Molecular rulers to measure membrane thickness in live cells

The plasma membrane defines the boundary of a cell, and many internal cellular processes are compartmentalised by membrane-bound organelles. Membranes are primarily composed of proteins and lipids, which are small greasy molecules that do not mix well with water, leading to the formation of separate membrane structures. Cells make many thousands of lipids and proteins that are chemically distinct. Biologists know why cells make so many different proteins - they have many functions within cells. However, it is less clear why cells invest so heavily into making many different types of lipids. One reason could be that lipids change the architecture of the membrane. This would affect how thick or thin the membrane is, how much it can bend and how far or rapidly proteins within the membrane can move. Changes in membrane architecture would affect, for example, how quickly a cell would be able to respond to a signal, or how much of a certain molecule it could make in a specific location. Although conceptually it is clear that membrane architecture, and specifically membrane thickness must be important in how the cell functions, it has so far not been possible to measure membrane thickness systematically in live human cells.

We are proposing to develop new chemical tools, called molecular rulers, that will allow us to measure how thick membranes are inside and at the exterior of cells. Our tools will use engineered DNA as a scaffold because it has precisely defined dimensions and it is possible to attach other chemicals to it. We will cloak part of the DNA with lipid-like molecules to allow it to move into membranes. We will also attach dyes at specific distances to each other. One of the dyes will change in brightness and colour depending on if it is inside or outside of a membrane. Since we will synthesise the molecular rulers with specific dimensions, we can use microscopy to detect changes in dye brightness and conclude how thick the membrane is at a specific location. We then plan to use the tools to understand how and why cells change their membrane thickness at different locations.

The physicochemical properties of membranes, including membrane thickness, are thought to be crucial for their functions. However, a lack of tools has hindered systematic investigations of membrane thickness in live mammalian cells. We propose here to develop and validate a set of DNA-based molecular rulers to measure membrane thickness in the plasma membrane as well as in internal organelles. These rulers will include hydrophobic belts to allow insertion of the DNA nanostructure into membranes, as well as fluorophores attached at defined distances, allowing quantitative analysis. Rulers designed to probe membranes in the interior of cells will carry SNAP-tag-reactive groups to direct them to SNAP-tag labelled organelle proteins. Using these molecular rulers, we will investigate how cells utilise changes in membrane thickness, for example, in the plasma membrane as they move or divide. This study will be the first time it will have been possible to investigate systematically and quantitatively how and why cells vary membrane thickness in different locations and during different processes.