Genetically encoded fluorescent reporter for polyamines

Abstract

Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson's disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a first-in-class genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conducted a genome-wide CRISPR screen and uncovered an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for Parkinson's disease. By offering a new lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.

Fig. 1 |. Polyamine sensor design and validation.

a, Chemical structures of mammalian polyamines. b, Schematic of the polyamime biosynthesis pathway in mammalian cells. c, Schematic of polyamine-responsive +1 ribosomal frameshifting during translation of OAZ1 mRNA. d-f, Design for the polyamine sensor (d), corresponding immunoblot (e) and micrographs under indicated treatments. g, Quantification of frameshift efficiency using flow-cytometry for cells expressing polyamine sensor under indicated treatments. DFMO: difluoromethylornithine, 1 mM (90 h) and Spd: spermidine, 5 μM (18 h). h-i, Representative fluorescence micrographs (h) and corresponding quantification by flow cytometry of cells (i) upon genetic knockdown of indicated polyamine biosynthesis enzymes. Each data point shown in (g) and (i) represents a single cell. Error bars in (g) and (i) denote the median ± interquartile range and are calculated from ≥ 10000 cells. 50 data points are shown. Flow cytometry quantification and the fluorescent micrographs are representative of ≥2 independent experiments. Significance values are calculated using Student’s t-test. *** denotes p<0.0001. Scale bars, 10 μm.

Fig. 2 |. Reporter calibration and reproducibility.

a, Schematic of LC/MS protocol for polyamine quantification. b-c, Intracellular spermidine concentration measured using LC/MS (b) and F/F₀ (c) in response to various concentrations of DFMO. d, Linear regression of the cellular spermidine concentration with F/F₀ (calculated from c). e, F/F₀ and the spermidine concentration measured using LC/MS upon treatment with ribavirin (100 μM for 72 h; SAT1 activator). The sensor value was calculated using the fit from d. f, Coefficient of variation (CV) was calculated for intra-assay and inter-day variability. CV is calculated as the ratio of standard deviation to the average value. Error bars in (b) and (e, left) denote the mean ± standard deviation (n ≥3 independent experiments). Error bars in (c) and (e, right) denote the median ± interquartile range and are calculated from ≥ 10000 cells. 50 data points are shown.

Fig. 3 |. Live-cell longitudinal imaging of polyamine dynamics.

a, Schematic of sensor design for real-time tracking of polyamines. b, Representative fluorescence images of cells expressing the degron-based sensor under indicated treatments. DFMO (2 mM), Spd (spermidine, 100 μM) and TMP (0.05 μM). Cells were grown in TMP starting 3 days before DFMO treatment to allow steady-state levels of the reporter expression. Media was replaced every two days with fresh reagents. c, Single-cell traces of F/F₀ upon indicated treatments. F is the miRFP670-2 to mCherry fluorescence. F₀ is the average frameshifting efficiency in untreated cells (at the time, t = 0). Arrows indicate the time-point of treatment. d, Mean F/F₀ over time from the single-cell traces shown in c. Shaded bands depict the 95% confidence intervals.

Fig. 4 |. Single-cell polyamine import assay.

a, Schematic of single-cell polyamine import assay. b, Representative fluorescence images of cells upon indicated treatments. AMXT-1501 (0.5 μM; polyamine import inhibitor), DFMO (1 mM), and spermidine (5 μM). c, Quantification of frameshift efficiency using flow-cytometry for treatments in (b).

Fig. 5 |. Genome-wide CRISPR screen identifies modifiers of polyamine import.

a, Schematic of the CRISPR-Cas9 FACS-based genome-wide screen to identify modulators of polyamine import. b, -log10 (MaGeCK score) versus median log2 (fold enrichment) for all genes from the spermidine import screen. c, Graph depicting the fold enrichment for sgRNAs targeting indicated genes. Each data point is for a distinct sgRNA sequence. d, Representative images of U-2OS cells corresponding to treatment with DFMO: 1 mM and spermidine: 5 μM. (e-g) Comparison of polyamine levels in wild-type and upon ATP13A3 knock-out in K562 (e), U-2OS (f), and U-2OS (g) cells under indicated treatments. h, Heatmap of enrichment scores for individual sgRNAs targeting genes previously proposed to be involved in polyamine import. i, Comparison of polyamine levels in U-2OS cells under indicated treatments. Rotenone (1 μM; complex I inhibitor) and antimycin (1 μM; complex III inhibitor) were added 24 h before reporter induction and spermidine addition (5 μM, 18 h). j, Total polyamine levels (see enzymatic total polyamine assay in Methods) for the indicated samples (relative abundance). Rotenone (1 μM, 48 h) and spermidine (5 μM, 16 h). 2 mM DFMO (48 h) was used as a positive control. Error bars in (c) denote the mean ± standard deviation (5 independent sgRNAs). Error bars in (e-g, i) denote the median ± interquartile range and are calculated from ≥ 5000 cells. 50 data points are shown. Error bars in (j) denote the mean ± standard deviation from 3 independent experiments. Significance values are calculated using Student’s t-test. Flow cytometry quantification and the fluorescent micrographs are representative of ≥2 independent experiments. Scale bars, 10 μm.