RESUMO
Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion.
RESUMO
Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.
Assuntos
DNA/análise , Receptores ErbB/metabolismo , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Pontos Quânticos , Imagem Individual de Molécula/métodos , Animais , Células CHO , Cricetulus , Imagem Óptica , Processos FotoquímicosRESUMO
Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (ß2-AR) in living cells, revealing the differential regulation of these receptors' dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells.
Assuntos
Imunoprecipitação/métodos , Mapeamento de Interação de Proteínas/métodos , Análise de Célula Única/métodos , Linhagem Celular , Membrana Celular/metabolismo , Receptores ErbB/fisiologia , Humanos , Proteínas de Membrana/fisiologia , Ligação Proteica/fisiologia , Receptores Adrenérgicos beta 2/fisiologia , Transdução de SinaisRESUMO
Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction.
Assuntos
Trifosfato de Adenosina/química , Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Estilbenos/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Antineoplásicos Fitogênicos/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luciferases/genética , Luciferases/metabolismo , Células MCF-7 , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Resveratrol , Transdução de Sinais , Estilbenos/química , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
We present a single-molecule diffusional-mobility-shift assay (smDIMSA) for analyzing the interactions between membrane and water-soluble proteins in the crowded membrane of living cells. We found that ligand-receptor interactions decreased the diffusional mobility of ErbB receptors and ß-adrenergic receptors, as determined by single-particle tracking with super-resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water-soluble binders that ranged from a few tens of kilodaltons to several hundred kilodaltons. This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants. smDIMSA enables the quantitative investigation of previously undetected ligand-receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single-molecule membrane proteins without ligand labeling.
Assuntos
Receptores ErbB/metabolismo , Ligantes , Animais , Anticorpos Monoclonais/imunologia , Células COS , Membrana Celular/metabolismo , Cetuximab/imunologia , Chlorocebus aethiops , Difusão , Receptores ErbB/química , Receptores ErbB/genética , Microscopia , MutaçãoRESUMO
We developed a process to produce novel interactions between two previously unrelated proteins. This process selects protein scaffolds and designs protein interfaces that bind to a surface patch of interest on a target protein. Scaffolds with shapes complementary to the target surface patch were screened using an exhaustive computational search of the human proteome and optimized by directed evolution using phage display. This method was applied to successfully design scaffolds that bind to epidermal growth factor receptor (EGFR) domain II, the interface of EGFR dimerization, with high reactivity toward the target surface patch of EGFR domain II. One potential application of these tailor-made protein interactions is the development of therapeutic agents against specific protein targets.
Assuntos
Simulação por Computador , Desenho de Fármacos , Receptores ErbB/química , Biblioteca de Peptídeos , Humanos , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pressão Osmótica , Serina-Treonina Quinases TOR/metabolismo , Sequência de Bases , Linhagem Celular , Imunoprecipitação da Cromatina , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Fosforilação , RNA Interferente Pequeno , Proteína Regulatória Associada a mTOR , Espectrometria de Massas em TandemRESUMO
Phospholipase D (PLD) is involved in diverse cellular processes including cell movement, adhesion, and vesicle trafficking through cytoskeletal rearrangements. However, the mechanism by which PLD induces cytoskeletal reorganization is still not fully understood. Here, we describe a new link to cytoskeletal changes that is mediated by PLD2 through direct nucleotide exchange on RhoA. We found that PLD2 induces RhoA activation independent of its lipase activity. PLD2 directly interacted with RhoA, and the PX domain of PLD2 specifically recognized nucleotide-free RhoA. Finally, we found that the PX domain of PLD2 has guanine nucleotide-exchange factor (GEF) activity for RhoA in vitro. In addition, we verified that overexpression of the PLD2-PX domain induces RhoA activation, thereby provoking stress fiber formation. Together, our findings suggest that PLD2 functions as an upstream regulator of RhoA, which enables us to understand how PLD2 regulates cytoskeletal reorganization in a lipase activity-independent manner.