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.

Organelles sequester biological phenomena within the cell, and allow an additional layer of complexity to life. The presence and maintenance of these organelles is crucial for cellular function. Two of the most expansive and complex organelles are the mitochondria and endoplasmic reticulum. These organelles contribute energy, protein folding and secretion, lipids, calcium regulation, and various other metabolites to the biology of the cell. Importantly, these organelles accumulate damage and cannot be derived de novo, therefore must be inherited and maintained in a functioning state. The study of these organelle quality control processes serves as the basis for my thesis. We use the budding yeast as a model organism to uncover conserved pathways affecting organelle, and ultimately cellular homeostasis. In yeast we find mitochondrial inheritance is critical for cell survival. Furthermore, not only is inheritance critical, but inheritance of a certain threshold of functional mitochondria appears critical in maintaining normal lifespan in yeast, identifying mitochondria as an aging determinant. By examining mutants that negatively affect mitochondrial inheritance in yeast, we established a role for phosphatidylcholine biosynthesis in organelle maintenance and inheritance. Glycerophospholipid biosynthesis plays a clear role not only in mitochondrial inheritance but also in that of the endoplasmic reticulum. We use insights gained from yeast to guide research into a human disease caused by similar glycerophospholipid biosynthetic deficiency.

Primary cilia are sensory organelles that facilitate early skeletal development, as well as maintenance and adaptation of bone later in life. These solitary, immotile organelles are known to be involved in cell differentiation, proliferation, and mechanotransduction, a process by which cells sense and covert external physical stimuli into intracellular biochemical signals. Bone is a metabolically active tissue that continuously recruits osteogenic precursors and relies on osteocytes, the sensory cells of bone, to coordinate skeletal maintenance. Overall bone quality is dependent on the integrity of the initial structure formed, as well as this organ’s ability to adapt to physical loads. Proper differentiation and controlled proliferation of osteogenic progenitors are critical to the initial formation of the skeleton, while osteocyte mechanotransduction is essential for adaptation of developed bone. These phenomena rely on primary cilia, but little is known about the origin of osteogenic precursors and the ciliary mechanisms that promote osteogenesis. In this thesis, we first characterize an osteochondroprogenitor (OCP) population that rapidly and extensively populates skeletal tissues during juvenile skeletal development (Chapter 2). We also demonstrate that the primary cilium is critical for these cells to differentiate and contribute to skeletogenesis. We then show this OCP population is required for adult bone adaptation and is mechanoresponsive (Chapter 3). Again, we demonstrate that primary cilia are necessary for these OCPs to sense physical stimuli and differentiate into active bone-forming cells. Finally, we identify a novel link between ciliary calcium and cAMP dynamics in the osteocyte primary cilium (Chapter 4). Specifically, we show that a calcium channel (TRPV4) and adenylyl cyclases, which produce cAMP, bind calcium to mediate calcium entry and cAMP production, respectively, and these phenomena are critical to fluid flow-induced osteogenesis. Collectively, our results demonstrate that an easily extracted progenitor population is pre-programmed towards an osteogenic fate and extensively contributes to bone generation through primary cilium-mediated mechanisms at multiple stages of life. Furthermore, we identified ciliary proteins that are potentially unique to the osteocyte and can be manipulated to encourage osteogenesis by tuning calcium/ cAMP dynamics. For these reasons, we propose that this OCP population and their primary cilia, as well as osteocyte ciliary proteins that coordinate calcium/ cAMP dynamics, are attractive therapeutic targets to encourage bone regeneration.

Thesis (M. Sc.)--York University, 2000. Graduate Programme in Kinesiology and Health Sciences. / Typescript. Includes bibliographical references (leaves 31-46, 67-72). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ59171.

Thesis (Ph. D.)--York University, 1999. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ56221.

The Chediak-Higashi Syndrome is a disorder affecting lysosome biogenesis. At the cellular level, the Chediak-Higashi syndrome is characterized by the presence of grossly enlarged lysosomes in every tissue. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. The Chediak-Higashi syndrome is caused by the loss of a BEACH protein of unknown function named Lyst. In this work, I have studied the function of the Dictyostelium LvsB protein, the ortholog of mammalian Lyst and a protein that is also important for lysosomal function. Using a knock-in approach we tagged LvsB with GFP and expressed it from its single chromosomal locus. GFP-LvsB was observed on endocytic and phagocytic compartments. Specific analysis of the endocytic compartments labeled by LvsB showed that they represented late lysosomes and postlysosomes. The analysis of LvsB-null cells revealed that loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. This work demonstrated that the abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of lysosomes with postlysosomal compartments. Furthermore, this work provided the first evidence that LvsB is a functional antagonist of the GTPase Rab14 in vesicle fusion events. In particular, we demonstrated that reduction of Rab14 activity suppressed the LvsB-null phenotype by reducing the enlarged post-lysosomes and the enhanced rate of heterotypic fusion. In contrast, expression of an active form of Rab14 enhanced the LvsB-null phenotype by causing an even more severe enlargement of endosome size. The results provided by this work support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early with late compartments and antagonize Rab GTPases in membrane fusion. The LvsB localization studies and the functional assessment of the LvsB-null phenotype helped make unique contributions to the understanding of the molecular function of Lyst proteins.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (PH. D.)--Michigan State University. Genetics, 2008. / Title from PDF t.p. (viewed on Sept. 2, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 82-90).

The lysosome, a multi-copy organelle, was chosen as an example to study intraorganelle dynamics. Lysosomal contents and membrane proteins were shown to intermix rapidly in fused mammalian cells, with a t_½ of ~30 min. Lysosomal content intermixing, shown by a sensitive invertase-lysosome/[¹⁴C]-sucrose-lysosome pairing assay, was inhibited greatly by ATP inhibitors and partially by cytochalasin D. Lysosomal membrane protein intermixing was shown by the transfer of LAMP-2, a mouse specific lysosomal membrane antigen, from mouse lysosomes to hamster sucrosomes, sucrose-swollen lysosomes. Lysosomal membrane protein intermixing was also shown by the co-localization of LIMP I, a rat specific lysosomal membrane antigen, and LAMP-1, a mouse specific lysosomal membrane antigen. Co-localization was assessed by both double immunofluorescent staining and double immunogold labeling of thin cryosections. Both lysosomal content and membrane protein intermixing were inhibited by nocodazole, a microtubule disruptor. In fused cells, lysosomes remained small, punctate and scattered throughout the cytoplasm. In comparison to lysosomes, the prelysosomal compartment (PLC), a single copy organelle which is related to the lysosome, congregated together to form an extended PLC complex associated with clustered nuclei. The intermixing of both resident and transient Golgi membrane proteins was studied in fused cells. Resident Golgi membrane protein intermixing was slow, with a t_½ of ~ 1.75 h; it was concomitant with the congregation of the Golgi units. In comparison, the transient Golgi membrane protein was transported much faster from Golgi units to the other Golgi units, with the t_½ ≤ 15 min. Transient Golgi membrane protein transport occurred between separate Golgi units. These results are consistent with two different pathways for resident and transient Golgi membrane protein transport: a slow, lateral diffusion along the Golgi connections transport pathway for resident Golgi membrane proteins; and a rapid, transient protein selective, vesicle-mediated transport pathway for transient Golgi membrane proteins. / Ph. D.