The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

SPARC is a collagen‐binding, matricellular glycoprotein with diverse roles in tissue remodeling and development. Previous studies have demonstrated that SPARC is required in Drosophila for larval development and maintenance of the fat body, an organ that incorporates endocrine, growth and immune functions. I have characterized effects of loss and knockdown of SPARC in the fat body. Loss‐of‐function analyses revealed remodeling of adipocytes demarcated by cell rounding and dense accumulation of extracellular matrix (ECM) beneath an abnormally thick basement membrane. Remodeling of adipocytes mediated by expression of matrix metalloproteinase 2 (MMP2) was found to cause ECM breakdown and accumulation of hemocytes, indicating endogenous fat body remodeling is mechanistically distinct from that which occurs upon silencing of SPARC. Knockdown of the lysyl hydroxylase, dPlod, in the fat body, revealed abnormal intracellular co‐localization of SPARC with Collagen IV, but not with Laminin. The data indicate SPARC is required for ECM homeostasis during development.

Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive. In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.

Microglia, immune cells of the central nervous system, activate in response to pathophysiological stimuli. One of their reactive phenotypes is to migrate to site of injury where they could have either beneficial or detrimental effects. However, little is known regarding the mechanisms underlying microglial migration and how they traverse the unique extracellular environment in brain tissue to reach their destination. Our laboratory first discovered that microglia express structures called podosomes, which can adhere to as well as degrade extracellular matrix. In this study, I further characterize microglial podosomes, and show that they associate with Iba1, Orai1 and calmodulin, proteins not yet observed in podosomes of other cell types. I also present evidence that podosome formation depends on Ca2+ and its entry through store-operated Ca2+ channels. The findings in this thesis contribute to a better understanding of podosome dynamics and their probable roles in microglia migration.

Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Microglia, immune cells of the central nervous system, activate in response to pathophysiological stimuli. One of their reactive phenotypes is to migrate to site of injury where they could have either beneficial or detrimental effects. However, little is known regarding the mechanisms underlying microglial migration and how they traverse the unique extracellular environment in brain tissue to reach their destination. Our laboratory first discovered that microglia express structures called podosomes, which can adhere to as well as degrade extracellular matrix. In this study, I further characterize microglial podosomes, and show that they associate with Iba1, Orai1 and calmodulin, proteins not yet observed in podosomes of other cell types. I also present evidence that podosome formation depends on Ca2+ and its entry through store-operated Ca2+ channels. The findings in this thesis contribute to a better understanding of podosome dynamics and their probable roles in microglia migration.

Cystic Fibrosis (CF) is a life-threatening autosomal recessive disease affecting 1:3600 children born in Canada. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. The most common disease causing mutation is a deletion of residue F508, resulting in a structurally compromised protein which is retained in the endoplasmic reticulum and targeted for proteasomal degradation. Therapeutic strategies currently being pursued to alleviate the afflictions caused by this and other mutants include the use of corrector compounds to modify the surface expression of the channel, and potentiator compounds to increase cAMP-mediated channel activity. Despite the discovery of a number of small molecules affecting CFTR, much is still unknown about the nature of these interactions. This thesis contains the investigation of two potentiators: VRT-532 and VX-770, and two correctors VX-809 and C18. We assessed the consequences of interactions with these drugs on CFTR channel activity, ATPase activity and phosphorylation. We demonstrated that VRT-532 binds directly to mutant CFTR to modify its channel and ATPase activity. VX-770, known to bind directly to CFTR, can stimulate channel activity in the absence of cAMP stimulation in baby hamster kidney (BHK) cells. Correctors VX-809 and C18, based off the same molecular scaffold, are both capable of acutely augmenting cAMP-stimulated channel activity, providing evidence for potentiator activities in these compounds. Quantitative mass spectrometry (MS) techniques demonstrate a defect in phosphorylation at Ser-660 in the regulatory (R) domain in the major mutant. Treatment with C18 was unable to repair this defect. These novel findings regarding interactions between several small molecules and CFTR contributes to the understanding of the mechanism of action of these compounds, and will help identify how they may be modified for greater efficacy to improve the treatment of CF disease.

Cystic Fibrosis (CF) is a life-threatening autosomal recessive disease affecting 1:3600 children born in Canada. CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel. The most common disease causing mutation is a deletion of residue F508, resulting in a structurally compromised protein which is retained in the endoplasmic reticulum and targeted for proteasomal degradation. Therapeutic strategies currently being pursued to alleviate the afflictions caused by this and other mutants include the use of corrector compounds to modify the surface expression of the channel, and potentiator compounds to increase cAMP-mediated channel activity. Despite the discovery of a number of small molecules affecting CFTR, much is still unknown about the nature of these interactions. This thesis contains the investigation of two potentiators: VRT-532 and VX-770, and two correctors VX-809 and C18. We assessed the consequences of interactions with these drugs on CFTR channel activity, ATPase activity and phosphorylation. We demonstrated that VRT-532 binds directly to mutant CFTR to modify its channel and ATPase activity. VX-770, known to bind directly to CFTR, can stimulate channel activity in the absence of cAMP stimulation in baby hamster kidney (BHK) cells. Correctors VX-809 and C18, based off the same molecular scaffold, are both capable of acutely augmenting cAMP-stimulated channel activity, providing evidence for potentiator activities in these compounds. Quantitative mass spectrometry (MS) techniques demonstrate a defect in phosphorylation at Ser-660 in the regulatory (R) domain in the major mutant. Treatment with C18 was unable to repair this defect. These novel findings regarding interactions between several small molecules and CFTR contributes to the understanding of the mechanism of action of these compounds, and will help identify how they may be modified for greater efficacy to improve the treatment of CF disease.

Calreticulin, an endoplasmic reticulum (ER)-resident protein, is a calcium buffering chaperone. In this study, with an optimized differentiation protocol from mouse R1 ES cells, we demonstrate a novel role of calreticulin in osteogenic commitment and differentiation. To enhance the efficacy of the method, we manipulated cell density and examined the addition of retinoic acid, dexamethasone and peroxisome proliferator-activated receptor γ. The regimen consisting of seeding 250 cells per embryoid body, with the addition of RA (from day 3 to 5) and Dex (from day 10 to 21) gave the most efficacious output. Using this optimized protocol, we investigated the potential involvement of calreticulin in osteogenesis. Calreticulin knock-out cells displayed impaired osteogenesis compared to wild-type cells. In particular, the nuclear translocation of the runt-domain related transcription factor 2 and Osterix, were impaired in the absence of calreticulin. The stimulatory effect of calreticulin on osteogenesis was mediated by its calcium buffering function.

Glycosylation, the addition of sugar moieties to nascent proteins, is one of the most common posttranslational modifications. Glycosylation regulates protein structure, function and localization. Most cell surface proteins and secreted proteins are glycosylated by the addition of Asparagine(N)-linked glycans (N-glycans). Aberrant N-glycosylation is a well-accepted feature of malignancy and is a potential prognostic marker for some types of cancer. For example, increased expression of complex N-glycans has been detected in cancers of breast, colon and has been correlated with reduced survival of the patients. Therefore, understanding the role of N-glycosylation in malignancy could be beneficial for developing novel therapeutic and prognostic strategies. To examine the role of N-glycosylation in malignancy, we applied chemical biology and genetic approaches. First, we conducted a high throughput screen to identify compounds that could block L-PHA-induced cell death. Our screen identified the cardiac glycoside Na+/K+-ATPase inhibitors as novel inhibitors of N-glycosylation. Further analysis of N-glycans consistently confirmed that inhibition of Na+/K+-ATPase impairs the N-glycosylation, as well as migration and invasion. Interestingly, other studies have shown antimetastatic effects of cardiac glycosides in patients. Thus, our high throughput screen identified Na+/K+-ATPase inhibition as a novel strategy to target the N-glycosylation pathway. In addition, we used a genetic approach to investigate the role of N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) in malignancy. Knockdown of GlcNAc-TI decreased the cell-surface expression of complex N-glycans. By confocal microscopy, knockdown of GlcNAc-TI decreased cell surface expression of β1 integrins and increased their localization around the nucleus. Moreover, GlcNAc-TI knockdown decreased the migration and invasion of malignant cells. Next, we investigated the effect of GlcNAc-TI in an orthotopic xenograft mouse model of metastasis. GlcNAc-TI knockdown significantly decreased the lung colony formation of the highly metastatic PC3N7 human prostate cancer cell line in mice. Our results suggest an important role for GlcNAc-TI in tumor metastasis. Interestingly, breast cancer patients with lower expression levels of Mgat1 had lower risk of disease relapse after therapy. Thus, GlcNAc-TI plays an important role in cancer progression and metastasis and GlcNAc-TI inhibitors could have therapeutic benefits for cancer patients. Moreover, expression levels of GlcNAc-TI could be used as a prognostic marker in patients with cancer.