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

La dynamique cellulaire des protéines marquées par fluorescence peut être étudiée à l’aide de techniques microscopiques avancées telles que FRAP, SPT et FRET.
Dans la récupération de fluorescence après photoblanchiment ou FRAP, une petite région contenant des protéines d’intérêt est irradiée avec un faisceau laser focalisé pour photoblanchir les fluorophores.
Au fur et à mesure que les protéines marquées se diffusent dans la zone blanchie, la région redevient fluorescente. Le taux de récupération de la fluorescence détermine le taux de diffusion des protéines.

Le suivi à particule unique ou SPT implique des protéines marquées avec des anticorps liés aux fluorophores. Le mouvement individuel des protéines est suivi à l’aide d’un microscope vidéo assisté par ordinateur.
Le transfert d’énergie par résonance de Forster ou FRET mesure la proximité de deux protéines marquées au fluorophore. Lorsque la distance est supérieure à dix nanomètres, il n’y a pas de fluorescence. Si les protéines se rapprochent, l’excitation du fluorophore donneur entraîne un transfert d’énergie vers le fluorophore accepteur.
 Le transfert d'énergie réduit la fluorescence du dono tout en augmentant l'émission de l'accepteur, permettant leur visualisation.

<ol>
	<li>Lippincott-Schwartz, J., Snapp, E.L. et Phair, R.D., 2018. Le développement et l’amélioration de FRAP en tant qu’outil clé pour l’étude de la dynamique des protéines. Revue biophysique, 115(7), pp.1146-1155.</li>
	<li>Takanishi, C.L., Bykova, E.A., Cheng, W. et Zheng, J., 2006. Analyse FRET basée sur la GFP dans des cellules vivantes. Recherche sur le cerveau, 1091(1), pp.132-139.</li>
	<li>Pantazis, A., Westerberg, K., Althoff, T., Abramson, J. et Olcese, R., 2018. Exploiter le transfert d’électrons photoinduit pour déterminer optiquement les distances atomiques subnanométriques des protéines. Nature communications, 9(1), pp.1-12.</li>
</ol>

dynamique des protéines dans les cellules vivantes

Protein Dynamics in Living Cells

Différentes techniques basées sur la fluorescence sont utilisées pour étudier la dynamique des protéines dans les cellules vivantes. Ces techniques incluent FRAP, FRET et PET.

La récupération fluorescente après photoblanchiment (FRAP) est une technique de détection basée sur les protéines fluorescentes utilisée pour quantifier les taux de mouvement des protéines dans la cellule. Cette méthode expose une petite partie de la cellule à un faisceau laser intense. Le faisceau laser provoque un photoblanchiment permanent des protéines marquées par un fluorophore dans la région exposée. À mesure que les protéines blanchies se diffusent au fil du temps et que des protéines marquées par fluorescence provenant d’autres parties de la cellule s’y installent, la région redevient fluorescente. La vitesse de mouvement est quantifiée en traçant l’intensité relative de fluorescence en fonction du temps nécessaire, ce qui permet de déterminer la vitesse à laquelle la protéine se déplace dans la cellule.

Le transfert d'énergie par résonance de Förster (FRET) est une technique moléculaire utilisée pour déterminer la distance entre deux protéines. Ainsi, FRET peut être utilisé comme règle moléculaire. La technique utilise deux protéines marquées par un fluorophore, l’une agissant comme donneur et l’autre comme accepteur. Lors d'une excitation à une longueur d'onde spécifiée, le fluorophore donneur émet une énergie de fluorescence qui est absorbée par le fluorophore accepteur. Le fluorophore donneur revient à l'état fondamental lors du transfert d'énergie, tandis que le fluorophore accepteur émet une fluorescence qui est visualisée avec un microscope à fluorescence. La technique FRET dépend de trois facteurs, la distance entre les protéines en interaction, l’étendue du chevauchement spectral entre les fluorophores donneur et accepteur et l’orientation du fluorophore donneur et accepteur pendant le transfert d’énergie. Le transfert d’énergie entre les fluorophores donneurs et accepteurs correctement orientés ne peut se produire que lorsque la distance entre les deux protéines en interaction est de 10 nm ou moins.

Le transfert d'électrons photoinduit ou PET détermine la distance subatomique entre les protéines d'une cellule. En PET, le fluorophore absorbe la lumière et émet un signal de fluorescence via un électron excité. L'électron excité est transféré au récepteur. Lors du transfert d'énergie, une réaction redox se produit, générant une séparation de charge entre les protéines donneuses et acceptrices.

Dynamique des Protéines dans les Cellules Vivantes

Différentes techniques basées sur la fluorescence sont utilisées pour étudier la dynamique des protéines dans les cellules vivantes. Ces techniques comprennent FRAP, FRET et PET.
Fluorescent...

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Visualizing Cells, Tissues, and Molecules

Visualisation des cellules, des tissus et des molécules

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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

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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...

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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 Biologie cellulaire illustre les processus cellulaires complexes dans des leçons vidéos complètes mais faciles à comprendre. Cette collection se concentre sur les composants et fonctions essentiels de la cellule ; cela inclut les organelles et les membranes ainsi que le transport et le métabolisme énergétique cellulaire. Elle est complétée par nos vidéos de scientifiques à l'action qui démontrent l'application de la biologie cellulaire dans des expériences concrètes.

Fluorescent Methods for Protein Movement

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