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FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

Doré K, Labrecque S, Tardif C, De Koninck P - PLoS ONE (2014)

Bottom Line: We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction.The activity of both CaMKII and calpain were essential for this effect in both developmental stages.Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

View Article: PubMed Central - PubMed

Affiliation: Institut Universitaire en Santé Mentale de Québec, Université Laval, Québec, QC, Canada.

ABSTRACT
Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

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GluN1-GFP and PSD95-mCherry are specific probes for measuring the interaction between PSD95 and the NMDAR with FRET-FLIM.(A) FLIM images of GluN1-GFP expressing spines, expressed alone (top row), with mCherry (second row), with PSD95-mCherry (third row) or Homer-mCherry (last row). Scale bar is 1 µm (placed in first spine). Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. Black crosses indicate the pixel selected for traces in B. Circles indicate ROI for panel C. (B) Fit curves of fluorescence intensity decays obtained from one pixel (black crosses in the spines shown in A) for a GluN1-GFP expressing spine (green curve) and GluN1-GFP/PSD95-mCherry expressing spine (black curve), see Fig. S1 in file S1 for raw data. (C) Distribution histograms of GluN1-GFP lifetimes in encircled spines in A (GluN1-GFP, green; GluN1-GFP/PSD95-mCherry, gray), the median (dotted line) of each distribution was averaged across samples to produce a mean lifetime. (D) The mean lifetime in GluN1-GFP/PSD95-mCherry expressing spines (14 neurons (N)/258 spines (s), gray) is significantly shorter than GluN1-GFP alone, indicating FRET between the NMDAR and PSD95. Spines expressing either GluN1-GFP (12 N/160 s, green), GluN1-GFP/mCherry (12 N/123 s, yellow) or GluN1-GFP/Homer-mCherry (13 N/182 s, blue) all have similar lifetimes. Statistical analysis was performed by Kruskal-Wallis test (p<0.0001) followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001. (E) FRET efficiency in the same spines as in D. Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001.
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pone-0112170-g001: GluN1-GFP and PSD95-mCherry are specific probes for measuring the interaction between PSD95 and the NMDAR with FRET-FLIM.(A) FLIM images of GluN1-GFP expressing spines, expressed alone (top row), with mCherry (second row), with PSD95-mCherry (third row) or Homer-mCherry (last row). Scale bar is 1 µm (placed in first spine). Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. Black crosses indicate the pixel selected for traces in B. Circles indicate ROI for panel C. (B) Fit curves of fluorescence intensity decays obtained from one pixel (black crosses in the spines shown in A) for a GluN1-GFP expressing spine (green curve) and GluN1-GFP/PSD95-mCherry expressing spine (black curve), see Fig. S1 in file S1 for raw data. (C) Distribution histograms of GluN1-GFP lifetimes in encircled spines in A (GluN1-GFP, green; GluN1-GFP/PSD95-mCherry, gray), the median (dotted line) of each distribution was averaged across samples to produce a mean lifetime. (D) The mean lifetime in GluN1-GFP/PSD95-mCherry expressing spines (14 neurons (N)/258 spines (s), gray) is significantly shorter than GluN1-GFP alone, indicating FRET between the NMDAR and PSD95. Spines expressing either GluN1-GFP (12 N/160 s, green), GluN1-GFP/mCherry (12 N/123 s, yellow) or GluN1-GFP/Homer-mCherry (13 N/182 s, blue) all have similar lifetimes. Statistical analysis was performed by Kruskal-Wallis test (p<0.0001) followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001. (E) FRET efficiency in the same spines as in D. Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001.

Mentions: Neuronal cultures were illuminated with a Chameleon Ultra IR laser (Coherent) at 80 MHz repetition rate tuned at 900 nm for GFP two-photon excitation. Fluorescence emission was detected with a cooled high speed PMT detector head (PMC-100-1, Becker and Hickl, Germany) between 505–545 nm by means of a GFP emission filter (510/42 nm BrightLine single-band bandpass filter, Semrock) coupled to a laser block filter (750 nm blocking edge BrightLine multiphoton short-pass emission filter). The acquisition of fluorescence lifetimes was synchronized by a time-correlated-single-photon-counting (TCSPC) module (SPC-830, Becker and Hickl, Germany). Measurements were performed on a Zeiss LSM 510 microscope using a 40x water immersion objective (Achroplan, Zeiss) for live experiments and a 60x water immersion objective (Olympus UPLSAPO 60XW, NA = 1.2) for fixed samples. The following parameters were kept constant for all acquired images: pixel size (90 nm; all 512×512 pixels) (a pixel size of 45 nm was used only for figure 1), pixel dwell time (1.6 µs), laser excitation intensity (a maximum of 2 mW after the microscope objective), and FLIM acquisition time (30–60 seconds/image). Reference green and red images in confocal mode were also recorded for each FLIM image (GFP excitation 488 nm, detection through a band-pass filter (500–530 nm), mCherry excitation 543 nm, detection through a band-pass filter (565–615 nm)).


FRET-FLIM investigation of PSD95-NMDA receptor interaction in dendritic spines; control by calpain, CaMKII and Src family kinase.

Doré K, Labrecque S, Tardif C, De Koninck P - PLoS ONE (2014)

GluN1-GFP and PSD95-mCherry are specific probes for measuring the interaction between PSD95 and the NMDAR with FRET-FLIM.(A) FLIM images of GluN1-GFP expressing spines, expressed alone (top row), with mCherry (second row), with PSD95-mCherry (third row) or Homer-mCherry (last row). Scale bar is 1 µm (placed in first spine). Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. Black crosses indicate the pixel selected for traces in B. Circles indicate ROI for panel C. (B) Fit curves of fluorescence intensity decays obtained from one pixel (black crosses in the spines shown in A) for a GluN1-GFP expressing spine (green curve) and GluN1-GFP/PSD95-mCherry expressing spine (black curve), see Fig. S1 in file S1 for raw data. (C) Distribution histograms of GluN1-GFP lifetimes in encircled spines in A (GluN1-GFP, green; GluN1-GFP/PSD95-mCherry, gray), the median (dotted line) of each distribution was averaged across samples to produce a mean lifetime. (D) The mean lifetime in GluN1-GFP/PSD95-mCherry expressing spines (14 neurons (N)/258 spines (s), gray) is significantly shorter than GluN1-GFP alone, indicating FRET between the NMDAR and PSD95. Spines expressing either GluN1-GFP (12 N/160 s, green), GluN1-GFP/mCherry (12 N/123 s, yellow) or GluN1-GFP/Homer-mCherry (13 N/182 s, blue) all have similar lifetimes. Statistical analysis was performed by Kruskal-Wallis test (p<0.0001) followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001. (E) FRET efficiency in the same spines as in D. Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001.
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Related In: Results  -  Collection

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Show All Figures
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pone-0112170-g001: GluN1-GFP and PSD95-mCherry are specific probes for measuring the interaction between PSD95 and the NMDAR with FRET-FLIM.(A) FLIM images of GluN1-GFP expressing spines, expressed alone (top row), with mCherry (second row), with PSD95-mCherry (third row) or Homer-mCherry (last row). Scale bar is 1 µm (placed in first spine). Color coding represents GluN1-GFP lifetime from 2 ns to 2.8 ns. Black crosses indicate the pixel selected for traces in B. Circles indicate ROI for panel C. (B) Fit curves of fluorescence intensity decays obtained from one pixel (black crosses in the spines shown in A) for a GluN1-GFP expressing spine (green curve) and GluN1-GFP/PSD95-mCherry expressing spine (black curve), see Fig. S1 in file S1 for raw data. (C) Distribution histograms of GluN1-GFP lifetimes in encircled spines in A (GluN1-GFP, green; GluN1-GFP/PSD95-mCherry, gray), the median (dotted line) of each distribution was averaged across samples to produce a mean lifetime. (D) The mean lifetime in GluN1-GFP/PSD95-mCherry expressing spines (14 neurons (N)/258 spines (s), gray) is significantly shorter than GluN1-GFP alone, indicating FRET between the NMDAR and PSD95. Spines expressing either GluN1-GFP (12 N/160 s, green), GluN1-GFP/mCherry (12 N/123 s, yellow) or GluN1-GFP/Homer-mCherry (13 N/182 s, blue) all have similar lifetimes. Statistical analysis was performed by Kruskal-Wallis test (p<0.0001) followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001. (E) FRET efficiency in the same spines as in D. Kruskal-Wallis test was performed (p<0.0001), followed by Dunn's test. GluN1-GFP/PSD95-mCherry group is different from all the other conditions p<0.001.
Mentions: Neuronal cultures were illuminated with a Chameleon Ultra IR laser (Coherent) at 80 MHz repetition rate tuned at 900 nm for GFP two-photon excitation. Fluorescence emission was detected with a cooled high speed PMT detector head (PMC-100-1, Becker and Hickl, Germany) between 505–545 nm by means of a GFP emission filter (510/42 nm BrightLine single-band bandpass filter, Semrock) coupled to a laser block filter (750 nm blocking edge BrightLine multiphoton short-pass emission filter). The acquisition of fluorescence lifetimes was synchronized by a time-correlated-single-photon-counting (TCSPC) module (SPC-830, Becker and Hickl, Germany). Measurements were performed on a Zeiss LSM 510 microscope using a 40x water immersion objective (Achroplan, Zeiss) for live experiments and a 60x water immersion objective (Olympus UPLSAPO 60XW, NA = 1.2) for fixed samples. The following parameters were kept constant for all acquired images: pixel size (90 nm; all 512×512 pixels) (a pixel size of 45 nm was used only for figure 1), pixel dwell time (1.6 µs), laser excitation intensity (a maximum of 2 mW after the microscope objective), and FLIM acquisition time (30–60 seconds/image). Reference green and red images in confocal mode were also recorded for each FLIM image (GFP excitation 488 nm, detection through a band-pass filter (500–530 nm), mCherry excitation 543 nm, detection through a band-pass filter (565–615 nm)).

Bottom Line: We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction.The activity of both CaMKII and calpain were essential for this effect in both developmental stages.Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

View Article: PubMed Central - PubMed

Affiliation: Institut Universitaire en Santé Mentale de Québec, Université Laval, Québec, QC, Canada.

ABSTRACT
Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.

Show MeSH
Related in: MedlinePlus