Investigating Spatial Organization and Physicochemical Interactions in Biomembranes: Tools and Insights

Abstract

The lipid membranes of cells are complex structural and functional landscapes. Beyond being a selective barrier separating the cell from its surroundings, the membrane serves also as a two-dimensional solvent dictating the thermodynamic environment in which membrane protein biochemistry takes place, and as a platform that facilitates and responds to the organization of membrane proteins into functional domains. Membranes including vesicles derived from eukaryotic plasma membranes also exhibit liquid-liquid phase coexistence. This dissertation aims to link the biochemical and organizational properties of membranes to their phase behavior.

The membrane's role as a thermodynamic platform is addressed in a chapter on the availability of cholesterol, specifically its chemical potential (Chapter 3). This work consists of measurements of the chemical potential of cholesterol in a family of synthetic lipid membrane compositions. This chemical potential describes the availability of cholesterol, and is a primary determinant of the occupancy of protein binding sites for cholesterol. The synthetic membranes used in this study are similar to mammalian plasma membranes in phase behavior and cholesterol concentration. The measurements show a close connection between the role of cholesterol in phase separation of these membranes and its availability. This finding suggests that treatments that modify the phase behavior of the membrane, of which many are known, may act through their effect on the availability of cholesterol. In addition, this study provides a framework for how to approach other questions about the biochemistry of cholesterol.

The remaining chapters describe methods that will enable more precise and robust measurement and analysis of the organization of membrane proteins using single molecule localization microscopy (SMLM). SMLM techniques produce location information of target molecules with precisions on the order of 10 nm, and so have been invaluable for characterizing protein organization in membranes. The methods contributions include direct improvements to the precision of these datasets through improved sample drift correction (Chapter 4), a novel method for characterizing SMLM measurement precision (Chapter 5), and a method for correcting spatially non-uniform labeling or detection artifacts in measurements of colocalization (Chapters 6 and 7). These methods extend the usefulness of SMLM so that it can detect more detailed and subtler structure in the organization of proteins on membranes. In particular, they will enable future experiments to measure the role of membrane phase behavior in biological systems where it has been too subtle to detect using past methods.

Overall, the developments described in this dissertation strengthen the connections between membrane phase behavior and biological function, by linking phase behavior to a new biochemical property of the membrane, and by enabling future investigations into how it organizes membrane proteins.