ABL1T315I+ tumor burden and significantly prolonged survival of mice with BCR-ABL1T315I+ CML treated with fibronectin or an ILK inhibitor in xenogeneic and syngeneic murine transplantation models, respectively. These data suggest that interactions with ECM proteins via the integrin ß3/ILK-mediated signaling pathway in BCR-ABL1T315I+ cells differentially and specifically influence leukemia progression. Niche targeting via modulation of the ECM may be a feasible therapeutic approach to consider in this setting.


Assuntos
Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Animais , Resistencia a Medicamentos Antineoplásicos , Fibronectinas/análise , Fibronectinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Proteínas de Fusão bcr-abl/análise , Proteínas de Fusão bcr-abl/fisiologia , Humanos , Imidazóis/farmacologia , Integrina beta3/fisiologia , Camundongos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Piridazinas/farmacologia
14.
Methods ; 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32389748

RESUMO

Fibroblast growth factor receptors (FGFRs) are a subfamily of receptor tyrosine kinases and central players in health and disease. Following ligand binding and the formation of homo- and heteromeric complexes, FGFRs initiate a cellular response. Challenges in studying FGFR activation are inner-subfamily interactions and a complex heterogeneity of these in the cell membrane, which demand for observation techniques that can resolve individual protein complexes and that are compatible with endogenous protein levels. Here, we established an imaging and analysis pipeline for multiplexed single-molecule localization microscopy (SMLM) of the FGFR network at the plasma membrane. Using DNA-labeled primary antibodies, we visualize all four FGFRs in the same cell with near-molecular spatial resolution. From the super-resolution imaging data, we extract information on FGFR density, spatial distribution, and inner-subfamily colocalization. Our approach is straightforward and easily adaptable to other multiplexed SMLM data of membrane proteins.

15.
Int J Mol Sci ; 21(8)2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32316583

RESUMO

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.


Assuntos
Membrana Celular/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Imagem Individual de Molécula/métodos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Células HeLa , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Ligantes , Complexos Multiproteicos/metabolismo , Transdução de Sinais
16.
Nat Commun ; 11(1): 1552, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32214101

RESUMO

Understanding the nano-architecture of protein machines in diverse subcellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While single-molecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D direct STORM (dSTORM) imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 targets in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision, allowing us to define distinct nano-architectural features of protein distribution within the presynaptic nerve terminal.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Proteínas/metabolismo , Animais , Humanos , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/instrumentação , Neurônios/metabolismo , Robótica , Software , Coloração e Rotulagem/instrumentação , Transmissão Sináptica
17.
Sci Signal ; 13(614)2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937565

RESUMO

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.


Assuntos
Membrana Celular/efeitos dos fármacos , Multimerização Proteica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Imagem Individual de Molécula/métodos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos Knockout , Camundongos Transgênicos , Modelos Moleculares , Mutação , NF-kappa B/metabolismo , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/química , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
18.
Nano Lett ; 19(11): 8245-8249, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31621335

RESUMO

Fluorescence methods are important tools in modern biology. Direct labeling of biomolecules with a fluorophore might, however, change interaction surfaces. Here, we introduce a competitive binding assay in combination with fluorescence correlation spectroscopy that reports binding affinities of both labeled and unlabeled biomolecules to their binding target. We investigated how fluorophore labels at different positions of a DNA oligonucleotide affect hybridization to a complementary oligonucleotide and found dissociation constants varying within 2 orders of magnitude. We next demonstrated that placing a fluorophore label at position Leu280 in the protein ligand internalin B does not alter the binding affinity to the MET receptor tyrosine kinase, compared to unlabeled internalin B. Our approach is simple to implement and can be applied to investigate the influence of fluorophore labels in a large variety of biomolecular interactions.


Assuntos
DNA/química , Corantes Fluorescentes/química , Oligonucleotídeos/química , Ligação Competitiva , Humanos , Modelos Moleculares , Hibridização de Ácido Nucleico/métodos , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Fluorescência/métodos
19.
Neurophotonics ; 6(3): 035008, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31637284

RESUMO

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.

20.
Nanoscale ; 11(39): 17981-17991, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31573593

RESUMO

Optical super-resolution microscopy has revolutionized our understanding of cell biology. Next to visualizing cellular structures with near-molecular spatial resolution, an additional benefit is the molecular characterization of biomolecular complexes directly in an intact cell. Single-molecule localization microscopy, as one technology out of the toolbox of super-resolution methods, generates images by detecting the position of single fluorophore labels and is particularly suited for molecular quantification. We review imaging and analysis methods employing single-molecule localization microscopy and extract molecule numbers.


Assuntos
Corantes Fluorescentes/química , Imagem Molecular , Proteínas , Animais , Humanos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Proteínas/química , Proteínas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...