Title: Visualizing cell surface interactions using cryogenic electron microscopy
Description: The study of the three-dimensional structures of biological macromolecules has given us significant insight into life and its mechanisms. Understanding these structures in their native contexts, a challenging but important goal, came closer to reality with the development of electron microscopy. After many years of technological development, we are now starting to understand previously intractable biological phenomena at an unprecedented resolution. One such phenomenon is how neighboring cells interact, both to communicate and send signals, and to adhere and form complex tissue structures. While the molecules that mediate such processes have long been studied in isolation, electron microscopy allows us to examine them in a more native biophysical environment; as hydrated, dynamic molecules tethered to opposed cellular membranes.Imaging unadulterated biological material using electron microscopy requires that the sample be embedded in a thin layer of vitreous ice to immobilize the molecules and protect them from the vacuum of the microscope, and thus is generally referred to as cryogenic electron microscopy (cryo-EM).

Samples can be imaged using two common cryo-EM modalities: single particle analysis (SPA), where many two-dimensional projection images of molecules in solution are collected, and cryo-electron tomography (cryo-ET), where the sample is tilted as it is imaged at multiple angles to reconstruct a three-dimensional volume. In this work, I will describe how I have used both SPA and cryo-ET to understand cell surface interactions involving a variety of proteins. The first chapter will look at the cell surface molecules known as the Toll receptors, a family of molecules found in Drosophila melanogaster, with orthologs in mammals known as the Toll-like receptors (TLRs). I will focus on their role in the development of the Drosophila embryo during germ band extension, a kind of convergent extension that is a conserved process through all metazoans. Biophysical assays of the three implicated Toll receptors, Toll-2, -6, and -8, revealed both homophilic and heterophilic interactions. SPA was used to determine the structure of monomeric Toll-2 which closely resembles the overall fold of Toll, whose structure was previously solved by x-ray crystallography.

Surface plasmon resonance (SPR) spectroscopy and analytical ultracentrifugation (AUC) showed Toll-6 is a dimer in solution, which I visualized using cryo-EM. The Toll-6 homodimer is a novel dimer interface for Tolls and TLRs, where molecules on the same cell surface have been shown to dimerize in the presence of a wide variety of ligands. In contrast, the Toll-6 dimer is formed in the absence of any ligand and exists in an antiparallel arrangement that could be formed by molecules on opposing cell surfaces. Together, these results provide a biochemical basis for germ band extension which may be further explored through the study of structure-based mutations. While cryo-EM SPA is a powerful tool, cryo-ET allows one to reconstruct three dimensional volumes of highly heterogeneous samples, such as the interior of cells, where molecules of interest may not exist in enough copies to facilitate averaging. This technique, where the sample is imaged multiple times as it is tilted to obtain three-dimensional information of a region of interest, was used to study cell adhesion of a different type: that mediated by the classical cadherins.

These calcium-dependent adhesion molecules cluster into adherens junctions, spot-like protein densities found in a wide variety of tissues. In the second chapter, these junctions are recapitulated between synthetic liposome membranes by tethering the adherent cadherin molecules to chemically functionalized lipids. They are then imaged using cryo-ET to reveal higher-order structural details. First, this method is applied to the clustered protocadherins, a family of cadherins that mediate neuronal self-avoidance in mammals. Cryo-ET in combination with x-ray crystallography revealed that clustered protocadherins form extended one-dimensional zippers between membranes, which are a combination of strictly homophilic trans interactions coupled with promiscuous cis interactions. Neurons express unique subsets of the ~50-60 possible isoforms, and when two neuronal processes express identical subsets, which happens only when those processes are a part of the same cell, these linear chains grow and initiate a repulsive signal.

If the subsets are different, the chains terminate and no repulsive signal is generated. The same technique has been used previously to study the type I classical cadherins, perhaps the most well-studied members of the cadherin superfamily. In the second half of this chapter, we extend our analysis to include the type II classical cadherins, which possess more complex expression patterns and binding specificities. Cryo-ET of type II cadherin ectodomains tethered to synthetic liposomes revealed that several representative members of this family form only moderately ordered arrays between liposomes, a finding in agreement with their role in cell sorting and migration. However, VE-cadherin, an outlier type II expressed in vascular endothelial cells where it withstands blood pressure, forms extraordinarily ordered junctions. Subtomogram averaging reveals the regularity of this two-dimensional array. In the final chapter, I describe my work on a membrane surface molecule of a different kind, one not involved in cell adhesion but viral infection.

The global COVID-19 pandemic gave me the opportunity to contribute to our understanding of SARS-CoV-2 by studying the structure of neutralizing antibodies bound to the viral spike protein, perhaps the most infamous membrane surface protein. The first subchapter describes the initial isolation, neutralization, and structural analysis of antibodies isolated from convalescent COVID-19 patients. This work revealed that patients with severe COVID-19 produce potently neutralizing antibodies that target two spike protein domains: the receptor binding domain (RBD) and the N-terminal domain (NTD). RBD-directed antibodies occlude binding to ACE2, the human receptor that mediates viral fusion, but the neutralization mechanism of NTD-directed antibodies is unknown. The following two subchapters are more detailed structural studies of two specific types of antibodies. The first looks at a class of RBD-directed antibodies derived from the VH1-2 gene, which are some of the most potent and common antibodies against SARS-CoV-2.

The heavy chains of these antibodies recognize almost identical epitopes, but the antibodies employ a modular approach to recognize the RBD in either of its possible conformations. The second class are antibodies that target the NTD, which our work revealed all bind to a single antigenic supersite. The final subchapter focuses on emerging SARS-CoV-2 variants and includes the structures of two antibodies that are still capable of neutralizing these new variants. They are also infrequent in the human antibody response to SARS-CoV-2, meaning they put little selective pressure on the virus to produce escape mutations, making them good candidates for monoclonal antibody therapies. Though Drosophila embryogenesis, adherens junction formation, and SARS-CoV-2 neutralization are seemingly unrelated systems, they are united by the incredible flexibility of cryo-EM to visualize biological molecules in more native environments. Whether it is the ability to study multiprotein complexes or assemblies formed between membranes, cryo-EM is a powerful technique that promises to help bridge the divide between structure and function.