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The developing brain requires an intricate network of signals to direct proliferation, differentiation and cell fate decisions. Primary cilia are vital organelles with an emerging role regulating several major signalling cascades, in particular the Hedgehog pathway. Talpid3 (Ta3) is 166.7 kD protein found at the distal tip of centrioles. It has been shown to interact with a number of key centriolar proteins and is essential for the formation of primary cilia. A recent mouse model has been designed to conditionally target the highly conserved coiled-coil domain of Ta3 using the Cre/loxP system. This project uncovers the role of Ta3 in the developing brain. It characterises in detail the phenotype of mice with conditional loss of Ta3 in the central nervous system using the Nestin-Cre deleter strain. Morphological and histological analyses demonstrate that significant defects occur postnatally with mice developing severe ataxia and hydrocephaly. Immunohistochemical techniques further characterise the distinct phenotypes of three key brain regions including the cerebellum, cortex and hippocampus. Ta3fl/fl;NesCre mutant mice exhibit defects in the proliferation, organisation, morphology and migration of both neuronal and glial cells. We have shown the mechanistic cause to be the result of widespread loss of primary cilia and a concomitant disruption in the transduction of the Hedgehog signalling pathway. The neural roles of Ta3 are explored further through the optimisation of an in vitro neurosphere system to culture postnatal hippocampal progenitors. The use of a tamoxifen inducible strain allows the timely recombination of Ta3 to study its role in a controlled environment. The cultured cells recapitulate many of the in vivo defects showing loss of primary cilia and reduced migration. Finally, characterisation of the phenotypes seen in the Ta3fl/fl;NesCre mice were shown to resemble neurological traits seen in human conditions with loss of Primary cilia, known as ‘human ciliopathies’. Through clinical collaboration this project demonstrated a human ciliopathy case of Joubert Syndrome with compound heterozygous mutations in TA3. This presents the Ta3fl/fl;NesCre mutant mice as a valuable model system to study a rare but clinically relevant condition.

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the hedgehog signaling, cAMP pathway, Wnt pathways, etc. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). IFT88, one of the important IFT proteins in complex B, is known to play a role in the formation and maintenance of cilia in various types of organisms. The ciliary transition zone (TZ), which is part of the gating apparatus at the ciliary base, is home to a large number of ciliopathy molecules. Recent studies have identified important regulating elements for TZ gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation processes. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a series of stimulated emission depletion (STED) superresolution images mapped to electron microscopy images, we analyzed the structural organization of the ciliary base. Subdiffraction imaging of TZ components defines novel geometric distributions of RPGRIP1L, MKS1, CEP290, TCTN2 and TMEM67, shedding light on their roles in TZ structure, assembly, and function. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227±18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205±20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165±8 nm diameter. MKS1 was situated between TMEM67 and RPGRIP1L, with an 186±21 nm diameter. Surprisingly, CEP290 was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to the width of the basal body, distant from the potential Y-links region of the TZ. Moreover, IFT88 was intriguingly distributed in two distinct patterns, forming three puncta or a Y shape at the ciliary base found in human retinal pigment epithelial cells (RPE), human fibroblasts (HFF), mouse inner medullary collecting duct (IMCD) cells and mouse embryonic fibroblasts (MEFs). We hypothesize that the two distribution states of IFT88 correspond to the open and closed gating states of the TZ, where IFT particles aggregate to form three puncta when the gate is closed, and move to form the branches of the Y-shape pattern when the gate is open. Two reservoirs of IFT particles, correlating with phases of ciliary growth, were localized relative to the internal structure of the TZ. These subdiffraction images reveal unprecedented architectural details of the TZ, providing a basic structural framework for future functional studies. To visualize the dynamic movement of IFT particles within primary cilia, we further conducted superresolution live-cell imaging of IFT88 fused to EYFP in IMCD cells. Our findings, in particular, show IFT88 particles pass through the TZ at a reduced speed by approximately 50%, implying the gating mechanism is involved at this region to slow down IFT trafficking. Finally, we report the distinct transport pathways of IFT88 and Smo (Smoothened), an essential player to hedgehog signaling, to support our hypothesis that two proteins are transported in different mechanisms at the ciliary base, based on dual-color superresolution imaging.

Mechanosensation, the ability for cells to sense and respond to physical cues, is a ubiquitous process among living organisms and its dysfunction can lead to devastating diseases, including atherosclerosis, osteoporosis, and cancer. The primary cilium is a solitary, immotile organelle that projects from the surface of virtually every cell in the human body and can function as a mechanosensor across diverse biological contexts, deflecting in response to fluid flow, pressure, touch and vibration. It can detect urinary flow rate in the kidney, monitor bile flow in the liver, and distinguish the direction of nodal flow in embryos. In this thesis, we examined the interplay of biology and mechanics in the context of this multifunctional sensory organelle from the tissue to subcellular scale. In the first part of this work, we examined the cilium at the tissue level. Primary cilia are just beginning to be appreciated in bone with studies recently reporting loss of cilia results in defects in skeletal development and adaptation. We disrupted primary cilia in osteocytes, the principal mechanosensing cells in bone, and demonstrated that loss of primary cilia in osteocytes impairs load-induced bone formation. Over the course of our work with primary cilia, we also identified the need for more standardized imaging approaches to the cilium and presented an improvement to distinguishing proteins within the cilium from the rest of the cell. In the later part of this work, we examined the primary cilium at the subcellular level. While deflection is integral to the cilium's mechanosensory function, it remains poorly understood and characterized. Using a novel experimental and computational approach to capture and determine the mechanical properties of the cilium, we demonstrated cilium deflection can be mechanically and chemically modulated. We revealed a mechanism, acetylation, through which this mechanosensor can adapt and regulate overall cellular mechanosensing. By modifying our combined experimental and computational approach, we analyzed cilium deflection in vivo for the first time. Collectively, this work uncovers new insights across biological scales in the primary cilium as an extracellular nexus integrating mechanical stimuli and cellular signaling. Understanding the mechanisms driving cilium mechanosensing has broad reaching implications and unlocks the cilium's potential as a therapeutic target to treat impaired cellular mechanosensing critical to a multitude of diseases.

Osteoporosis is a devastating condition characterized by decreased bone mass, and affects over 50% of the population over 50 years old. Progression of osteoporosis results in significantly heightened risk of fracture leading to loss of mobility, prolonged rehabilitation, and even mortality due to extended hospitalization. Current therapeutic options exist to combat low bone mass, but these treatments are being met with increasing concern as reports emerge of atypical fractures and necrosis. Thus, new therapeutic strategies are required. Bone is highly dynamic, and it has long been known that physical load is a potent stimulus of bone formation. Despite this, none of the current treatments for bone disease leverage the inherent mechanosensitivity of bone – the ability of bone cells to sense and respond to mechanical forces such as exercise. One potential therapeutic target is the primary cilium. Primary cilia are solitary antenna-like organelles, and over the last 20 years have been identified as a critical cellular mechanosensor. Primary cilia and cell mechanotransduction are critical to the function of numerous cells and tissues. Thus, understanding primary cilia-mediated mechanotransduction has potential applications in treating kidney and liver disease, atherosclerosis, osteoarthritis, and even certain cancers. Previous work from our group has demonstrated that disruption of the cilium impairs bone cell mechanosensitivity, resulting in abrogated whole bone adaptation in response to physical load. In this thesis we examine the potential of targeting the primary cilium to enhance bone cell mechanosensitivity and promote whole bone formation. First, we demonstrate the pharmacologically increasing primary cilia length significantly enhances cell mechanotransduction. Next, we expand our list of candidate compounds to manipulate ciliogenesis through the use of high-throughput drug screening. We developed an automated platform for culturing, staining, imaging, and analyzing nearly 7000 small molecules with known biologic activity, and classify them based on mechanism of action. One of these compounds is then used in a co-culture model to study the effects of manipulating osteocyte primary cilia-mediated mechanosensing on pro-osteogenic paracrine signaling to promote the activity of bone-forming osteoblasts and osteogenic differentiation of mesenchymal stem cells. Finally, we translate our in vitro findings into an in vivo model of load-induced bone formation using the same compound to enhance cell mechanotransduction. We demonstrate that we can sensitize bones to mechanical stimulation to enhance load-induced bone formation in healthy and osteoporotic animals, with minimal adverse effects. Together, this work demonstrates the therapeutic potential and viability of targeting primary cilia-mediated mechanotransduction for treating bone diseases.

Mutations of the gene encoding the inositol polyphosphate 5-phosphatase OCRL1 are responsible for causing two disorders in humans: Lowe syndrome and type 2 Dent's disease (Dent-2). Lowe syndrome (oculocerebrorenal syndrome of Lowe) is an X-linked genetic disorder that causes multisystem defects affecting predominantly the eyes, brain and kidneys. Dent-2 disease is very similar to Lowe syndrome, but it affects primarily the kidneys with little or no symptoms in the brain and eyes. The enzymatic activity, structure and binding partners of the OCRL1 protein have been described and progress on the cellular functions of OCRL1 has been made. However the studies to date have not provided the necessary insight to explain the tissue-specific defects observed in Lowe syndrome and Dent-2 patients. In order to investigate the role of OCRL1 and the consequences of its deficiency in a physiological context an animal model is required. We have chosen the zebrafish for this study due to its suitability for investigating vertebrate early development and the abundance of research techniques available for this model organism. We have studied the expression of OCRL1 in zebrafish and its role in the early embryonic development. We have also investigated its role in the endocytic function of the zebrafish larval pronephric kidney. Finally we have investigated its role in ciliogenesis and function of pronephric cilia. Our studies show that OCRL1 depletion does not cause gross developmental defects, nor affects the development of pronephros, but impairs their endocytic activity. We have also shown, that efficient pronephric uptake requires OCRL1 interactions with clathrin, Rab GTPase family proteins, APPL1 and IPIP27A/B. Our studies link the reduced uptake with lowered levels of megalin receptor, which is responsible for the bulk of protein reabsorption in the kidney. Together our results strongly suggest that defects in this process are responsible for low molecular weight proteinuria present in Lowe syndrome and Dent-2 patients and zebrafish is a suitable model to study the renal aspect of these diseases.

During embryogenesis, airway epithelial cells possess primary cilia, and HH signaling guides lung development. As epithelial cells mature, they produce hundreds of motile cilia and continue to produce the sonic hedgehog (SHH) ligand, which is found apically in the thin layer of liquid covering airways. However, whether ciliated airway cells express apical HH signaling components and what their function might be have remained unknown. Here we show that motile cilia are enriched for HH signaling proteins, including patched 1 and smoothened. These cilia are also enriched for proteins affecting cAMP-dependent signaling, including Gαi and adenylyl cyclase 5/6. Surprisingly, SHH in differentiated airway epithelia did not elicit the canonical SHH signaling pathway that regulates transcription during development. But instead, activating HH signaling decreases intracellular levels of cAMP, which reduces ciliary beat frequency and airway surface liquid pH, similar to changes that have been observed in the airway of people with chronic obstructive pulmonary disease (COPD). Furthermore, we observed that significant increase of SHH ligand expression in differentiated airway epithelia with COPD, suggesting a potential role of SHH signaling in the pathogenesis of airway disease. Collectively, our study indicates that airway cilia detect apical SHH to mediate airway physiology through noncanonical HH signaling. SHH may dampen defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. This may suggest a potential role of SHH signaling in the pathogenesis of airway disease, such as COPD.

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Typewritten copy Includes bibliographical references (leaves 141-144) Pt. 1. Historical review -- pt. 2. Techniques used in electron microscopy -- pt. 3. Experiments on cilia from Hydrdella australis -- pt. 4. Electron microscope experiments on cilia from the rat trachea -- pt. 5. Electron microscope experiments on cilia from paramecium -- pt. 6. Electron microscope experiments on the trichocysts of paramecium -- pt. 7. Discussion An account of experimental investigations carried out from January 1952 to September 1954.