Revealing Single-Cell Heterogeneity of Labile Cu(I) Accumulation and Metabolism in Microalgae by Image-Enabled Flow Cytometry

Heterogeneity is an intrinsic characteristic of living organisms, yet the single-cell heterogeneity in metal uptake and toxicity remains poorly understood. Here, we investigated the single-cell heterogeneity of labile Cu(I) metabolism in the model microalga <i>Chlamydomonas reinhardtii</i> using an image-enabled flow cytometer platform. Algal cells exposed to chronic Cu stress exhibited distinct labile Cu(I) bioaccumulation patterns, forming two subpopulations: "LCu(I) cells" and "HCu(I) cells," differentiated by intracellular labile Cu(I) content. These results provide direct evidence of heterogeneous Cu(I) distribution at the single-cell level in microalgae. Higher Cu stress induced a shift from LCu(I) cells to HCu(I) cells, suggesting differential cellular sensitivity to Cu stress and varying labile Cu(I) accumulation. At the molecular level, multiomics analyses identified Ctr3p as a potential key regulator of labile Cu(I) homeostasis in algal cells. Confocal imaging revealed abnormal aggregation of glutathione (GSH) in granules within HCu(I) cells. Complementary GSEA results indicated an aberrant GSH compartmentalization in HCu(I) cells that might contribute to Cu(I) hyperaccumulation. At the physiological level, hyperaccumulated Cu(I) in HCu(I) cells likely caused cytotoxicity and photosynthesis inhibition. This study highlights the complexity and variability of labile Cu metabolism at the single-cell level, emphasizing the importance of accounting for subpopulation-specific responses in metal toxicity assessments, rather than relying solely on bulk-level analyses.