BPS2026 – Visualization of lipid dynamics in the ER using new functionalized ceramide analogs and single molecule tracking

The endoplasmic reticulum is the primary site of lipid synthesis and trafficking in the cell, yet little is known about how lipids navigate this network and their kinetics in the ER membrane. This gap is largely due to the challenges that come with imaging lipids in their native membranes, including their relatively small size and sensitivity to tagging-induced perturbations. To overcome these limitations, we combined bioorthogonal lipid labeling approaches with live cell imaging techniques, including single molecule tracking (SMT). We developed an azido-modified 1-deoxysphingosine derivative, which allows for strain-promoted azide-alkyne cycloaddition (SPAAC) “click” reactions at the C1 hydroxyl of sphingosine. In cells, the probe is metabolized by ER-localized ceramide synthases and can thus be used to track dynamics of ceramide, the building block for sphingolipids. We have found BODIPY-labeled products of the probe to show exquisite brightness and ER localization in RPE-1 cells, especially on the cell periphery. Through fluorescence recovery experiments, we have measured differences in mobility of the 1-azido-1-deoxyceramide probe as compared to an N-Boc-1-azido-1-deoxysphingosine probe, the latter of which is unable to be N-acylated into deoxyceramide. Additionally, the peripheral ER localization and demonstrated brightness of our lipid probes have made them suitable for lipid SMT in live cells, informing on the mobility of ceramides in different regions of ER. Overall, this work combines development of new lipid probes with complementary imaging techniques to characterize the movement of ceramide in the ER, informing how this organelle carries out nanoscale lipid regulation and trafficking.