Fluorescence imaging of lipid trafficking pipelines in mammalian cells
Juan Manuel Iglesias Artola
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Germany
Lipid composition determines organelle identity and function, yet it remains unclear how cells maintain lipid homeostasis across their membranes, largely because native lipids cannot be fluorescently tagged using conventional genetic tools and chemical modifications disrupt protein–lipid interactions. To overcome these limitations, we developed a library of bifunctional (photoactivatable and clickable) lipids and built an imaging and mass spectrometry pipeline to quantify intracellular lipid transport and link it to lipid metabolism. Our data reveal that non-vesicular lipid transport provides both higher molecular specificity and faster kinetics than vesicular trafficking or metabolism, suggesting it is the primary mechanism maintaining organelle membrane composition and intracellular lipid homeostasis.