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Tracking the activity-dependent diffusion of synaptic proteins using restricted photoconversion of Dendra2.

Cassé F, Martin S - Front Cell Neurosci (2015)

Bottom Line: Here we describe an elegant method to measure the activity-dependent diffusion of synaptic proteins using Dendra2 photoconversion.We provide a successful method to obtain Dendra2-photoconverted images and a step-by-step procedure to analyze the data.Once the imaging system is set up, data can be acquired in 1-30 min and analyzed in approximately 1-4 h.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique UMR7275 - Laboratory of Excellence "Network for Innovation on Signal Transduction, Pathways in Life Sciences, " Institut de Pharmacologie Moléculaire et Cellulaire, University of Nice - Sophia Antipolis Valbonne, France.

ABSTRACT
Spines are small protrusions on dendritic membranes receiving inputs from axonal termini. They consist in a head connected to the dendritic shaft by a narrow neck and contain multiple synaptic proteins that interact in a coordinated manner to allow for synaptic communication. This process involves many proteins that are moving in and out spines. However, comparing this synaptodendritic movement in basal and stimulated conditions is very challenging. Here we describe an elegant method to measure the activity-dependent diffusion of synaptic proteins using Dendra2 photoconversion. We provide a successful method to obtain Dendra2-photoconverted images and a step-by-step procedure to analyze the data. This live-imaging approach may also apply to investigate the diffusion of proteins across other subcellular compartments or organelles including but not restricted to, nucleus, nucleolus, ER, or vesicular structures. Once the imaging system is set up, data can be acquired in 1-30 min and analyzed in approximately 1-4 h.

No MeSH data available.


Related in: MedlinePlus

Workflow diagram showing the main steps of the photoconversion protocol used to assess the activity-dependent synaptic transport of Dendra2-tagged proteins.
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Figure 1: Workflow diagram showing the main steps of the photoconversion protocol used to assess the activity-dependent synaptic transport of Dendra2-tagged proteins.

Mentions: Measuring the diffusional properties of synaptic proteins in living neurons is still quite challenging experimentally but is essential to assess the regulation of a specific target protein in basal and stimulated conditions. We designed a protocol that provides a basis to directly visualize and compare the synaptic exit of a tagged protein before and after cell activation. We fused the photoactivatable Dendra2 protein to the sole conjugating enzyme of the SUMO system, Ubc9 (Dendra2-Ubc9) in order to visualize in real time, the synaptic exit of this essential enzyme. We then achieved the photoconversion of Dendra2-Ubc9-expressing spines using the photoactivation unit of a spinning disk confocal microscope (UltraView Vox, Perkin Elmer). The photoactivation unit triggers the irradiation of a region of interest (ROI) thereby allowing the spatially restricted photoconversion of Dendra2 proteins. The main steps of our imaging protocol are depicted in Figure 1.


Tracking the activity-dependent diffusion of synaptic proteins using restricted photoconversion of Dendra2.

Cassé F, Martin S - Front Cell Neurosci (2015)

Workflow diagram showing the main steps of the photoconversion protocol used to assess the activity-dependent synaptic transport of Dendra2-tagged proteins.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585026&req=5

Figure 1: Workflow diagram showing the main steps of the photoconversion protocol used to assess the activity-dependent synaptic transport of Dendra2-tagged proteins.
Mentions: Measuring the diffusional properties of synaptic proteins in living neurons is still quite challenging experimentally but is essential to assess the regulation of a specific target protein in basal and stimulated conditions. We designed a protocol that provides a basis to directly visualize and compare the synaptic exit of a tagged protein before and after cell activation. We fused the photoactivatable Dendra2 protein to the sole conjugating enzyme of the SUMO system, Ubc9 (Dendra2-Ubc9) in order to visualize in real time, the synaptic exit of this essential enzyme. We then achieved the photoconversion of Dendra2-Ubc9-expressing spines using the photoactivation unit of a spinning disk confocal microscope (UltraView Vox, Perkin Elmer). The photoactivation unit triggers the irradiation of a region of interest (ROI) thereby allowing the spatially restricted photoconversion of Dendra2 proteins. The main steps of our imaging protocol are depicted in Figure 1.

Bottom Line: Here we describe an elegant method to measure the activity-dependent diffusion of synaptic proteins using Dendra2 photoconversion.We provide a successful method to obtain Dendra2-photoconverted images and a step-by-step procedure to analyze the data.Once the imaging system is set up, data can be acquired in 1-30 min and analyzed in approximately 1-4 h.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique UMR7275 - Laboratory of Excellence "Network for Innovation on Signal Transduction, Pathways in Life Sciences, " Institut de Pharmacologie Moléculaire et Cellulaire, University of Nice - Sophia Antipolis Valbonne, France.

ABSTRACT
Spines are small protrusions on dendritic membranes receiving inputs from axonal termini. They consist in a head connected to the dendritic shaft by a narrow neck and contain multiple synaptic proteins that interact in a coordinated manner to allow for synaptic communication. This process involves many proteins that are moving in and out spines. However, comparing this synaptodendritic movement in basal and stimulated conditions is very challenging. Here we describe an elegant method to measure the activity-dependent diffusion of synaptic proteins using Dendra2 photoconversion. We provide a successful method to obtain Dendra2-photoconverted images and a step-by-step procedure to analyze the data. This live-imaging approach may also apply to investigate the diffusion of proteins across other subcellular compartments or organelles including but not restricted to, nucleus, nucleolus, ER, or vesicular structures. Once the imaging system is set up, data can be acquired in 1-30 min and analyzed in approximately 1-4 h.

No MeSH data available.


Related in: MedlinePlus