Unveiling the Molecular Architecture of Mammalian Ciliary Doublet Microtubules and Mechanisms of von Willebrand Factor Concatemerization

Abstract

Cilia are molecular appendages of cells that accomplish tasks requiring stunning coordination. Remarkable examples in humans include motile cilia in the respiratory tract that utilize motility to escalate allergens and pathogens out of our airways, motile cilia on ependymal cells that circulate cerebrospinal fluid about our brain ventricles, and motile ciliated cells in oviducts that utilize motile cilia to facilitate movement of an ovum to the uterus. More than fifty years before this dissertation, the gross protein ultrastructure within cilia was observed as a “9+2” arrangement of microtubules: 9 doublet microtubules surrounding a central pair of microtubules. Since then, advances in electron microscopy have allowed increasingly detailed structural analysis of ciliary components. These advances led to the residue-resolution reconstruction of the doublet microtubule (DMT) from Chlamydomonas reinhardtii – a model organism for studying ciliary biology. This approach reached resolutions that allowed identification of proteins bound to the DMT from native cilia purifications using cryo-EM density, mass-spectrometry, and homology model docking strategies. Utilizing these technological advances, this thesis extends this high-resolution structural analysis to mammalian cilia. In Chapter 1, an average of the 48-nm repeating biological unit of the Bos taurus DMT from respiratory cilia is presented, illustrating protein components unique, and similar, to those observed in C. reinhardtii. In Chapter 2, the protein network of the 48-nm repeat of the human female oviduct ciliary DMT is described and compared to human respiratory and sperm DMTs. In parallel, I use a subtomogram averaging workflow to determine an intermediate resolution (9.5 – 11.1 Å) reconstruction of the porcine oviduct DMT, allowing comparisons between human and porcine oviduct MIP repertoires. Work that leverages high-resolution cryo-EM density to distinguish tubulin isotypes in averages of the 48-nm repeat from human respiratory and oviduct DMTs is also included in Chapter 2. Distinct from DMTs in biological function and structure is von Willebrand Factor (VWF). VWF is a crucial hemostatic protein. VWF functions in primary hemostasis by tethering platelets to sites of endothelial damage. VWF also has a role in secondary hemostasis through its stabilization of Factor VIII. VWFs hemostatic function is accomplished through a striking biogenesis pathway that results in VWF forming a tubule like structure at low pH on its way to forming “high molecular weight concatemers” (HMWC). Despite the centrality of VWF to hemostasis in humans, the exact residues that mediate the covalent linkage that endows VWF with the ability to form hemostatic HMWC has not been determined conclusively. Chapter 3 focuses on several of these issues – identifying the residues involved in the concatemerization of VWF into HMWC and clarifying the tubule architecture VWF takes on in the low pH of the trans-Golgi and in the Weibel Palade body. In low pH conditions similar to those that allow VWF tubule formation, the C-terminal portion of VWF has been shown to zipper between VWF polypeptides to form a structural arrangement termed a “dimeric bouquet”, thought to extend extraluminally from the VWF tubule. Experiments focused to determine the domain architecture and residue arrangement of the dimeric bouquet is also presented in Chapter 3.