Lipid droplet diversity: mobility and mitochondrial interaction

Every cell must maintain a balance between lipid availability and lipid toxicity, storing enough to fuel metabolism while preventing accumulation that disrupts membrane integrity and cell function. Lipid droplets (LDs) are the organelle at the heart of this balance. In recent decades, advances in microscopy, proteomics, and cell biology have fundamentally reshaped our understanding of these structures from how they form and grow to how they communicate with partner organelles. Still, many questions remain unanswered, particularly at the level of individual organelles - how they move, why they differ from one another within the same cell, and what determines the outcome of their contacts with mitochondria. LD mobility is considered a key enabler of formation of organelle contacts. What governs this mobility and how it shifts with the metabolic state of the cell, remained an open question in the field, and mobility seems to change across different cell types. Using quantitative trajectory analysis, we identified two coexisting LD subpopulations with distinct motion signatures, suggesting that LDs do not move as a uniform population but follow different modes of motion. The balance between these two populations shifts depending on the metabolic condition, with LD size, LD crowding, and mitochondrial tethering each contributing to the observed differences. Using a contact reporter, I further show that LD-mitochondria contact frequency is regulated by metabolic demand and cellular stress. As an additional line of investigation on LD mobility, we asked how cell shape influences LD dynamics. Our findings suggest that differences in cell geometry were not sufficient to alter LD transport. Beyond mobility, I examine structural heterogeneity within the LD population. Polarization microscopy shows that cholesterol feeding drives a liquid-crystalline phase transition across the entire LD population; yet Contact-FP imaging shows that only a subset of these crystalline LDs retain competence for mitochondrial engagement, indicating that liquid-crystalline LDs are themselves heterogeneous. Finally, to address the challenge of capturing transient LD-mitochondria contacts without phototoxic damage, I exploited a hybrid event-driven acquisition (hybrid-EDA) approach. Phase-contrast surveillance continuously monitors the cells and triggers targeted fluorescence acquisition only when a contact event is detected. Applying this framework, I find that brief LD-mitochondria contacts do not produce detectable changes in mitochondrial membrane potential, suggesting that a minimum contact duration may be required for productive metabolic coupling. Together, these findings converge on a common theme: LDs are not a uniform population. Their mobility, structural state, and capacity for mitochondrial engagement all vary within a single cell, suggesting that individual LDs may carry distinct functional identities. These observations were made possible by recent advances in micro