Name: fluorescent methods for protein movement
Uploaded: 2023-04-30T12:00:21.000Z
Duration: 1 min 19 s
Channel: JoVE

A dinâmica celular de proteínas marcadas com fluorescência pode ser estudada usando técnicas microscópicas avançadas como FRAP, SPT e FRET.
Na recuperação de fluorescência após fotobranqueamento ou FRAP, uma pequena região contendo proteínas de interesse é irradiada com um feixe de laser focalizado para fotobranquear os fluoróforos.
À medida que as proteínas marcadas se difundem na área branqueada, a região torna-se fluorescente novamente. A taxa de recuperação de fluorescência determina a taxa de difusão das proteínas.

O rastreamento de partícula única ou SPT envolve proteínas marcadas com anticorpos ligados ao fluoróforo. O movimento individual da proteína é rastreado usando um microscópio de vídeo aprimorado por computador.
A transferência de energia de ressonância de Forster ou FRET mede a proximidade de duas proteínas marcadas com fluoróforo. Quando a distância é superior a dez nanômetros, não ocorre fluorescência. Se as proteínas se aproximarem, excitar o fluoróforo doador leva à transferência de energia para o fluoróforo aceptor.
 A transferência de energia reduz a fluorescência do dono enquanto aumenta a emissão do aceptor, permitindo sua visualização.

<ol>
	<li>Lippincott-Schwartz, J., Snapp, E.L. e Phair, R.D., 2018. O desenvolvimento e aprimoramento do FRAP como uma ferramenta chave para investigar a dinâmica de proteínas. Revista biofísica, 115(7), pp.1146-1155.</li>
	<li>Takanishi, C.L., Bykova, E.A., Cheng, W. e Zheng, J., 2006. Análise de FRET baseada em GFP em células vivas. Pesquisa do cérebro, 1091(1), pp.132-139.</li>
	<li>Pantazis, A., Westerberg, K., Althoff, T., Abramson, J. e Olcese, R., 2018. Aproveitando a transferência de elétrons fotoinduzida para determinar opticamente as distâncias atômicas em sub-nanoescala de proteínas. Comunicações da natureza, 9(1), pp.1-12.</li>
</ol>

dinâmica de proteínas em células vivas

Protein Dynamics in Living Cells

Diferentes técnicas baseadas em fluorescência são usadas para estudar a dinâmica das proteínas em células vivas. Essas técnicas incluem FRAP, FRET e PET. A recuperação fluorescente após fotobranqueamento (FRAP) é uma técnica de detecção baseada em proteínas fluorescentes usada para quantificar as taxas de movimento de proteínas dentro da célula. Este método expõe uma pequena porção da célula a um intenso feixe de laser. O feixe de laser causa fotodegradação permanente das proteínas marcadas com fluoróforo na região exposta. À medida que as proteínas branqueadas se difundem ao longo do tempo e as proteínas marcadas com fluorescência de outras partes da célula se movem, a região torna-se fluorescente novamente. A taxa de movimento é quantificada traçando a intensidade relativa da fluorescência versus o tempo necessário, o que permite determinar a taxa com que a proteína se move dentro da célula. A transferência de energia de ressonância de Förster (FRET) é uma técnica molecular usada para determinar a distância entre duas proteínas. Assim, a FRET pode ser usada como régua molecular. A técnica utiliza duas proteínas marcadas com fluoróforo, uma atuando como doadora e outra como aceitadora. Após excitação em um comprimento de onda especificado, o fluoróforo doador emite energia de fluorescência que é absorvida pelo fluoróforo aceitador. O fluoróforo doador retorna ao estado fundamental após a transferência de energia, enquanto o fluoróforo aceitador emite fluorescência que é visualizada com um microscópio de fluorescência. A técnica FRET depende de três fatores: a distância entre as proteínas que interagem, a extensão da sobreposição espectral entre os fluoróforos doadores e aceitadores, e a orientação do fluoróforo doador e aceitador durante a transferência de energia. A transferência de energia entre os fluoróforos doador e aceitador corretamente orientados só pode ocorrer quando a distância entre as duas proteínas que interagem é de 10 nm ou menos. A transferência de elétrons fotoinduzida, ou PET, determina a distância subatômica entre as proteínas em uma célula. Na PET, o fluoróforo absorve a luz e emite um sinal de fluorescência através de um elétron excitado. O elétron excitado é transferido para o receptor. Durante a transferência de energia, ocorre uma reação redox, gerando separação de carga entre as proteínas doadoras e receptoras.

Dinâmica de Proteínas em Células Vivas

Diferentes técnicas baseadas em fluorescência são usadas para estudar a dinâmica de proteínas em células vivas. Essas técnicas incluem FRAP, FRET e PET.
Fluorescente...

Education

JoVE Core

Cell Biology

Biologia Celular

Unit 1

Unidade 1

Visualizing Cells, Tissues, and Molecules

Visualizando Células, Tecidos e Moléculas

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

fluorescent methods for protein movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell.

Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away and fluorescent proteins from other parts of the cell move in. The region then becomes fluorescent again, and the change in intensity is plotted against time to calculate how fast the protein moves.

Fӧrster resonance energy transfer, or FRET, measures the distance between two proteins and can act like a molecular ruler. It uses two fluorophore-tagged proteins, a donor and an acceptor. When the donor is excited at a specific wavelength, it transfers fluorescence energy to the acceptor. The donor returns to the ground state, and the acceptor emits fluorescence that can be seen with a fluorescence microscope. FRET depends on distance between the proteins, spectral overlap between the fluorophores, and their orientation during energy transfer. This transfer occurs only when the interacting proteins are 10 nm apart or less.

Photoinduced electron transfer, or PET, measures sub-atomic distance between proteins in a cell. In PET, the fluorophore absorbs light and sends out a fluorescence signal through an excited electron. That electron is transferred to a receptor. During this transfer, a redox reaction creates charge separation between the donor and acceptor proteins.

Fluorescent Methods for Protein Movement

imaging biological samples with optical microscopy

imaging-biological-samples-with-optical-microscopy

How Light Microscopes Bring Samples Into View

Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution. A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The...

phase contrast and differential interference contrast microscopy

phase-contrast-and-differential-interference-contrast-microscopy

Microscope Contrast for Live, Unstained Cells

Phase-contrast and differential interference contrast microscopy help scientists see live cells without stains. Both methods boost contrast so thin, transparent structures become easier to study under the microscope. Phase-contrast microscopes use interference between light that passes straight through a cell and light that is refracted by cell parts. The microscope uses an annular stop in the condenser to make a hollow cone of light. The objective lens also contains a phase plate with a phase...

fixation and sectioning

Microscope Slide Prep and Staining

Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures. A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes...

immunofluorescence microscopy

Fluorescent Antibody Staining in Cells

Fluorescent antibody staining uses a fluorescence microscope to make cells and microbes visible in bright colors against a dark background. The microscope works with fluorochromes, which are fluorescent dyes that absorb energy from a light source and then give off visible light. Some fluorochromes are natural, such as chlorophylls. Others are added to the specimen to improve contrast. Common examples of fluorochromes include Texas red and FITC. The method can also use nucleic acid dyes such as...

immunocytochemistry and immunohistochemistry

immunocytochemistry-and-immunohistochemistry

Antibody Staining in Cells and Tissues

Immunocytochemistry (ICC) and immunohistochemistry (IHC) use antibodies to detect specific proteins or antigens in samples. ICC focuses on individual cells, such as blood cells and stem cells. IHC is used for tissue samples. These methods were first published by Albert Coons in 1941, when they were used to detect pneumococcal antigen in mouse tissue sections from mice infected with Pneumococcus. Both techniques can produce either a colored signal or a fluorescent signal. Enzyme-conjugated...

confocal fluorescence microscopy

Confocal Microscopy for Sharp Cell Images

Confocal microscopy is an advanced imaging method used to capture sharp fluorescence images. Its main advantage is that it blocks out-of-focus light from the sample with pinholes. That helps produce high-resolution images with strong contrast. Unlike a standard optical microscope, a confocal microscope uses a focused laser beam to scan the sample surface at different z-planes. This makes it especially useful for thick specimens, such as biofilms. These samples can often be examined alive and...

protein dynamics in living cells

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

total internal reflection fluorescence microscopy

total-internal-reflection-fluorescence-microscopy

Surface-Only Imaging with TIRF Microscopy

Total internal reflection fluorescence microscopy, or TIRF, is a microscope method used to view fluorophores near a solid surface with a higher refractive index, such as a glass coverslip. It is useful when scientists want to study signals close to the cell surface. TIRF works when light from a medium with a lower refractive index, such as air, hits the glass coverslip at a critical angle. Instead of entering the glass, the light undergoes total internal reflection. This reflection creates an...

atomic force microscopy

Atomic Force Microscopy for Surface Mapping

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images. The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends...

super-resolution fluorescence microscopy

Sharper Cell Images with Fluorescence Microscopy

Super-resolution fluorescence microscopy gives sharper images than standard fluorescence microscopy by reducing the point spread function, or PSF. The PSF is the light pattern from a point source that makes it look blurred. Because of this blur, each fluorescent spot appears larger than it really is. Nearby fluorophores can also overlap through their PSFs and make the image less clear. Several super-resolution methods have been developed to improve image detail. Photoactivated localization...

overview of electron microscopy

Electron Microscopy: TEM vs SEM

Electron microscopy uses electrons instead of visible light to make images with much higher detail than a light microscope. Visible light limits the resolution of light microscopes, which usually magnify up to 1000X, and sometimes up to 1500X. Electrons have a much shorter wavelength, about 0.005 nm, so they can produce resolution as fine as 0.05 nm. An electron microscope can also magnify images up to 2,000,000X and reveal subcellular structures and some molecular structures, including single...

scanning electron microscopy

How SEM Reveals Surface Details

A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

Transmission electron microscopy uses electrons to reveal very small structures inside a specimen. The technique grew from the first electron microscope prototype developed in 1931 by physicist Ernst Ruska. He built on the idea that magnetic fields can direct an electron beam, just as lenses direct light in an optical microscope. In a transmission electron microscope, a hot tungsten element produces electrons. An electron gun then accelerates them with a potential difference, giving the beam...

preparation of samples for electron microscopy

preparation-of-samples-for-electron-microscopy

Electron Microscopy Sample Prep Methods

Electron microscopy sample prep methods help biological samples survive imaging in TEM and SEM. Before a specimen can be viewed, it must be fixed, or stabilized, so the electron beam does not damage it. It also must be dried well, or desiccated, because the microscope uses a vacuum. Fixation should happen as quickly as possible. Once a sample leaves its natural environment, its properties begin to change. In tissue samples, oxygen levels can drop and change the appearance of mitochondria. That...

immunogold electron microscopy

Immunogold Labeling in Electron Microscopy

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions. The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins...

cryo-electron microscopy

Cryo-EM for Native Biomolecule Imaging

Cryo-EM, or cryo-electron microscopy, lets scientists image biomolecules in their frozen, native state. It avoids the dehydration, fixation, and staining used in conventional electron microscopy, which can distort samples and create artifacts. It also reduces damage from the electron beam by using gentler imaging conditions. This method is useful when other structure tools have limits. X-ray diffraction needs a molecule to form a crystal, and that is not always possible. Even when...

electron microscope tomography and single-particle reconstruction

electron-microscope-tomography-and-single-particle-reconstruction

3D Imaging with Electron Microscopy

Transmission electron microscopy (TEM) can build 3D images of biological samples. It does this with electron microscope tomography and single-particle reconstruction. Tomography can show cell parts or small cells in vivo, while single-particle reconstruction is used for macromolecules and macromolecular complexes in vitro only. Electron tomography can be done in TEM or in STEM, which stands for scanning transmission electron microscopy. STEM is mainly used for thick biological specimens. It...

key terms and concepts

Learn how FRAP, FRET, and PET use fluorescence to measure protein movement, protein distance, and protein interactions in living cells.

Fluorescent Methods for Protein Movement | JoVE K12

cell biology

JoVE Core - Biologia Celular ilustra processos celulares complexos em vídeos de animação fáceis de entender e, ao mesmo tempo, abrangentes. Esta coleção foca nos componentes e funções essenciais das células, incluindo organelas e membranas, transporte celular e energia celular. É complementado por vídeos de Cientistas em ação que demonstram a aplicação da biologia celular em experimentos de pesquisa do mundo real.

Fluorescent Methods for Protein Movement

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