A non-fluorescent HaloTag blocker for improved measurement and visualization of protein synthesis in living cells

Abstract

Background: HaloTag is a modified bacterial enzyme that binds rapidly and irreversibly to an array of synthetic ligands, including chemical dyes. When expressed in live cells in conjunction with a protein of interest, HaloTag can be used to study protein trafficking, synthesis, and degradation. For instance, sequential HaloTag labeling with spectrally separable dyes can be used to separate preexisting protein pools from proteins newly synthesized following experimental manipulations or the passage of time. Unfortunately, incomplete labeling by the first dye, or labeling by residual, trapped dye pools can confound interpretation. Methods: Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. To that end, we synthesized a non-fluorescent, cell permeable blocker (1-chloro-6-(2-propoxyethoxy)hexane; CPXH), essentially the HaloTag ligand backbone without the reactive amine used to attach fluorescent groups. Results: High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise2 and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 µM at 30 min. No overt effects on cell viability were observed at any concentration or treatment duration. The ability of CPXH to improve the reliability of newly synthesized protein detection was then demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. Practically no labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in live cells using HaloTag technology.

Synapse integrity and function: Dependence on protein synthesis and identification of potential failure points

“…Then, for each inhibitor, we quantified the fold reduction in mean JF635HT/mTurquoise2 fluorescence ratio in comparison to controls, providing a quantitative assessment of protein synthesis suppression in these preparations ( Figure 1 ; Supplementary Figure S1 ). As shown in Figures 1B , D , protein synthesis inhibition was extremely effective for CHX (data regarding CHX effects and Figure 1B present previously published results shown here in sake of completeness; Cohen et al, 2020 ) and quite effective for ANI, resulting in ~39.2x and ~ 4.2x reductions in JF635HT labeling, respectively. Inhibition was, however, unsatisfactory for puromycin at the selected concentrations and measurement duration (~1.9x, Figure 1F ).…”

“…We thus set out to measure the efficacy of several commonly used PSIs, and then select the most efficient ones for subsequent experiments. To that end, we used an assay we previously described ( Cohen et al, 2020 ) based on the HaloTag technology and a fusion protein of HaloTag and the fluorescent protein mTurquoise2 (HaloTag-mTurq2). Here, cortical neurons in primary culture expressing this fusion protein were first exposed at saturating concentrations to the non-fluorescent, cell permeable HaloTag ligand CPXH (1-chloro-6-(2-propoxyethoxy) hexane), essentially the HaloTag ligand backbone without a fluorescent group ( Cohen et al, 2020 ).…”

“…To that end, we used an assay we previously described ( Cohen et al, 2020 ) based on the HaloTag technology and a fusion protein of HaloTag and the fluorescent protein mTurquoise2 (HaloTag-mTurq2). Here, cortical neurons in primary culture expressing this fusion protein were first exposed at saturating concentrations to the non-fluorescent, cell permeable HaloTag ligand CPXH (1-chloro-6-(2-propoxyethoxy) hexane), essentially the HaloTag ligand backbone without a fluorescent group ( Cohen et al, 2020 ). The neurons were then washed and exposed to the fluorescent HaloTag ligand JF635HT ( Grimm et al, 2017 ).…”

“…SEM and t test based on numbers of neurons (analyses by replicates provided in Supplementary Figure S1 ). Data in A and B taken from Cohen et al (2020) .…”

“…HaloTag-mTurq2 was generated as described previously ( Cohen et al, 2020 ) as was the non-fluorescent HaloTag blocker CPXH [1-chloro-6-(2-propoxyethoxy)hexane]. Halo-Tag mTurq2 was expressed by calcium phosphate transfection as described previously ( Bresler et al, 2004 ), or by adding HaloTag-mTurq2 lentiviral particles to the neuronal cultures (as detailed below).…”