Aknine, N. | bioRxiv Science

bioRxiv2025

Prodan-based solvatochromic probes for polarity imaging of organelles

Solvatochromic probes provide microscopic, structural and functional information on their targeted cellular compartments. In this field, the challenge lies in designing probes that are both sufficiently sensitive to environment, and specific in their subcellular localization. Here, we design Prodan-based polarity probes targeting the following organelles: mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes and lipid droplets. The new probes provide robust organelle targeting, except for the mitochondrial probe, whose targeting ability is cell type dependent. Due to operating range of Prodan fluorophore in the UV-Blue region, our probes can be easily combined with other fluorescent tags in the visible range. Therefore, polarity of sub-cellular compartments can be studied together with additional fluorescent reporters. We used these probes to show the polarity of organelle membranes in healthy cells and under starvation condition where we observed organelle-specific polarity remodeling. These probes are important addition to the repertoire of smart cellular probes and will find critical use in understanding spatiotemporal regulation of cellular physiology.

biophysics↗

bioRxiv2024

Lipid-directed covalent fluorescent labeling of plasma membranes for long-term imaging, barcoding and manipulation of cells

Fluorescent probes for cell plasma membrane (PM) are generally based on amphiphilic anchors that incorporate non-covalently into biomembranes. Therefore, they are not compatible with fixation and permeabilization, presence of serum, or cell co-culture because of their exchange with the medium. Here, we report a concept of lipid-directed covalent labeling of PM, which exploits transient binding to lipid membrane surface generating high local dye concentration, thus favoring covalent ligation to random proximal membrane proteins. This concept yielded a class of fluorescent probes for PM (MemGraft), where a cyanine dye (Cy3 and Cy5) bears at its two ends low-affinity membrane anchor and reactive group: an activated ester or a maleimide. We found that MemGraft probes with these reactive groups provide efficient PM labelling, in contrast to a series of control compounds, including commercial Cy3-based labels of amino and thiol groups, revealing the crucial role of the membrane anchor combined with high reactivity of activated ester and a maleimide groups. In contrast to conventional PM probes, based on non-covalent interactions, MemGraft labelling approach is compatible with cell fixation, permeabilization, trypsinization and presence of serum. The latter allows long-term cell tracking and video imaging of cell PM dynamics without signs of phototoxicity. The covalent strategy also enables staining and long-term tracking of co-cultured cells labelled in different colors without probes exchange. Moreover, combination of different ratios of MemGraft-Cy3 and MemGraft-Cy5 probes enabled long-term cell barcoding in at least 5 color codes, important for tracking and visualizing multiple cells populations. Ultimately, we found that MemGraft strategy enables efficient biotinylation of cell surface, opening the path to cell surface engineering and cell manipulation.

biochemistry↗