Imaging and Quantifying Lipid Membrane Asymmetry in Living Cells with Sum-Frequency Vibrational Microscopy

University Of Utah, Salt Lake City UT

Investigators

Abstract

In this project funded by the Chemical Structure, Dynamics and Mechanisms A (CSDM-A), Chemistry of Life Processes (CLP) and Chemical Measurement and Imaging (CMI) Programs of the Chemistry Division, Professors John Conboy and Markus Babst of the University of Utah are exploring the structure of cell membranes. Cell membranes are composed of various types of molecules, but their major constituents are lipid molecules, which themselves are composed of long oily hydrocarbon chains that avoid contact with water (hydrophobic), and one end that contains phosphorus and or oxygen atoms that make that end prefer to be in the presence of water (hydrophilic). Cell membranes are actually two layers of lipid molecules; the long hydrophobic chains on the two layers face each other inside the membrane, and the hydrophilic head groups are exposed to watery environments inside and outside the cell. In living cells, the membrane can be asymmetric; in other words, the number and specific kinds of lipids in the inner and outer lipid layers are different. The current theory of cell membrane structure assumes that this asymmetry is due to the action of another class of molecules present in cell membranes called flippases and floppases. These are proteins that are thought to control movement of lipid molecules from one side of the cell membrane to the other. Professors Conboy and Babst hypothesize that membrane asymmetry can arise without flippases and floppases in the membrane, and simply through inner membrane wall interactions with proteins inside the cell. In order to test their hypothesis, Professors Conboy and Babst are using a special technique called sum-frequency vibrational microscopy that can reveal the molecular structure of the lipid molecules in a membrane as well as the motion of these molecules from one layer to the other. One goal of the project is to directly measure and generate images of lipid asymmetry in living cells for the first time. This research project seeks to reveal new fundamental insights into how large complex molecules interact with each other to form even larger assembled structures. The findings of this project are likely to influence how we think about living systems. This project is the vehicle for advanced training of two graduate students and two undergraduate researchers. In addition to formal training activities, the Conboy and Babst research groups are participating in public outreach activities to bring science to a wider audience. Our current view of the cell membrane was established in the early 1970’s by Singer and Nicolson. The fluid-mosaic model they proposed portrays the membrane as a “liquid-like” bilayer of lipids, cholesterol, and proteins, exhibiting rapid and free diffusion in the two dimensions parallel to the membrane surfaces. In stark contrast, the exchange of lipids between the leaflets of a bilayer was presumed to be prohibited by the large energetic barrier associated with translocating the hydrophilic lipid headgroup, through the hydrophobic membrane core. This static picture of lipid translocation (or flip-flop) has been a long-held belief in the study of membrane dynamics and is the basis for the current theories regarding the bilateral organization of cell membrane components, particularly phosphatidylserine (PS) asymmetry. We hypothesize that spontaneous PS flip-flop is a common, facile process in vivo. This prediction raises the question: How does the cell maintain PS asymmetry in the plasma membrane? Our hypothesis is that in vivo PS localization to the cytosolic leaflet of the membrane is driven by electrostatic interactions with cytoplasmic proteins, in particular, the proteins comprising the cytoskeleton. Our proposed model of PS asymmetry and the currently accepted view differ in that our model suggests a very dynamic membrane system, over the more conventional “static” picture of lipid asymmetry. This dynamic behavior changes the current theories regarding the creation and maintenance of lipid asymmetry; however, it also simplifies and unifies the explanation of many known lipid phenomena. Our hypothesis is being tested by construction of a unique sum-frequency vibrational microscope capable of directly quantifying and imaging lipid asymmetry in living cells and measuring native lipid flip-flop in vivo for the first time. The students engaged in this project are gaining knowledge and experience in rigorous physical and biophysical concepts as well as advanced imaging techniques based on nonlinear optical processes. Outreach activities include engagement of K-12 schools in the Salt Lake Valley, especially Catholic schools that serve higher percentages of underrepresented groups. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.