Live cell imaging can be used to study dynamic cellular systems at single cell resolution. In heterogeneous cell populations, analyzing cell properties at the single cell level reduces the generalization of results caused by population-based assays. This thesis details the implementation of live cell imaging and single cell tracking to characterize heterogeneous cell systems undergoing dynamic processes over multiple generations. This approach enables the consideration of both spatial and temporal variables as well as the mapping of cell phenotype trajectories along their generational lineages. Cell-, lineage-, and colony-level properties are used as descriptors of the underlying molecular mechanisms that they are produced by. These may be unexpected, emergent properties that can not be predicted or completely characterized at the molecular level. Analysis of these properties can reveal and characterize the properties and processes of dynamic, heterogeneous cell systems. Live cell imaging culture strategies were developed to enable characterization of both two- and three-dimensional cell systems. Computational modeling was performed to evaluate the conditions imposed by a confined imaging chamber that enables single cell resolution imaging of monolayer and multilayer cell systems. Imaging chamber dimensions and cell colony/aggregate sizes were calculated that would prevent the introduction of metabolite transport limitations and allow for stable, long term imaging. Methods for single cell tracking and analysis were also developed, which produces a database detailing the tracked, observed and extracted properties of every cell and colony, while maintaining the lineage structure of the data. Visualizations such as lineages, histograms and scatter plots were implemented to enable interactive data analysis and querying. These methods were used to characterize heterogeneity in two separate cell systems: human islet of Langerhans-derived progenitor cells, and human embryonic stem cells. Islet-derived progenitors are an expandable source of cells with potential for treatment of diabetes. Here, it was shown that there is an unequal contribution of islets to the progenitor derivation process. Islet-derived progenitors consist of two distinct sub-populations of cells that were distinguished by morphological identification during live cell image analysis. These sub-populations possess unique proliferation profiles and appear to exist in a dynamic state with each other. Three-dimensional tracking of islet progenitor derivation was implemented, but suffered from a lack of resolution to capture the dynamic nature of the transformation process. However, entire islets were imaged and tracked successfully under maintenance conditions, suggesting that this system may be useful for other cell types. These results highlight that live cell imaging and cell tracking may not be suitable for all cell systems and that inclusion of other analytical information, such as immunocytochemistry, would improve the power of cell tracking analysis. Human embryonic stem cell cultures were studied using live cell imaging to identify the mechanisms by which they differentiate to produce supportive niche cells. Cell tracking, morphology scoring and lineage analysis revealed a previously unappreciated level of heterogeneity within human embryonic stem cell colonies. The results show that a sub-population of human embryonic stem cells exist that are precursors to niche cell differentiation. However, these cells exist in a dynamic equilibrium with self-renewing stem cells, which is dependant on the presence of existing local niche cells. Sub-optimal niche conditions leads to the production of niche differentiation-competent cells and, significantly, considerable cell death. The effect of cell death is the clonal selection of self-renewing cells that contribute to colony expansion. Overall, these results highlight the importance of the co-transfer of existing niche cells and the dynamic balance that regulates human embryonic stem cell self-renewal and differentiation. This thesis displays the utility of live cell imaging, cell tracking and cell, colony and lineage analysis for studying dynamic heterogeneous systems. Furthermore, it highlight the fact that cell-, lineage- and colony-level analysis can uncover previously unappreciated heterogeneity and unknown sub-populations of cells. The system does not rely on characterization at the molecular level, but uses higher order measures to generalize them. However, future incorporation of cell, lineage and colony information with molecular-level information may results in analytical power not possible from either level alone. Such systems will be valuable tools in the growing fields of stem cell biology and systems biology.

Cells utilize various signal transduction networks to regulate metabolism. Nevertheless, a quantitative understanding of the relationship between growth factor signaling and metabolic state at the single cell level has been lacking. The signal transduction and metabolic states could vary widely among individual cells. However, such cell-to-cell variation might be masked by the bulk measurements obtained from conventional biochemical methods. To assess the spatiotemporal dynamics of metabolism in individual intact cells, we developed genetically encoded biosensors based on fluorescent proteins. As a key redox cofactor in metabolism, NADH has been implicated in the Warburg effect, the abnormal metabolism of glucose that is a hallmark of cancer cells. To date, however, sensitive and specific detection of NADH in the cytosol of individual live cells has been difficult. We engineered a fluorescent biosensor of NADH by combining a circularly permuted green fluorescent protein variant with a bacterial NADH-binding protein Rex. The optimized biosensor Peredox reports cytosolic \(NADH:NAD^+\) ratios in individual live cells and can be calibrated with exogenous lactate and pyruvate. Notably pH resistant, this biosensor can be used in several cultured and primary cell types and in a high-content imaging format. We then examined the single cell dynamics of glycolysis and energy-sensing signaling pathways using Peredox and other fluorescent biosensors: AMPKAR, a sensor of the AMPK activity; and FOXO3-FP, a fluorescently-tagged protein domain from Forkhead transcription factor FOXO3 to report on the PI3K/Akt pathway activity. With perturbation to growth factor signaling, we observed a transient response in the cytosolic \(NADH:NAD^+\) redox state. In contrast, with partial inhibition of glycolysis by iodoacetate, individual cells varied substantially in their responses, and cytosolic \(NADH:NAD^+\) ratios oscillated between high and low states with a regular, approximately half-hour period, persisting for hours. These glycolytic NADH oscillations appeared to be cell-autonomous and coincided with the activation of the PI3K/Akt pathway but not the AMPK pathway. These results suggest a dynamic coupling between growth factor signaling and metabolic parameters. Overall, this thesis presents novel optical tools to assess metabolic dynamics – and to unravel the elaborate and complex integration of glucose metabolism and signaling pathways at the single cell level.

Gene expression is a fundamental process in the cell and is made up of two parts – the information flow from DNA to RNA, and from RNA to protein. Here, we examined specific sub-processes in Escherichia coli gene expression using newly available tools that permit genome-wide analysis. We begin our studies measuring mRNA and protein abundances in single cells by single-molecule fluorescence microscopy, and then focus our attention to studying RNA generation and degradation by high throughput sequencing. The details of the dynamics of gene expression can be observed from fluctuations in mRNA and protein copy numbers in a cell over time, or the variations in copy numbers in an isogenic cell population. We constructed a yellow fluorescent fusion protein library in E. coli and measured protein and mRNA abundances in single cells. At below ten proteins per cell, a simple model of gene expression is sufficient to explain the observed distributions. At higher expression levels, the distributions are dominated by extrinsic noise, which is the systematic heterogeneity between cells. Unlike proteins which can be stable over many hours, mRNA is made and degraded on the order of minutes in E. coli. To measure the dynamics of RNA generation and degradation, we developed a protocol using high throughput sequencing to measure steady-state RNA abundances, RNA polymerase elongation rates and RNA degradation rates simultaneously with high nucleotide-resolution genome-wide. Our data shows that RNA has similar lifetime at all positions throughout the length of the transcript. We also find that our polymerase elongation rates measured in vivo on a chromosome are generally slower than rates measured on plasmids by other groups. Studying nascent RNA will allow further understanding of RNA generation and degradation. To this end, we have developed a labeling protocol with a nucleoside analog that is compatible with high throughput sequencing.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by over expression of the transcription factors OCT4, SOX2, KLF4 and c-MYC. Using serial live cell immunofluorescence imaging of human fibroblasts undergoing reprogramming, we traced the emergence of nascent iPS cell colonies among heterogeneous cell populations and defined the kinetics of marker expression. We identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters, and differentiation into teratomas, we determined that only one colony type represented bona fide iPS cells, whereas the others represented reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B, and REX1 distinguished the fully reprogrammed state, whereas Alkaline Phosphatase, SSEA-4, GDF3, hTERT and NANOG proved insufficient as markers. Reprogramming in chemically defined medium favored formation of bona fide iPS cell colonies relative to partially reprogrammed colonies. These data highlight the need for rigorous characterization and standardization of putative iPS cells.

A cell's ability to sense and respond to mechanical signals highlights the significance of physical forces in biology; however, to date most biomedical research has focused on genetics and biochemical signaling. We sought to further understand the physical mechanisms that guide the cellular migrations that occur in a number of biological processes, such as tissue development and regeneration, bacterial infections and cancer metastasis. We investigated the migration of single cells and determined whether the biomechanics of these cells could be used to elucidate multi-cellular mechanisms. We first studied Borrelia burgdorferi (Bb), the bacterium that causes Lyme disease. We created a mathematical model based on the mechanical interactions between the flagella and cell body that explained the rotation and undulation of the cell body that occurs as the bacterium swims. This model further predicts how the swimming dynamics could be affected by alterations in flagellar or cell wall stiffnesses. Fitting the model to experimental data allowed us to calculate the flagellar torque and drag for Bb, and showed that Treponema pallidum (Tp), the syphilis pathogen, is biomechanically similar to Bb. Next, we used experimentally-determined parameters of Bb's motility to develop a population-level model that accounts for the morphology and spreading of the "bulls-eye" rash that is typically the first indicator of Lyme disease. This work supported clinical findings on the efficacy of antibiotic treatment regimes. Finally, we investigated the dynamics of epithelial monolayers. We found that intracellular contractile stress is the primary driving force behind collective dynamics in epithelial layers, a result previously predicted from a biophysical model. Taken together, these findings identify the relevance of physics in cellular migration and a role of mechanical signaling in biomedical science.

The myelin basic protein (MBP) family arises from different transcription start sites of the Golli (gene of oligodendrocyte lineage) gene, with further variety generated by differential splicing. The “classic” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath, but also have multiple protein interactions. As an intrinsically disordered protein, MBP has proven to have complex structural and functional relationships with proteins in vitro including actin, tubulin, Ca2+-calmodulin, and multiple protein kinases. The investigations reported in this thesis were to further examine the multifunctionality, and protein:protein interactions of MBP with cytoskeletal and SRC homology 3 domain (SH3) proteins in cells using an oligodendrocyte (OLG) model system to better understand MBP’s contributions to membrane structure, formation, and elaboration in the developing OLG. A new function of MBP has been described showing that classic MBPs can modulate voltage operated calcium channels (VOCCs) by direct or indirect protein-protein interactions at the OLG cytoplasmic leaflet. These interactions contribute to global calcium homeostasis, and may play a complex developmental and spatiotemporal role in the regulation of oligodendrocyte precursor cell (OPC) migration and OLG differentiation. The importance of MBPs SH3 ligand binding domain within its central amino acid region was investigated with the protein-tyrosine kinase Fyn. Co-expression of MBP with a constitutively-active form of Fyn in OLGs resulted in membrane process elaboration, a phenomenon that was abolished by amino acid substitutions within MBP’s SH3-ligand domain. These results suggest that MBP’s SH3-ligand domain plays a key role, and may be required for proper membrane elaboration of developing OLGs. Lastly, interactions of MBP with the OLG cytoskeleton were investigated in OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it had increased co-localization with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. The results presented here advance our understanding regarding protein:protein interactions of MBP, and its multifunctionality in OLGs with regards to membrane formation and elaboration. / This work was supported by the Canadian Institutes of Health Research (MOP #86483, J.M.B. and G.H.), and Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, G.H., RG121541). G.S.T.S. was a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada

In recent years, semiconductor quantum dots (QDs) have arisen as a new class of fluorescent probes that possess unique optical and electronic properties well-suited for single-molecule imaging of dynamic live cell processes. Nonetheless, the large size of conventional QD-ligand constructs has precluded their widespread use in single-molecule studies, especially on cell interiors. A typical QD-ligand construct can range upwards of 35 nm in diameter, well exceeding the size threshold for cytosolic diffusion and posing steric hindrance to binding cell receptors. The objective of this research is to develop tagging strategies that allow QD-ligand conjugates to specifically bind their target proteins while maintaining a small overall construct size. To achieve this objective, we utilize the HaloTag protein (HTP) available from Promega Corporation, which reacts readily with a HaloTag ligand (HTL) to form a covalent bond. When HaloTag ligands are conjugated to size-minimized multidentate polymer coated QDs, compact QD-ligand constructs less than 15 nm in diameter can be produced. These quantum dot-HaloTag ligand (QD-HTL) conjugates can then be used to covalently bind and track cellular receptors genetically fused to the HaloTag protein. In this study, size-minimized quantum dot-HaloTag ligand conjugates are synthesized and evaluated for their ability to bind specifically to purified and cellular HTP. The effect of QD-HTL surface modifications on different types of specific and nonspecific cellular binding are systematically investigated. Finally, these QD-HTL conjugates are utilized for single-molecule imaging of dynamic live cell processes. Our results show that size-minimized QD-HTLs exhibit great promise as novel imaging probes for live cell imaging, allowing researchers to visualize cellular protein dynamics in remarkable detail.

Live single-cell imaging has emerged as an advanced single-cell study tool for approaching a quantitative understanding of many biological questions in recent years. In previous cell studies using bulk cell measurements, the population averages can miss the information from cell to cell variability and mask the underlying signaling networks and mechanisms. Currently, some single cell analysis methods, including but not limited to, live single-cell imaging experiments that built around a fluorescent imaging setup and microfluidic devices enable the measurement and analysis of cell dynamics and responses of single cells across a population and across time. Furthermore, by changing the cells’ environmental conditions in well controlled ways, e.g. balanced steady growth, or temporal pulses, live single-cell imaging can record the cellular behaviors corresponding to these changes in exquisite details. An important question of current interest in both developmental, stem cell and cancer biology is the question of epigenetic differentiation. Continuous long-term live single-cell observations offer insights into the molecular control of cell fate. However, maintaining the imaged cells in a healthy state remains a major challenge. One of our aims in this work was to develop a semi-automated single-cell live imaging and analysis platform to obtain dynamic information of the cellular processes. An imaging incubator that controls and regulates the environmental conditions of the imaged cells also had to be designed and tested. In this thesis, I address the key design considerations of developing a single-cell live imaging platform and demonstrate the capability of this technology through three case studies. To test the design and fabrication of microfluidic devices and micro-valves in imaging malaria infected red blood cells (iRBCs), I recorded the flow of iRBCs through microfluidic channels and constrictions in Chapter 3. Our results illustrate the behaviors of iRBCs with different flow rates and the potential to offer dynamic control in studying the infection probability of iRBCs by implementing the micro-valve system. In order to develop a more adaptable live cell imaging platform, we further developed our semi-automated imaging software and in house built imaging incubator to explore the link between proliferation and differentiation of CD4+ T cells in Chapter 4. By using cells expressing an IL-13-GFP reporter, we distinguished between differentiating and non-differentiating CD4+ T cell population and demonstrated a positive association between cycling differentiation of CD4+ T cells. In Chapter 5, we incorporated the FUCCI cell reporter system in our single cell live imaging system to reveal the effect of different media conditions on the cell cycle progression and cell fate choices of mouse embryonic stem (mES) cells. By improving different factors such as longer pre-incubation time before imaging and exchanging media during the experiments, we maintained a healthy state of mES cells during live cell imaging for extended periods. We observed significant differences in time between divisions of mES cells cultured in 2i +LIF and serum + LIF media, and also small but significant differences in durations of sub-cell cycle phases (G1,G1/S,S/G2/M) between the two media conditions. We further applied this imaging setup to study the behaviors of differentiating mES cells in vitro, and observed lengthening of the G1 phase for both 2i-LIF and serum-LIF cells in agreement with literature. Overall, our semi-automated single cell imaging platform not only offers adjustable intervals between fluorescent imaging, but also provides a constant temperature and gas feeding devices that allows the cells to proliferate for extended microscope imaging. Commercially produced incubators that fit onto the microscope stage and satisfied all requirements in restriction of the cell movement, gas feeding, temperature regulation and optical accessibility are not easily available. Thus, there exists a significant potential for our imaging setup to provide a versatile and adaptable live cell imaging platform for both academia and industrial researchers.

The endothelial cells lining the inner surface of the tissue capillaries and post-capillary venules form a semi-permeable barrier between the blood circulation and interstitial compartments. The semi-permeable barrier in these vessels is the major site of blood-tissue exchange. A compromised endothelial barrier contributes to the pathological process such as edema, acute respiratory distress syndrome (ARDS) and tumor metastasis. Sphingosine-1-phosphate (S1P), an endogenous, bioactive lipid present in all cells, is a potential therapeutic agent that can restore compromised endothelial barrier function. On the other hand, S1P also has pleotropic effects and can either increase or decrease arterial tone and tissue perfusion under different conditions. The detailed mechanisms underlining S1P’s endothelial barrier protective effect are still largely unknown, but are suggested to depend on cell spreading termed “lamellipodia”. Therefore, to fully take advantage of the beneficial properties of S1P, it is important to first understand how S1P-induced lamellipodia protrusions correlate with its effect on endothelial barrier function. It is also important to know the underlining mechanisms that S1P enhances endothelial barrier function, including intracellular signaling and receptor signaling. To study local lamellipodia activities, we acquired time-lapse images of live endothelial cells expressing GFP-actin, and subsequently analyzed different lamellipodia parameters. Experiments were performed under baseline conditions, and during endothelial barrier disruption or enhancement. The compounds used in these experiments included thrombin and S1P. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function for in vitro studies. Changes of local lamellipodia dynamics and endothelial barrier function within the same time frame were studied. For mechanistic studies, we combined biochemical, immunological and pharmacological approaches. Rho family small GTPase activities were measured with an ELISA pull-down assay. Fluorescence Resonance Energy Transfer (FRET) was also used to study the localization of RhoA activation. Pharmacological compounds targeting intracellular signaling messengers were used to test the involvement of Rac1, RhoA, MLC-2 in endothelial local lamellipodia activity and S1P-mediated endothelial barrier enhancement. Receptor agonists and antagonists were used to study the involvement of S1P receptor signaling. Finally, for cell behavior and cytoskeleton studies, we utilized immunofluorescence labeling that enables direct visualization of changes in cytoskeleton, cell-cell junction and focal adhesions. We found that S1P increases both local lamellipodia protrusions and TER. The rapid increase in local lamellipodia protrusion frequencies also corresponded to the rapid increase in TER seen within the same time frame. Under the microscope, local lamellipodia protrusions from adjacent cells overlapped with each other and extended beyond junctional cell-cell contacts. Strikingly, S1P-induced lamellipodia protrusions carry VE-cadherin molecules to the cell-cell contact, established junctional adhesions. Combined with our previous published studies on thrombin induced lamellipodia activity changes, we think lamellipodia protrusions are a major component that regulates endothelial barrier function. Combined, our imaging studies revealed the mechanisms on how lamellipodia regulates endothelial barrier function: 1) lamellipodia overlap and increase the apical to basal diffusion distance, which in turn decreases permeability and upregulates endothelial barrier function. 2) Local lamellipodia protrusions contain VE-cadherin, which is delivered the to the cell-cell contact by the lamellipodia to increase junctional stability. S1P is effective for rescuing thrombin-induced endothelial barrier dysfunction. The known barrier disruptor thrombin, decreased local lamellipodia protrusions, disrupted VE-cadherin integrity, and caused a drop in TER. S1P increased local lamellipodia protrusions after thrombin challenge, and resulted in faster recovery towards baseline TER compared with vehicle controls. Interestingly, we also found that both thrombin and S1P increased MLC-2 phosphorylation at Thr18/Ser19. We subsequently accessed Rho family GTPase activity after thrombin and S1P. As expected, thrombin rapidly increased GTP-bound RhoA levels, and decreased GTP-bound Rac1 levels. Unexpectedly, S1P not only increased GTP-bound Rac1, but also increased GTP-bound RhoA to a more prominently levels (4-fold). Since Rac1 has been implicated in promoting lamellipodia protrusions, we tested the role of Rac1 on the local lamellipodia activities first. We found that Wild-Type (WT) Rac1 group had the highest local lamellipodia protrusion frequencies, protrusion distances, withdraw time and highest percentage of protrusions that lasted more than 5 min. WT Rac1 overexpression had greatest protrusion frequencies and lowest monolayer permeability to FITC-albumin compared to GFP and DN-Rac1 overexpression monolayers. These results suggest that Rac1 is important for baseline endothelial barrier function. This is also confirmed by the finding that pharmacological inhibition of Rac1 significantly decreased baseline TER. Although Rac1 is important for baseline endothelial barrier function, we noticed that it is dispensable in S1P-mediated endothelial barrier enhancement. Rac1 inhibitors, DN-Rac1 overexpression, and Rac1 siRNA knockdown all failed to abolish the S1P-mediated increase in TER. This is partially explained by the findings that S1P-induced Rac1 activation is short-lived and less pronounced in contrast to RhoA activation. We subsequently tested the role of RhoA in S1P-mediated endothelial barrier enhancement, based on our findings that both S1P and thrombin significantly activated RhoA and induced MLC-2 phosphorylation. Significant RhoA activation was found to be mainly at cell periphery and lamellipodia protrusions in HUVEC on FRET, after S1P was given. In addition, RhoA inhibitors significantly decreased the amplitude of S1P-induced MLC-2 phosphorylation, vinculin redistribution and barrier enhancement. The data suggest that the mechanisms involved in S1P-mediated endothelial barrier enhancement depend on RhoA activation and subsequent cytoskeletal rearrangement. We next investigated which receptor is responsible for the endothelial barrier enhancement of S1P. However, antagonism of S1P1, S1P2 or S1P3 alone with W146, JTE-013 or TY-52156 respectively all failed to attenuate S1P-mediated increase in TER. While agonism of S1P1 with CYM-5442 hydrochloride alone produced significant increase in TER, neither S1P2 nor S1P3 activation (CYM 5520 & CYM 5541) produced any change on TER. Interestingly, S1P1 antagonist failed to block the effect of S1P1 agonist on TER. This could be due to that the S1P1 agonist may not be very selective at concentrations tested. We also identified that S1P4 and S1P5 are present on endothelial cells. Further studies would be necessary to elucidate the roles of newly identified S1P4 or S1P5 alone on endothelial barrier function. It is also worth investigating in the future if multiple S1P receptors are involved in its endothelial barrier enhancing effect. In conclusion, we found that lamellipodia protrusions contribute to the endothelial barrier enhancement of S1P. While Rac1 is important for the maintenance of endothelial barrier function, it is dispensable in S1P-mediated endothelial barrier enhancement. On the other hand, RhoA activation appears to be, at least in part, responsible for the endothelial barrier enhancement of S1P. It is currently still unclear if S1P’s endothelial barrier enhancing effect is through one single receptor activation or activation of multiple receptors. Future studies are needed to elucidate the receptor signaling that contributes to S1P-mediated endothelial barrier enhancement.

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