Exploring the biophysical regulation of lipoprotein-membrane interactions

This thesis investigates the biophysical mechanisms underlying lipoprotein- membrane interactions, with a focus on receptor-independent pathways, while also advancing versatile tools for studying membrane dynamics. By integrating foundational research with methodological innovations, it provides a comprehensive framework for understanding lipid transport, membrane remodeling, and their implications in health and disease. The receptor-independent dynamics of high-density lipoproteins (HDL), low- density lipoproteins, and very low-density lipoproteins were explored using advanced imaging and analytical techniques. Structural characterization confirmed the integrity of these lipoproteins, while functional studies demonstrated their ability to transfer cargo directly to both synthetic and biological membranes. These interactions significantly altered membrane properties, increasing order and rigidity. Further analysis of phase-specific behavior revealed HDL's preferential partitioning into liquid-disordered membrane phases and its enhanced interactions with ether lipid-enriched membranes. These findings underscore the influence of lipid composition and membrane order in modulating interaction dynamics, revealing a critical role of these processes in cholesterol efflux and cellular signaling. In parallel, methodological advancements enhanced the precision and scalability of biophysical research. The single-particle profiling technique enabled high- throughput analysis of lipoprotein subpopulations, uncovering heterogeneity in lipid packing and particle mobility. Spectral imaging with environment-sensitive probes established a standardized approach to quantifying membrane fluidity and lipid order. Complementing these methods, the VISION software provided an open-source platform for multidimensional image analysis, resolving nanoscale lipid heterogeneity and protein clustering with unprecedented detail. Collectively, these studies highlight the versatility of lipoproteins as dynamic modulators of membrane properties and provide new insights into lipid transport. By integrating receptor-independent mechanisms with advanced analytical methodologies, this thesis advances our understanding of lipoprotein functionality and its broader implications for cellular processes, metabolic regulation, and therapeutic interventions targeting lipid-related disorders. List of scientific papers This thesis is a compilation of five research papers, each contributing to the field of membrane biophysics by exploring lipoprotein interactions and methodological advancements. Below is an overview of each paper, including publication details and a summary of my contributions. I. Lipoprotein particles interact with membranes and transfer their cargo without receptors. https://doi.org/10.1021/acs.biochem.0c00748 II. High-throughput measurement of the content and properties of nano-sized bioparticles with single-particle profiler. https://doi.org/10.1038/s41587-023-01825-5 III. Measuring plasma membrane fluidity using confocal microscopy. https://doi.org/10.1038/s41596-024-01122-8 IV. VISION - an open-source software for automated multi- dimensional image analysis of cellular biophysics. https://doi.org/10.1242/jcs.262166 V. "Head-to-toe" lipid properties govern the binding and cargo transfer of high-density lipoprotein. https://doi.org/10.3390/membranes14120261