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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.

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.

We design actuated cilia that can maneuver microscopic particles normal to a microfluidic channel wall and transport microscopic particles parallel to the channel wall. For identifying the design specifications, we employ a hybrid LBM/LSM computational model, to simulate hydrodynamic interactions between oscillating elastic cilia and microscopic particles in a microfluidic channel. The oscillating synthetic cilia are elastic filaments tethered to the channel wall and actuated by sinusoidal force acting at their free ends. The cilia are arranged in a square pattern. The microscopic particle is a neutrally buoyant solid sphere, which is sufficiently small compared to the cilium length and inter-cilium distances, so that the particle can move freely inside the ciliated layer. We study the effect of actuation frequency on the particle motion inside the ciliated layer. We show that depending on the frequency, particles can be either driven away from the ciliated channel wall or drawn towards the wall. We also examine how to use inclined cilia to transport particles along the ciliated layer. We show that the particle transport along the ciliated layer can be regulated by the frequency of cilium oscillation. The results uncover a new route for regulating particle position and transport in microfluidic devices.

The vertebrate planar cell polarity (PCP) pathway has been previously found to control polarized cell behaviors rather than cell fate. We report here that disruption of Xenopus laevis orthologs of the Drosophila melanogaster PCP genes Xint or Xfy affected not only PCP-dependent convergent extension but also caused embryonic phenotypes consistent with defective Hedgehog signaling. These defects in Hedgehog signaling resulted from a broad requirement for Inturned and Fuzzy in ciliogenesis. We show that these proteins are necessary for the formation of both primary cilium in the neural tube and multi-cilia in the epidermis. Also, using Xenopus muco-ciliary epidermis, we demonstrated that one of the core PCP genes Dishevelled performs dual functions in ciliogenesis, basal body docking and planar polarization of ciliary beating. To this end, we showed that Dishevelled works in concert with the PCP effector protein Inturned and Rho GTPase to mediate the docking of basal bodies to the apical cell surface. We suggest that this docking involves a Dvl-dependent association of basal bodies with vesicles, and with the vesicle-trafficking protein Sec8. Finally, we showed that independent of their roles in apical docking, Dvl/PCP signaling is required again for directional ciliary beating. For the first time, this study uncovered the mechanism for controlling the apical docking of basal bodies. Moreover, the results suggest that the same Dvl/PCP signaling is also important for the planar polarization of ciliary beating in a vertebrate muco-ciliary epithelium. / text

published_or_final_version / Medicine / Master / Master of Research in Medicine

Typewritten copy / Includes bibliographical references (leaves 141-144) / [5], 144 leaves : ill. ; 27 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / An account of experimental investigations carried out from January 1952 to September 1954. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physics, 1956