UC Berkeley

The cell membrane is a complex mixture of various lipids, proteins and other biomolecules that are all organized into a fluid 2-dimensional bilayer. A rather unique trait of this organelle is the lateral mobility of the component molecules. Surprisingly, these molecules are not necessarily distributed homogeneously in the membrane. From a physical perspective, these inhomogeneities are interesting because they indicate some level of organization in the membrane. From a biological perspective, this organization is interesting because it might be a key regulatory element in the enzymatic processes and cell signaling events that occur at the cell membrane. Due to the difficulty of studying membrane organization, not much is known about the spatiotemporal scale of these organized domains, nor is it clear what the physical driving forces are, although there are models based on observations from a variety of different methods. The key factor to overcome in studying membrane organization is the ability to probe the membrane in an informative way that does not perturb the native organization of the membrane.

Membrane anchors are lipid moieties covalently conjugated to various membrane proteins and have been implicated in the lateral sorting of anchored proteins in the membrane. Most studies on lipid anchors focus either on identifying what molecules anchored proteins colocalize with or observing how anchored proteins diffuse in the cell membrane. Truncated anchored proteins with just the anchor domain remaining can be genetically fused to fluorescent proteins and also studied to determine the extent to which the anchor-membrane interactions, as opposed to protein-protein interactions, influence their distribution in the membrane. The methods used to study these behaviors are varied and, subsequently, the observations that result from these studies are also varied and the conclusions are conflicting.

Time-resolved spectroscopy of fluorescently labeled anchor domains in living cells offers a non-invasive method to extract a wealth of information about the spatiotemporal localization of anchored proteins in a live cell. More specifically, fluorescence cross-correlation spectroscopy (FCCS) analysis and fluorescence lifetime analysis can be derived from the same data stream. In this dissertation, I review the current model of how membranes are organized and present observations made by myself and coworkers, of two instances of homogeneous colocalization of the same anchors but no heterogeneous colocalization of different anchors in Jurkat cell membranes. We conclude that the observation of two distinct non-overlapping domains existing in the same cell membrane indicate a more complex organization than the current model allows.