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

Die zelluläre Dynamik fluoreszenzmarkierter Proteine kann mit fortschrittlichen mikroskopischen Techniken wie FRAP, SPT und FRET untersucht werden.
Bei der Fluoreszenzwiederherstellung nach Photobleichen oder FRAP wird eine kleine Region, die Proteine von Interesse enthält, mit einem fokussierten Laserstrahl bestrahlt, um die Fluorophore zu photobleichen.
Wenn markierte Proteine in den gebleichten Bereich diffundieren, wird die Region wieder fluoreszierend. Die Rate der Fluoreszenzrückgewinnung bestimmt die Diffusionsrate der Proteine.

Beim Single-Particle Tracking (SPT) werden Proteine verwendet, die mit fluorophorgebundenen Antikörpern markiert sind. Die Bewegung einzelner Proteine wird mit einem computergestützten Videomikroskop verfolgt.
Der Forster-Resonanzenergietransfer (FRET) misst die Nähe von zwei Fluorophor-markierten Proteinen. Wenn der Abstand mehr als zehn Nanometer beträgt, findet keine Fluoreszenz statt. Nähern sich die Proteine, führt die Anregung des Donorfluorophors zu einem Energietransfer zum Akzeptorfluorophor.
 Der Energietransfer reduziert die Fluoreszenz des Donos und erhöht gleichzeitig die Emission des Akzeptors, wodurch deren Visualisierung ermöglicht wird.

<ol>
	<li>Lippincott-Schwartz, J., Snapp, E.L. und Phair, RD, 2018. Die Entwicklung und Weiterentwicklung von FRAP als Schlüsselwerkzeug zur Untersuchung der Proteindynamik. Biophysikalische Zeitschrift, 115(7), S.1146-1155.</li>
	<li>Takanishi, C.L., Bykova, E.A., Cheng, W. und Zheng, J., 2006. GFP-basierte FRET-Analyse in lebenden Zellen. Hirnforschung, 1091(1), S.132-139.</li>
	<li>Pantazis, A., Westerberg, K., Althoff, T., Abramson, J. und Olcese, R., 2018. Nutzung des photoinduzierten Elektronentransfers zur optischen Bestimmung von atomaren Abständen von Proteinen im Sub-Nanobereich. Naturkommunikation, 9(1), S.1-12.</li>
</ol>

proteindynamik in lebenden zellen

Protein Dynamics in Living Cells

Zur Untersuchung der Proteindynamik in lebenden Zellen werden verschiedene fluoreszenzbasierte Techniken eingesetzt. Zu diesen Techniken gehören FRAP, FRET und PET.

Fluorescent Recovery After Photobleaching (FRAP) ist eine auf Fluoreszenzproteinen basierende Detektionstechnik, die zur Quantifizierung der Proteinbewegungsraten innerhalb der Zelle verwendet wird. Bei dieser Methode wird ein kleiner Teil der Zelle einem intensiven Laserstrahl ausgesetzt. Der Laserstrahl bewirkt eine dauerhafte Photobleichung der Fluorophor-markierten Proteine im belichteten Bereich. Wenn die gebleichten Proteine im Laufe der Zeit herausdiffundieren und fluoreszierend markierte Proteine aus anderen Teilen der Zelle eindringen, wird die Region wieder fluoreszierend. Die Bewegungsgeschwindigkeit wird durch Auftragen der relativen Fluoreszenzintensität gegen die benötigte Zeit quantifiziert, wodurch die Geschwindigkeit bestimmt werden kann, mit der sich Protein innerhalb der Zelle bewegt.

Der Förster-Resonanzenergietransfer (FRET) ist eine molekulare Technik zur Bestimmung des Abstands zwischen zwei Proteinen. Somit kann FRET als molekulares Lineal verwendet werden. Bei der Technik werden zwei mit einem Fluorophor markierte Proteine verwendet, von denen eines als Donor und das andere als Akzeptor fungiert. Bei Anregung mit einer bestimmten Wellenlänge emittiert der Donor-Fluorophor Fluoreszenzenergie, die vom Akzeptor-Fluorophor absorbiert wird. Der Donor-Fluorophor kehrt bei der Energieübertragung in den Grundzustand zurück, während der Akzeptor-Fluorophor Fluoreszenz emittiert, die mit einem Fluoreszenzmikroskop sichtbar gemacht wird. Die FRET-Technik hängt von drei Faktoren ab: dem Abstand zwischen interagierenden Proteinen, dem Ausmaß der spektralen Überlappung zwischen den Donor- und Akzeptor-Fluorophoren und der Ausrichtung der Donor- und Akzeptor-Fluorophore während der Energieübertragung. Der Energietransfer zwischen den korrekt ausgerichteten Donor- und Akzeptor-Fluorophoren kann nur erfolgen, wenn der Abstand zwischen den beiden interagierenden Proteinen 10 nm oder weniger beträgt.

Der photoinduzierte Elektronentransfer oder PET bestimmt den subatomaren Abstand zwischen Proteinen in einer Zelle. Bei der PET absorbiert der Fluorophor das Licht und sendet über ein angeregtes Elektron ein Fluoreszenzsignal aus. Das angeregte Elektron wird auf den Rezeptor übertragen. Während der Energieübertragung findet eine Redoxreaktion statt, die eine Ladungstrennung zwischen den Donor- und Akzeptorproteinen erzeugt.

Proteindynamik in lebenden Zellen

Verschiedene fluoreszenzbasierte Techniken werden verwendet, um die Proteindynamik in lebenden Zellen zu untersuchen. Zu diesen Techniken gehören FRAP, FRET und PET.
Fluoreszierend...

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Lehre

JoVE Core

Cell Biology

Zellbiologie

Unit 1

Einheit 1

Visualizing Cells, Tissues, and Molecules

Visualisierung von Zellen, Geweben und Molekülen

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

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

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Fluorescent Methods for Protein Movement

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