In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons

Abstract

Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click chemistry'. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous.

Figure 1. Chemical components and FUNCAT procedure

(a) Chemical structures of the modified amino acids azidohomoalanine (AHA) and homopropargylglycine (HPG), and the two fluorescent tags TexasRed–PEO2–Alkyne (TRA) and 5–carboxyfluorescein–PEO8–Azide (FLA) used in this study to visualize newly synthesized proteins. (b) Cartoon illustrating the Cu(I)–catalyzed [3+2] azide–alkyne cycloaddition (CuAAC) principle.

Figure 2. Visualization of newly synthesized proteins in dissociated primary hippocampal neurons

(a) Dissociated hippocampal neurons (DIV 17) were incubated with either 2 mM AHA or 2 mM HPG in the presence or absence of 40 µM anisomycin (Aniso) for 1 h, tagged with 1 mM TRA or FLA tag and immunostained for the dendritic marker protein MAP2. Scalebar = 20 µm. (b) Dissociated neurons were incubated with 2 mM AHA for 2 h followed by tagging with 1 mM TRA tag and immunostaining for Bassoon as a synaptic marker. Arrowheads denote spine–like protrusions. Scalebar = 10 µm in the left panel images, scalebar = 5 µm in the magnified images.

Figure 3. Sequential labeling of 2 newly synthesized protein pools with two metabolic markers

(a) Dissociated hippocampal neurons (DIV 16–18) were incubated in 2 mM AHA for 1.5 h followed by 4 mM HPG for 1.5 h and vice versa, and sequentially tagged with either 1 mM TRA then 1 mM FLA tag, or FLA tag followed by TRA tag for 12 hours each. Scalebar = 20 µm. Color lookup table indicates fluorescence intensity (pixel intensities 0–255). Labeling and detection sequences: A,H, F,T: first AHA then HPG, first FLA then TRA tag; H,A, T,F: first HPG then AHA, first TRA then FLA tag. Images were acquired and analyzed using identical parameters on a Zeiss Meta 510 confocal microscope using a 40x objective, postprocessing and analysis was done with ImageJ. Graph (b) represents mean intensities ± SEM of somata for the indicated labeling and detection sequences. Graph (c) represents mean intensities ± SEM of dendrites (0–100 µm) for the indicated tagging and detection sequences. White boxes correspond to TRA signals, grey boxes indicate FLA signals. n = 6–10 neurons. P–values: ** p < 0.005; * p < 0.05.

Figure 4. Time–course for the detection of newly synthesized proteins in somata and dendrites

(a) Cultures (DIV 16) were incubated for the time points indicated with 2 mM AHA, 2 mM methionine (Met) or 2 mM AHA in the presence of 40 µM anisomycin (Aniso). After cycloaddition with the fluorescent TRA tag, images were acquired with identical parameters on a Zeiss Meta 510 confocal microscope using a 63x objective. Scalebar = 5 µm. Graph (b) represents mean intensities ± SEM of the somata. Per time point, data from 20–50 cells were collected and analyzed using ImageJ. Representative examples are shown in the right panel. (c) Representative straightened dendrites of neurons incubated with 2 mM AHA, 2 mM Methionine (Met) or AHA plus 40 µM anisomycin (Aniso) for time points indicated. Color lookup table indicates fluorescence intensity (pixel intensities 0–255). Left: proximal; right: distal. Scalebar = 10 µm. Graph (d) represents mean intensities ± SEM of the dendrites in 20 µm bins. Per time point data from 25–40 dendrites were analyzed. Note, that somatic signals are saturated in some cases, in order to optimize the imaging parameters for signal detection in distal dendrite segments.

Figure 5. BDNF–induced increases in protein synthesis

(a) Hippocampal neurons (DIV 16) were incubated for 1 h with 2 mM AHA alone (vehicle control) or 2 mM AHA in the presence of 50 ng/ml BDNF. After a 15 min chase with 2 mM methionine, cells were fixed, tagged with 1 mM fluorescent tag and immunostained for the dendritic marker protein MAP2. For quantitative analysis, dendrites of both groups were straightened and fluorescent intensities of binned 40 µm segments were measured using ImageJ. Representative images for both groups are shown. Color lookup table indicates fluorescence intensity (pixel intensities 0–255). Note the increase in signal intensity in both cell body and dendrites in BDNF–treated versus control cells. Left: proximal; right: distal. Scalebar = 20 µm. (b) Graph shows the change of TRA–signal of BDNF–treated cells ± SEM normalized to controls (vehicle). P–values: *** p < 0.000005; ** p < 0.005; * p < 0.05.

Figure 6. BDNF–induced increase in dendritic protein synthesis

(a) Somata of neurons were perfused with 40µM anisomycin before and during bath-application of 2 mM AHA, or 2 mM AHA + BDNF (50 ng/ml) for 30 min, scalebar = 50 µm. (b) Images showing signals of newly synthesized proteins from nonperfused somata (top left, AHA (I); top right, AHA + BDNF (II)), anisomycin–perfused somata (bottom left, AHA (III); bottom center, AHA + BDNF (IV)) and anisomycin–bath cells (bottom right, (V)). n = 6–9 cells. Large grid tick marks = 20 µm. Graph shows TRA–signal to volume ratios ± SEM. (c,d) Analysis of newly synthesized proteins in dendrites of groups I–IV. Images show signal intensity of representative dendrites beginning at the soma. Left, proximal; right, distal. Color lookup table indicates fluorescence intensity. Note, that somatic signals are saturated in some cases, in order to optimize the imaging parameters for signal detection in distal dendrite segments. Large grid tick marks = 10 µm. Summary graphs shows signal to volume ratios ± SEM. n= 6–9 dendrites. P–values: *** p < 0.001; ** p < 0.01; * p < 0.05, one–way ANOVA with Tukey testing. In graph (c) significance values for group (I) vs. (V) dendrites are indicated in green, for group (II) vs. (V) in magenta, for group (I) vs. (II) dendrites in grey; in graph (d) significance values for group (III) vs. (V) dendrites are indicated in violet, for group (IV) vs. (V) in red, for group (III) vs. (IV) in grey.

Figure 7. Local BDNF–induced increase in dendritic protein synthesis

(a) Hippocampal neurons for dendritic local perfusions in combination with anisomycin bath application were pre–incubated in 40 µM anisomycin for 20 min during which time the micropipettes were positioned. Dendritic local perfusion with 2 mM AHA in the presence or absence of BDNF (50 ng/ml) in HibA (with no anisomycin in the perfusion pipette) was initiated, and constantly monitored over the perfusion period of 30 min. Scalebar = 50 µm. Images (b) show TRA signals of representative dendrites from groups (I, top) no perfusion, (II, center) AHA perfusion, (III, bottom) BDNF plus AHA perfusion. Grey bars mark the location and extension of the perfusion stream. Large grid tick marks = 10 µm. Color lookup table indicates fluorescence intensity. Graph (c) shows TRA–signal to volume ratios ± SEM. Colored ovals represent the positions of the perfusion spots. n= 4–5 dendrites. P–values: *** p < 0.001; ** p < 0.01; * p < 0.05 using one–way ANOVA statistical analysis with Tukey testing. Significance values are indicated for the dendritic segments before, within and beyond the perfusion spots; violet labels indicate significances for dendrites derived from group (I) vs. (II), red labels indicate significance values for group (I) vs. (III) dendrites, grey labels indicate significances for group (II) vs. (III) dendrites.

Figure 8. Diffusion properties of newly synthesized proteins at the surface of dissociated primary hippocampal neurons

(a) Strategy for labeling newly synthesized proteins inserted in the membrane of living neurons. (b) Neurons were incubated with either 2mM AHA in the presence or the absence of anisomycin, or 2mM Methionine for 2–4 hours and labeled with DIFO–biotin and QD–SA. DIC (left) and QD labeling (right) of live neurons from each condition. Scalebar = 5µm. (c) Example of trajectories of newly synthesized proteins labeled with QDs. Left, DIC and QD labeling (green); active synapses are visualized with FM4–64; right, Individual trajectories reconstructed from QDs. Synaptic (yellow) and extrasynaptic (blue) trajectories were classified in relation to active presynaptic terminals labeled with FM4–64. Scalebar = 2µm. (d) Average MSD of synaptic and extrasynaptic QDs as a function of time. Values are mean ±SEM. (e) Cumulative distribution of newly synthesized proteins diffusion coefficients at (n=101) and outside (n=262) synapses (Kolmogorov–Smirnov test, p<0,001). The diffusion coefficients of the γ₂–GABA_AR subunit at synapses (n=212) and at extrasynaptic sites (n=77) (KS test, p<0,001) were plotted for comparison. (f) Frequency distribution of extrasynaptic diffusion coefficients of newly synthesized proteins. Colored bars correspond to the 75% of the values (12,5%–87,5% range, black squares correspond to the mode of the distribution) of D values for AMPAR (purple, 50), GABA_AR (red), mGluR5 (green, 31,32) and GFP–GPI (grey, 30). (g) Frequency distribution of extrasynaptic diffusion coefficients of γ₂–GABA_AR. The red bar on the x-axis corresponds to 75% of the values (12,5%–87,5% range) of γ₂–GABA_AR diffusion coefficients.