Direct visualization of newly synthesized target proteins in situ

Abstract

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.

Figure 1. Labeling specific newly synthesized proteins

(a) The working principle. Newly synthesized proteins incorporate either AHA or puromycin. AHA is biotinylated by click chemistry. Antibody (pink Y) recognition of the ‘newly synthesized’ tag (biotin or puromycin, pink triangle) and recognition of a POI by a protein-specific antibody (blue Y) detects close proximity when PLA^minus and PLA^plus oligonucleotides (yellow and green squiggles) coupled to secondary antibodies (grey Y) are close enough to template linker oligo ligation to a circle. Signal is amplified by binding of fluorescently-coupled detection probes (green circles). (b) Example images of FUNCAT-PLA signal (green) for newly synthesized Camk2a (2 h AHA) in cultured hippocampal neurons (left) and control without anti-Camk2a antibody (right). (c) Puro-PLA images of newly synthesized Camk2a (green) after 15 min of puromycin labeling without (left) or with (right) the protein synthesis inhibitor Anisomycin. Scale bars (b,c) = 15 μm. (d) High-magnification images of FUNCAT-PLA (grey-scale) for newly synthesized Bassoon, TGN38 or LaminB (as indicated, 2 h AHA) in somata and principal dendrites of cultured hippocampal neurons (MAP2 outlines in magenta). Scale bar = 10 μm. (e) Micrograph of newly synthesized TGN38 after 5 min puromycylation. Inset: soma signal converted as in (d). Scale bar = 20 μm. (f) Correlation of TGN38 FUNCAT-PLA and Puro-PLA signal (integrated PLA intensity in the soma) dependence on incubation time with AHA and puromycin. (R² = 0.99 and 0.98 from two and three independent experiments, n = 20 – 27 and n = 15 – 20 respectively. Mean ± SEM and the linear regression is shown).

Figure 2. Assessing intramolecular labeling of Puro-PLA

(a) Scheme depicting the binding sites for N- and C-terminal anti-Bassoon antibodies (blue Y, upper panel). In Puro-PLA experiments, an N-terminal Bassoon antibody and an anti-puromycin antibody (pink Y) are predicted to generate a larger signal compared to a C-terminal Bassoon and puromycin antibody pair since puromycin (pink triangle) blocks the elongation of the nascent chain. If both antibodies recognize epitopes in the same protein, the Puro-PLA signal generated with N-terminal antibodies is expected to be greater than that generated with C-terminal antibodies in contrast to binding to neighbouring proteins. (b) Representative fluorescence images and close-ups (upper panel, PLA-signal green, MAP2 magenta and DAPI-labeled nuclei blue, lower panel grey scale PLA-signal) showing Puro-PLA signal for the Bassoon protein detected after labeling with puromycin for four minutes or without puromycin, using either N- or C-terminal anti-Bassoon antibodies. Scale bar = 30 μm and 10 μm respectively. (c) Quantification of signal shown in (b). For each experiment, the background-corrected Puro-PLA density for each antibody was normalized to the mean value for the C-terminal antibody (means ± SEM from three independent experiments, n = 10 – 11). Statistical significance was tested using an unpaired Student’s t-test (*P < 0.05, P = 0.014).

Figure 3. Following protein lifetime, distribution changes and synthesis rate changes with FUNCAT-PLA

(a) Scheme depicts two hypothetical FUNCAT-PLA labeled proteins with different lifetimes. (b) Graph of Bassoon and TrkB FUNCAT-PLA signal dependence on chase time following a 2 h AHA pulse. The FUNCAT-PLA signal area overlapping with the dilated MAP2 signal was normalized to the MAP2 signal area (normalized means ± SEM from three (TrkB, n = 12 – 14) and five (Bassoon, n = 23 – 39 cells per condition) independent experiments. (c) Scheme depicting the spatio-temporal dynamics of a hypothetical FUNCAT-PLA-labeled protein population (green) and images of neuronal somata and dendrites (green MAP2 outline) labeled for Bassoon FUNCAT-PLA (grey-scale) following a 2 h AHA incubation with either a 10 min or 24 h chase. Scale bar = 10 μm; dendrite length = 135 μm. (d) Analysis graph of (c). Values displayed are percent of the maximal signal density in the respective compartment with chase time (normalized means ± SEM from three independent experiments, n = 11 – 13). (e) Micrographs showing GluA1 FUNCAT-PLA (cell overview and a corresponding dendrite below) following 3 h AHA labeling without (control) and with induction of homeostatic plasticity by blocking action potentials (TTX) alone or in combination with a block of the NMDA-component of miniature synaptic events (TTX/APV). GluA1 FUNCAT-PLA (green), MAP2 (magenta), Scale bar = 20 (overview) and 10 μm (close-ups). (f) Analysis of (e). GluA1 FUNCAT-PLA signal density normalized to control in neuronal somata (upper panel). Treatment with TTX/APV significantly enhanced the population of newly synthesized GluA1 (*P < 0.05, P = 0.0349). Lower panel: Treatment with either TTX or TTX/APV significantly enhanced the amount of newly synthesized GluA1 in the dendrites even more than in somata (*P < 0.05, **P < 0.01, normalized means ± SEM, n = 16 – 25 cells per condition from three different experiments, ANOVA, Tukey’s multiple comparisons test on raw values).