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Related Concept Videos

SNAREs and Membrane Fusion01:43

SNAREs and Membrane Fusion

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Once a transport vesicle has recognized its target organelle, the vesicular membrane needs to fuse with the target membrane to unload the cargo. Transmembrane proteins called SNAREs present on organelle membranes and their vesicles, mediate vesicle fusion.
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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Once a ligand binds to a receptor, the signal is transmitted through the membrane and into the cytoplasm. The continuation of a signal in this manner is called signal transduction. Signal transduction only occurs with cell-surface receptors, which cannot interact with most components of the cell, such as DNA. Only internal receptors can interact directly with DNA in the nucleus to initiate protein synthesis. When a ligand binds to its receptor, conformational changes occur that affect the...
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Total Internal Reflection Fluorescence Microscopy01:05

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
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Related Experiment Video

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Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy
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Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy.

Daniëlle R J Verboogen1, Maksim V Baranov1, Martin Ter Beest1

  • 1Department of Tumor Immunology, Radboud University Medical Center.

Journal of Visualized Experiments : Jove
|January 25, 2018
PubMed
Summary

This study introduces a novel FRET-FLIM method to visualize Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex formation in live cells, overcoming challenges of protein abundance and redundancy.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biophysics

Background:

  • Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are crucial for membrane fusion and intracellular trafficking in eukaryotic cells.
  • Studying SNARE protein function is challenging due to their high abundance, functional redundancy, and overlapping roles.
  • Specific SNARE protein sets mediate distinct intracellular transport routes, ensuring specificity and fidelity.

Purpose of the Study:

  • To develop and present a new protocol for visualizing SNARE complex formation in live cells.
  • To enable the study of SNARE protein interactions and their role in membrane trafficking.
  • To overcome technical challenges associated with SNARE protein abundance and functional redundancy.

Main Methods:

  • Expressing SNARE proteins C-terminally fused to fluorescent proteins.
  • Utilizing Förster resonance energy transfer (FRET) in conjunction with fluorescence lifetime imaging microscopy (FLIM).
  • Analyzing FRET-FLIM data by fitting fluorescence lifetime histograms with a multicomponent decay model for quantitative estimation.

Main Results:

  • Successfully visualized SNARE complex formation at the plasma membrane and endosomal compartments in mammalian cells.
  • Enabled semi-quantitative estimation of SNARE complex formation fractions at different vesicles.
  • Demonstrated the applicability in mammalian cell lines and primary immune cells.

Conclusions:

  • The developed FRET-FLIM protocol provides a powerful tool for visualizing SNARE complex formation in live cells.
  • This method facilitates the study of SNARE protein function and membrane trafficking dynamics.
  • The protocol is adaptable for investigating SNAREs in various organelles across different species, including plants and fungi.