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1.
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
2.
Front Synaptic Neurosci ; 13: 671288, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220481

RESUMO

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.

3.
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.

4.
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
5.
iScience ; 24(1): 101895, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33364584

RESUMO

Single-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in vivo in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.

6.
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
7.
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.

8.
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
9.
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
10.
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
11.
Mol Biol Cell ; 30(12): 1369-1376, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969885

RESUMO

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.


Assuntos
Dimerização , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Probabilidade , Imagem Individual de Molécula
12.
Nano Lett ; 18(7): 4626-4630, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29943993

RESUMO

DNA-PAINT is an optical super-resolution microscopy method that can visualize nanoscale protein arrangements and provide spectrally unlimited multiplexing capabilities. However, current multiplexing implementations based on, for example, DNA exchange (such as Exchange-PAINT) achieves multitarget detection by sequential imaging, limiting throughput. Here, we combine DNA-PAINT with single-molecule FRET and use the FRET efficiency as parameter for multiplexed imaging with high specificity. We demonstrate correlated single-molecule FRET and super-resolution on DNA origami structures, which are equipped with binding sequences that are targeted by pairs of dye-labeled oligonucleotides generating the FRET signal. We futher extract FRET values from single binding sites that are spaced just ∼55 nm apart, demonstrating super-resolution FRET imaging. This combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging with low background and opens the door for accurate distance readout in the 1-10 nm range.


Assuntos
DNA/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Nanotecnologia/métodos , Imagem Individual de Molécula , Sítios de Ligação , DNA/química , Oligonucleotídeos/química
13.
Biophys J ; 114(10): 2432-2443, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29650369

RESUMO

The dynamics of biomolecules in the plasma membrane is of fundamental importance to understanding cellular processes. Cellular signaling often starts with extracellular ligand binding to a membrane receptor, which then transduces an intracellular signal. Ligand binding and receptor-complex activation often involve a complex rearrangement of proteins in the membrane, which results in changes in diffusion properties. Two widely used methods to characterize biomolecular diffusion are single-particle tracking (SPT) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS). Here, we compare the results of recovered diffusion coefficients and mean-square displacements of the two methods by simulations of free, domain-confined, or meshwork diffusion. We introduce, to our knowledge, a new method for the determination of confinement radii from ITIR-FCS data. We further establish and demonstrate simultaneous SPT/ITIR-FCS for direct comparison within living cells. Finally, we compare the results obtained by SPT and ITIR-FCS for the receptor tyrosine kinase MET. Our results show that SPT and ITIR-FCS yield complementary information on diffusion properties of biomolecules in cell membranes.


Assuntos
Membrana Celular/metabolismo , Espectrometria de Fluorescência , Difusão
14.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(6): 614-624, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29526665

RESUMO

ACSL3 is the only long chain fatty acyl-CoA synthetase consistently found on growing and mature lipid droplets (LDs), suggesting that this specific localization has biological relevance. Current models for LD growth propose that triglycerides are synthesized by enzymes at the LD surface, with activated fatty acids provided by LD localized ACSL3, thus allowing growth independent of the ER. Here, we tested this hypothesis by quantifying ACSL3 on LDs from human A431 cells. RNAi of ACSL3 reduced the oleoyl-CoA synthetase activity by 83%, suggesting that ACSL3 is by far the dominant enzyme of A431 cells. Molar quantification revealed that there are 1.4 million ACSL3 molecules within a single cell. Metabolic labeling indicated that each ACSL3 molecule contributed a net gain of 3.1 oleoyl-CoA/s. 3D reconstruction of confocal images demonstrated that 530 individual lipid droplets were present in an average oleate fed A431 cell. A representative single lipid droplet with a diameter of 0.66 µm contained 680 ACSL3 molecules on the surface. Subcellular fractionation showed that at least 68% of ACSL3 remain at the ER even during extensive fatty acid supplementation. High resolution single molecule microscopy confirmed the abundance of cytoplasmic ACSL3 outside of LDs. Model calculations for triglyceride synthesis using only LD localized ACSL3 gave significant slower growth of LDs as observed experimentally. In conclusion, although ACSL3 is an abundant enzyme on A431 LDs, the metabolic capacity is not sufficient to account for LD growth solely by the local synthesis of triglycerides.


Assuntos
Coenzima A Ligases/metabolismo , Retículo Endoplasmático/enzimologia , Gotículas Lipídicas/enzimologia , Triglicerídeos/biossíntese , Linhagem Celular Tumoral , Humanos
15.
Angew Chem Int Ed Engl ; 57(20): 5620-5625, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29464841

RESUMO

Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His6 - and His12 -tagged proteins as well as single-molecule-based super-resolution imaging.


Assuntos
Quelantes/química , Corantes Fluorescentes/química , Ácido Nitrilotriacético/química , Proteínas/análise , Células HeLa , Humanos , Cinética , Estrutura Molecular , Imagem Óptica
16.
FEBS Open Bio ; 7(9): 1422-1440, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28904870

RESUMO

The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto-oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single-particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B-bound MET. We show that internalin B-bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B-bound receptors than for resting MET. Results of single-particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.

17.
EMBO Rep ; 18(9): 1572-1585, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28784601

RESUMO

Ubiquitylation is one of the cardinal post-translational modifications in the cell, balancing several distinct biological processes and acting as a pathogen recognition receptor during bacterial pathogen invasion. A dense layer of polyubiquitin chains marks invading bacteria that gain access to the host cytosol for their selective clearance via xenophagy. However, the enzymes that mediate recognition of cytosolic bacteria and generate this ubiquitin (Ub) coat remain largely elusive. To address this, we employed an image-based RNAi screening approach to monitor the loss of Ub on Salmonella upon depletion of human Ub E3 ligases in cells. Using this approach, we identified ARIH1 as one of the ligases involved in the formation of Ub coat on cytosolic bacteria. In addition, we provide evidence that the RING-between-RING ligase ARIH1, together with LRSAM1 and HOIP, forms part of a network of ligases that orchestrates recognition of intracellular Salmonella and participates in the activation of the host cell immune response.


Assuntos
Proteínas de Transporte/metabolismo , Citosol/microbiologia , Interações Hospedeiro-Patógeno , Salmonella typhimurium/fisiologia , Ubiquitina/metabolismo , Proteínas de Transporte/genética , Células HeLa , Humanos , Poliubiquitina/metabolismo , Interferência de RNA , Salmonella typhimurium/imunologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
18.
Photochem Photobiol ; 93(3): 881-887, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28500697

RESUMO

Plant cryptochromes are photoreceptors that regulate flowering, circadian rhythm and photomorphogenesis in response to blue and UV-A light. It has been demonstrated that the oxidized flavin cofactor is photoreduced to the neutral radical state via separate electron and proton transfer. Conformational changes have been found in the C-terminal extension, but few studies have addressed the changes in secondary structure in the sensory photolyase homology region (PHR). Here, we investigated the PHR of the plant cryptochrome from the green alga Chlamydomonas reinhardtii by light-induced infrared difference spectroscopy in combination with global 13 C and 15 N isotope labeling. Assignment of the signals is achieved by establishing a labeling strategy for cryptochromes that preserves the flavin at natural abundance. We demonstrate by UV/vis spectroscopy that the integrity of the sample is maintained and by mass spectrometry that the global labeling was highly efficient. As a result, difference bands are resolved at full intensity that at natural abundance are compensated by the overlap of flavin and protein signals. These bands are assigned to prominent conformational changes in the PHR by blue light illumination. We postulate that not only the partial unfolding of the C-terminal extension but also changes in the PHR may mediate signaling events.


Assuntos
Criptocromos/química , Desoxirribodipirimidina Fotoliase/química , Luz , Marcação por Isótopo , Espectrometria de Massas , Conformação Proteica , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta
19.
Biosci Rep ; 35(4)2015 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-26181364

RESUMO

CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6 in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs.


Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Receptores de Hialuronatos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
20.
Chemphyschem ; 16(4): 713-21, 2015 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-25521567

RESUMO

Membrane receptors control fundamental cellular processes. Binding of a specific ligand to a receptor initiates communication through the membrane and activation of signaling cascades. This activation process often leads to a spatial rearrangement of receptors in the membrane at the molecular level. Single-molecule techniques contributed significantly to the understanding of receptor organization and rearrangement in membranes. Here, we review four prominent single-molecule techniques that have been applied to membrane receptors, namely, stepwise photobleaching, Förster resonance energy transfer, sub-diffraction localization microscopy and co-tracking. We discuss the requirements, benefits and limitations of each technique, discuss target labeling, present a selection of applications and results and compare the different methodologies.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Fotodegradação , Multimerização Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Humanos , Microscopia de Fluorescência
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