Last spring, Assistant Professor Khalid Salaita‘s lab was awarded a grant from the National Institute of General Medical Science (part of the NIH) to study the Notch signaling pathway and develop techniques to look at the forces applied at the interface of cell membranes. Originally named for its role in the formation of notched Drosophila wings, the Notch receptor play a crucial role in cell to cell communication, cell development and differentiation. Mutations in this transmembrane protein result in dysfunction of the entire pathway, which can lead to various types of cancers including T-cell acute lymphoblastic leukemia (T-ALL). Because Notch is so crucial in cell differentiation, having too much or too little present in the membrane can also cause tumor growth.
Notch’s ligand-binding domain exists outside the cell membrane and when it locates a ligand molecule on the surface of an adjacent cell they bind, and the signal pathway begins. Once the ligand is bound, a protease comes in and snips off the extracellular domain from the transmembrane domain. However, the site that gets cut is buried within the folded protein, suggesting there must be a conformational change to allow access to the site. The Salaita Lab hypothesizes this conformational change occurs via a mechanical force; the cell pulling back on Notch, exposing the cleavage site. Their lab is working on tagging ligands with chromophores and quenchers so they can use fluorescence to see the protein stretching as it is being pulled by the cell. By calibrating the fluorescence of a given chromophore/quencher pair to the amount of force being applied to stretch them apart, they can quantitatively look at mechanical force exerted by the cell.
The Notch receptor with a green fluorescent protein tag on the intracellular domain is overexpressed in mammalian cells and seeded onto a membrane surface functionalized with the ligand (DLL4-mCherry). The two proteins bind and the extracellular domain gets snipped, the domain inside the cell can then be cut and act as a transcription factor, starting the signaling pathway. (Fig. 1)
By labeling the ligand and Notch with fluorescence tags they can not only establish that the two are binding based on the overlap (Fig 2), but by studying the intensity of the fluorescence, they can determine the density of molecules at the interface and their binding stoichiometry.
Unlike other membrane proteins that have been more heavily studied, only parts of the Notch structure are known by either NMR or X-ray crystallography, which makes it even more difficult to work with. The Salaita lab is up for the challenge though: “Having a five year grant allows you to take a breath and really dive in to solving some hard problems,” said Salaita.
Notch receptor mechanotransduction could be just the tip of the iceberg, there could be thousands of other receptors where a similar mechanism could be in play. Developing the methodology to explore these forces will open up new avenues for understanding and ultimately controlling membrane proteins and the diseases to which they contribute.