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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

Synaptic Dendra2-Ubc9 photoconversion experiments. (A) Time-lapse series of confocal images of photoconverted Dendra2-Ubc9 red fluorescence in spines before and after pharmacological stimulations. Images of spine before Dendra2-Ubc9 photoconversion are shown in green (left). Following the synaptic photoconversion, neurons were incubated with the mGlu5R agonist DHPG (50 μM), with the PKC activator PMA (2 μm) or in control (vehicle) solution for 10 min at 37°C as indicated. Dendra2-Ubc9 from the same spine was then photoconverted a second time and imaged for 20 s. Scale bar, 1 μm. (B). Representative sample paired recording traces of normalized fluorescence values obtained from individual photoconverted Dendra2-Ubc9 expressing spines before (Pre) and after (Post) vehicle, DHPG or PMA treatment as shown in (A). The thin black curves represent the corresponding fits. Note that some parts of this figure derived from Loriol et al. (2014).
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Figure 3: Synaptic Dendra2-Ubc9 photoconversion experiments. (A) Time-lapse series of confocal images of photoconverted Dendra2-Ubc9 red fluorescence in spines before and after pharmacological stimulations. Images of spine before Dendra2-Ubc9 photoconversion are shown in green (left). Following the synaptic photoconversion, neurons were incubated with the mGlu5R agonist DHPG (50 μM), with the PKC activator PMA (2 μm) or in control (vehicle) solution for 10 min at 37°C as indicated. Dendra2-Ubc9 from the same spine was then photoconverted a second time and imaged for 20 s. Scale bar, 1 μm. (B). Representative sample paired recording traces of normalized fluorescence values obtained from individual photoconverted Dendra2-Ubc9 expressing spines before (Pre) and after (Post) vehicle, DHPG or PMA treatment as shown in (A). The thin black curves represent the corresponding fits. Note that some parts of this figure derived from Loriol et al. (2014).

Mentions: Identify a spiny Dendra2-Ubc9-expressing hippocampal neuron (Figures 2, 3). Dendra2-Ubc9 is partly located within the nucleus and also distributed in dendrites and spines (Figure 2). Be sure to select spines of similar shape in you experiments since spine morphology may affect the diffusion rate of synaptic proteins (Simon et al., 2014).


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

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

Synaptic Dendra2-Ubc9 photoconversion experiments. (A) Time-lapse series of confocal images of photoconverted Dendra2-Ubc9 red fluorescence in spines before and after pharmacological stimulations. Images of spine before Dendra2-Ubc9 photoconversion are shown in green (left). Following the synaptic photoconversion, neurons were incubated with the mGlu5R agonist DHPG (50 μM), with the PKC activator PMA (2 μm) or in control (vehicle) solution for 10 min at 37°C as indicated. Dendra2-Ubc9 from the same spine was then photoconverted a second time and imaged for 20 s. Scale bar, 1 μm. (B). Representative sample paired recording traces of normalized fluorescence values obtained from individual photoconverted Dendra2-Ubc9 expressing spines before (Pre) and after (Post) vehicle, DHPG or PMA treatment as shown in (A). The thin black curves represent the corresponding fits. Note that some parts of this figure derived from Loriol et al. (2014).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Synaptic Dendra2-Ubc9 photoconversion experiments. (A) Time-lapse series of confocal images of photoconverted Dendra2-Ubc9 red fluorescence in spines before and after pharmacological stimulations. Images of spine before Dendra2-Ubc9 photoconversion are shown in green (left). Following the synaptic photoconversion, neurons were incubated with the mGlu5R agonist DHPG (50 μM), with the PKC activator PMA (2 μm) or in control (vehicle) solution for 10 min at 37°C as indicated. Dendra2-Ubc9 from the same spine was then photoconverted a second time and imaged for 20 s. Scale bar, 1 μm. (B). Representative sample paired recording traces of normalized fluorescence values obtained from individual photoconverted Dendra2-Ubc9 expressing spines before (Pre) and after (Post) vehicle, DHPG or PMA treatment as shown in (A). The thin black curves represent the corresponding fits. Note that some parts of this figure derived from Loriol et al. (2014).
Mentions: Identify a spiny Dendra2-Ubc9-expressing hippocampal neuron (Figures 2, 3). Dendra2-Ubc9 is partly located within the nucleus and also distributed in dendrites and spines (Figure 2). Be sure to select spines of similar shape in you experiments since spine morphology may affect the diffusion rate of synaptic proteins (Simon et al., 2014).

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