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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

Computational analyses of synaptic Dendra2-Ubc9 photoconversion data. (A) Scatter plots of computed half-time of photoconverted Dendra2-Ubc9 fluorescence diffusion in spines pre- and post- pharmacological treatment. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (B). Histograms showing the mean ± s.e.m. of the fluorescence half-time constant in seconds calculated for the exponential decay fit from independent synaptic Dendra2-Ubc9 photoconversion experiments as shown in Figure 3B. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (C). Scatter plots showing the mean of half-time constant ratio of (Post/Pre) in control Vehicle (1.001 ± 0.025; n = 21), DHPG (1.824 ± 0.156; n = 23) and PMA (1.511 ± 0.078; n = 22) conditions. One-Way ANOVA were performed with a Newman-Keuls post-test for multiple comparison data sets. **P < 0.01; ***P < 0.001. These data indicate that the activation of the PKC pathway is sufficient to transiently promote the diffusional trapping of Dendra2-Ubc9 in spines. Note that some parts of this figure derived from Loriol et al. (2014).
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Figure 4: Computational analyses of synaptic Dendra2-Ubc9 photoconversion data. (A) Scatter plots of computed half-time of photoconverted Dendra2-Ubc9 fluorescence diffusion in spines pre- and post- pharmacological treatment. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (B). Histograms showing the mean ± s.e.m. of the fluorescence half-time constant in seconds calculated for the exponential decay fit from independent synaptic Dendra2-Ubc9 photoconversion experiments as shown in Figure 3B. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (C). Scatter plots showing the mean of half-time constant ratio of (Post/Pre) in control Vehicle (1.001 ± 0.025; n = 21), DHPG (1.824 ± 0.156; n = 23) and PMA (1.511 ± 0.078; n = 22) conditions. One-Way ANOVA were performed with a Newman-Keuls post-test for multiple comparison data sets. **P < 0.01; ***P < 0.001. These data indicate that the activation of the PKC pathway is sufficient to transiently promote the diffusional trapping of Dendra2-Ubc9 in spines. Note that some parts of this figure derived from Loriol et al. (2014).

Mentions: Y is the F/F0 value; Plateau is the Y-value at infinite time; K is the rate constant, expressed in reciprocal of the X-axis time units. The half-time is in the time units of the X axis and is computed as ln(2)/K. To compare stimulated to unstimulated control conditions, calculate the ratio of half-time constant for each experiment by dividing the T1/2 obtained after treatment by the control T1/2 [Ratio = T1/2 (post-treatment)/T1/2 (pre-treatment); Figure 4].


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

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

Computational analyses of synaptic Dendra2-Ubc9 photoconversion data. (A) Scatter plots of computed half-time of photoconverted Dendra2-Ubc9 fluorescence diffusion in spines pre- and post- pharmacological treatment. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (B). Histograms showing the mean ± s.e.m. of the fluorescence half-time constant in seconds calculated for the exponential decay fit from independent synaptic Dendra2-Ubc9 photoconversion experiments as shown in Figure 3B. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (C). Scatter plots showing the mean of half-time constant ratio of (Post/Pre) in control Vehicle (1.001 ± 0.025; n = 21), DHPG (1.824 ± 0.156; n = 23) and PMA (1.511 ± 0.078; n = 22) conditions. One-Way ANOVA were performed with a Newman-Keuls post-test for multiple comparison data sets. **P < 0.01; ***P < 0.001. These data indicate that the activation of the PKC pathway is sufficient to transiently promote the diffusional trapping of Dendra2-Ubc9 in spines. Note that some parts of this figure derived from Loriol et al. (2014).
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Figure 4: Computational analyses of synaptic Dendra2-Ubc9 photoconversion data. (A) Scatter plots of computed half-time of photoconverted Dendra2-Ubc9 fluorescence diffusion in spines pre- and post- pharmacological treatment. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (B). Histograms showing the mean ± s.e.m. of the fluorescence half-time constant in seconds calculated for the exponential decay fit from independent synaptic Dendra2-Ubc9 photoconversion experiments as shown in Figure 3B. Paired t-test: Control Vehicle, n = 21; DHPG, n = 23; PMA, n = 22. n.s., not significant; ***P < 0.0001 compared with unstimulated (Pre) conditions. (C). Scatter plots showing the mean of half-time constant ratio of (Post/Pre) in control Vehicle (1.001 ± 0.025; n = 21), DHPG (1.824 ± 0.156; n = 23) and PMA (1.511 ± 0.078; n = 22) conditions. One-Way ANOVA were performed with a Newman-Keuls post-test for multiple comparison data sets. **P < 0.01; ***P < 0.001. These data indicate that the activation of the PKC pathway is sufficient to transiently promote the diffusional trapping of Dendra2-Ubc9 in spines. Note that some parts of this figure derived from Loriol et al. (2014).
Mentions: Y is the F/F0 value; Plateau is the Y-value at infinite time; K is the rate constant, expressed in reciprocal of the X-axis time units. The half-time is in the time units of the X axis and is computed as ln(2)/K. To compare stimulated to unstimulated control conditions, calculate the ratio of half-time constant for each experiment by dividing the T1/2 obtained after treatment by the control T1/2 [Ratio = T1/2 (post-treatment)/T1/2 (pre-treatment); Figure 4].

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