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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

Assessing the potential deleterious effect of the Dendra2 protein tagging. (A) Representative distribution of the endogenously expressed Ubc9 in a 20 DIV rat hippocampal neuron. Fixed cells were permeabilised for 20 min at RT in PBS containing 0.1% Triton X100 and 10% Horse Serum (HS). Neurons were then immunolabelled with mouse anti-Ubc9 (1/50, BD Bioscience) and rabbit anti-Homer1 (1/200; Synaptic System, Germany) antibodies overnight at 4°C in PBS containing 0.05% Triton X100 and 5% HS. Cells were washed three times in PBS and incubated with the appropriate secondary antibodies conjugated to either Alexa488 (post-synaptic Homer staining) or Alexa594 (endogenous Ubc9) in PBS containing 5% HS with 0.05% Triton X100 for 1 h at RT. Merge color (yellow) shows the colocalisation between the proteins indicating that part of Ubc9 is expressed at Homer-positive post-synaptic sites (arrowheads). Note that, as expected, a significant proportion of Ubc9 immunoreactivity is present in the nucleus. Scale bar, 20 μm. (B) Representative image of a 20 DIV Dendra2-Ubc9-expressing rat hippocampal neuron. Note that Dendra2-Ubc9 is partly localized in the nucleus (inset) indicating that the Dendra2 tag does not impair its nuclear translocation. Dendra2-Ubc9 fluorescence is also distributed in dendrites and spines (circle). Scale bar, 20 μm.
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Figure 2: Assessing the potential deleterious effect of the Dendra2 protein tagging. (A) Representative distribution of the endogenously expressed Ubc9 in a 20 DIV rat hippocampal neuron. Fixed cells were permeabilised for 20 min at RT in PBS containing 0.1% Triton X100 and 10% Horse Serum (HS). Neurons were then immunolabelled with mouse anti-Ubc9 (1/50, BD Bioscience) and rabbit anti-Homer1 (1/200; Synaptic System, Germany) antibodies overnight at 4°C in PBS containing 0.05% Triton X100 and 5% HS. Cells were washed three times in PBS and incubated with the appropriate secondary antibodies conjugated to either Alexa488 (post-synaptic Homer staining) or Alexa594 (endogenous Ubc9) in PBS containing 5% HS with 0.05% Triton X100 for 1 h at RT. Merge color (yellow) shows the colocalisation between the proteins indicating that part of Ubc9 is expressed at Homer-positive post-synaptic sites (arrowheads). Note that, as expected, a significant proportion of Ubc9 immunoreactivity is present in the nucleus. Scale bar, 20 μm. (B) Representative image of a 20 DIV Dendra2-Ubc9-expressing rat hippocampal neuron. Note that Dendra2-Ubc9 is partly localized in the nucleus (inset) indicating that the Dendra2 tag does not impair its nuclear translocation. Dendra2-Ubc9 fluorescence is also distributed in dendrites and spines (circle). Scale bar, 20 μm.

Mentions: Note: It is essential to verify the functionality of the tagged protein and to show that the selected tag does not impair the subcellular localization (Figure 2) and the overall activity of the studied protein (Loriol et al., 2014).


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

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

Assessing the potential deleterious effect of the Dendra2 protein tagging. (A) Representative distribution of the endogenously expressed Ubc9 in a 20 DIV rat hippocampal neuron. Fixed cells were permeabilised for 20 min at RT in PBS containing 0.1% Triton X100 and 10% Horse Serum (HS). Neurons were then immunolabelled with mouse anti-Ubc9 (1/50, BD Bioscience) and rabbit anti-Homer1 (1/200; Synaptic System, Germany) antibodies overnight at 4°C in PBS containing 0.05% Triton X100 and 5% HS. Cells were washed three times in PBS and incubated with the appropriate secondary antibodies conjugated to either Alexa488 (post-synaptic Homer staining) or Alexa594 (endogenous Ubc9) in PBS containing 5% HS with 0.05% Triton X100 for 1 h at RT. Merge color (yellow) shows the colocalisation between the proteins indicating that part of Ubc9 is expressed at Homer-positive post-synaptic sites (arrowheads). Note that, as expected, a significant proportion of Ubc9 immunoreactivity is present in the nucleus. Scale bar, 20 μm. (B) Representative image of a 20 DIV Dendra2-Ubc9-expressing rat hippocampal neuron. Note that Dendra2-Ubc9 is partly localized in the nucleus (inset) indicating that the Dendra2 tag does not impair its nuclear translocation. Dendra2-Ubc9 fluorescence is also distributed in dendrites and spines (circle). Scale bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Assessing the potential deleterious effect of the Dendra2 protein tagging. (A) Representative distribution of the endogenously expressed Ubc9 in a 20 DIV rat hippocampal neuron. Fixed cells were permeabilised for 20 min at RT in PBS containing 0.1% Triton X100 and 10% Horse Serum (HS). Neurons were then immunolabelled with mouse anti-Ubc9 (1/50, BD Bioscience) and rabbit anti-Homer1 (1/200; Synaptic System, Germany) antibodies overnight at 4°C in PBS containing 0.05% Triton X100 and 5% HS. Cells were washed three times in PBS and incubated with the appropriate secondary antibodies conjugated to either Alexa488 (post-synaptic Homer staining) or Alexa594 (endogenous Ubc9) in PBS containing 5% HS with 0.05% Triton X100 for 1 h at RT. Merge color (yellow) shows the colocalisation between the proteins indicating that part of Ubc9 is expressed at Homer-positive post-synaptic sites (arrowheads). Note that, as expected, a significant proportion of Ubc9 immunoreactivity is present in the nucleus. Scale bar, 20 μm. (B) Representative image of a 20 DIV Dendra2-Ubc9-expressing rat hippocampal neuron. Note that Dendra2-Ubc9 is partly localized in the nucleus (inset) indicating that the Dendra2 tag does not impair its nuclear translocation. Dendra2-Ubc9 fluorescence is also distributed in dendrites and spines (circle). Scale bar, 20 μm.
Mentions: Note: It is essential to verify the functionality of the tagged protein and to show that the selected tag does not impair the subcellular localization (Figure 2) and the overall activity of the studied protein (Loriol 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