Kinesins are motor proteins responsible for the anterograde transport on microtubules. Kinesin-1 is the first characterized kinesin, and it consists of two heavy chains and two light chains. KIF5B is a form of Kinesin-1 heavy chains that is ubiquitously expressed in mammals. The head domain of KIF5B is responsible for ATP-dependent mechanical movement along microtubules, while the tail region is well-known for its interaction with cell specific cargos. Recent studies reveal a second microtubule binding site in the tail, suggesting special functions of KIF5B in microtubule sliding and bundling. To understand the role of KIF5B in long bone development, a conditional knockout mouse model was generated, in which Kif5b is deleted in early limb mesenchyme using Prx1-cre/LoxP mediated recombination. Unlike Col2a1-cre directed Kif5b knockout in chondrocytes, the expression of Prx1-cre in limb mesenchyme results in Kif5b knockout in both chondrocyte and osteoblast lineages. The Prx1-cre mediated Kif5b conditional knockout mice develop malformed long bones characterized by their bowed shape, shortened length and multiple fractures, which reflects a combination of defects in bone matrix and growth plate. The mutant mice demonstrate impaired bone matrix formation, as indicated by both collagen density reduction and collagen matrix disorganization. Also, the growth plate does not retain its normal organization, and the hypertrophic zone is absent. The KIF5B deficient chondrocytes not only lose planar cell polarity, but also undergo early apoptosis and fail in terminal differentiation. Interestingly, the binucleation rate is significantly increased in these chondrocytes, suggesting a severe cytokinesis defect. Besides, the intracellular retention of extracellular matrix (ECM) molecules and the uneven distribution of ECM in the cartilage imply both blockage and inappropriate direction of secretion. Cytokinetic defect in chondrocytes is closely associated with growth plate abnormality and growth retardation. In Kif5b knockout chondrocytes, cytokinetic defect is also one of the earliest and principal phenotypes. Therefore the underlying mechanism of cytokinetic defect was further investigated at cellular level. Since Kif5b knockout chondrocytes cannot survive in primary culture, RNA interference approach was adopted to generate a Kif5b-knockdown chondrogenic cell line. As expected, the Kif5b knockdown cells demonstrate cytokinetic defects characterized by increased binucleation rate and prolonged cytokinesis phase. In control cells, KIF5B becomes concentrated in the midbody during cytokinesis, and the midbody organization is disrupted in Kif5b knockdown cells. Furthermore, transient expression of full-length KIF5B significantly reduces the binucleation rate of these KIF5B deficient cells, whereas over-expression of a truncated KIF5B (without microtubule binding sites in tail region) cannot rescue the defect. Additionally, KIF5B is found to interact with midbody components PRC1 and Aurora B kinase by GST pull-down assay. This study demonstrates the multiple functions of KIF5B in long bone development and emphasizes its significant role as a key modulator in chondrocyte cytokinesis. More importantly, the study also brings new insights into the mechanisms of cytokinesis: we propose that KIF5B may participate in cytokinesis by regulating the midbody organization and stability via microtubule bundling and transporting or anchoring important components to the midbody. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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To obtain a cell source for cartilage tissue engineering primary cells are passaged on polystyrene dishes to increase cell number however, this stiff environment results in dedifferentiation. This study evaluates the role of microenvironment stiffness on regulation of passaged chondrocyte phenotype. Results show passaged cells on soft polyacrylamide gels (0.5kPa) become round, less proliferative, less contractile, have higher levels of globular actin (g-actin) compared to filamentous actin (f-actin), MRTF localization in the cytoplasm and down-regulation of MRTF associated genes such as type I collagen, alpha-smooth muscle actin, transgelin, tenascin C and vinculin. This suggests that the chondrogenic phenotype during passaging is regulated by actin polymerization and activation of MRTF signaling that induces expression of non-chondrogenic genes, and has functional effects as the cells become proliferative and contractile. Modulating substrate stiffness maybe a way to influence aspects of the chondrogenic phenotype in order to obtain sufficient cells suitable for cartilage tissue engineering.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The application of prolonged mechanical loading to cartilage alters the osmolality of the extracellular environment, with osmotic challenge known to alter the gene expression and the metabolic activity of chondrocytes. However, the mechanisms by which osmolality controls chondrocyte activity remain unclear. Previous study on various cell types, including chondrocytes, showed that hyper-osmotic challenge induces the condensation of chromatin, with highly condensed chromatin often associated with gene poor regions of DNA and gene silencing. The present study investigated the effect of osmotic challenge on chromatin organisation, genome wide gene-expression and the cellular and nuclear deformability of chondrocytes. In order to observe a broad effect of osmotic challenge on the nuclei, the chondrocytes were subjected to a range of hypo- and hyper-osmotic challenge and imaged by confocal microscopy. Chromatin condensation was quantified by the Sobel edge algorithm in MATLAB. Hyper-osmotic challenge on chondrocytes induced an increase in chromatin condensation. Interestingly, the most marked condensation occurred within the osmolality range of articular cartilage in vivo. The effect of osmotic challenge varied between the monolayer cultured and agarose seeded chondrocytes, which may be due to the differences in cytoskeleton organisation between the two culture conditions. Additionally, chromatin condensation induced by hyper-osmotic challenge was shown to be reversible. Marked differences were observed in the deformability of the cell and nucleus in chondrocytes post osmotic challenge, compared to the 300 mOsm/kg conditions typically used for in vitro isolated chondrocyte studies. From the microarray study, the application of 500 mOsm/kg for both 1 and 5 hours altered the gene expression, including the expression of histone related genes, with a higher number of genes affected by the 5 hours hyper-osmotic challenge. The findings of this study suggest that osmotically-induced alterations in nuclei morphology and chromatin structure may provide a direct biophysical mechanism that controls chondrocytes activity.

Experimental studies indicate that culturing chondrocytes on biodegradable polymeric scaffolds may yield“engineered" cartilage for the replacement of tissue lost to injury or diseases such as osteoarthritis. A method of estimating the outcome of cell-polymer cultures would aid in the design and evaluation of engineered tissue for therapeutic use. The goals of this project were to develop, validate, and apply first-generation mathematical models that describe the kinetics of extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs cultured in vitro. The ECM deposition model is based on a product-inhibition mechanism and predicts an asymptotic, exponential increase in the concentration of ECM molecules found in cartilage, including collagen and glycosaminoglycans (GAG). The scaffold degradation model uses first-order kinetics to describe the hydrolysis of biodegradable polyesters in systems not limited by diffusion. Each model was fit to published data describing the accumulation of GAG and collagen, as well as the degradation of poly glycolic acid (PGA) and poly lactic acid (PLA), respectively. As experimental validation, cell-polymer constructs (n = 24) and unseeded scaffolds (n = 24) were cultured in vitro, and biochemical assays for GAG and collagen content, as well as scaffold mass measurements, were performed at 1, 2, 4, 6, 8, or 10 weeks of culture (n = 8 per time point). The mathematical models demonstrate a moderate to strong goodness of fit with the previously published data and our experimental results (R2=0.75-0.99). These models were also combined to predict the temporal evolution of total construct mass with reasonable accuracy (30% RMS deviation). In ongoing work, estimates of biochemical composition derived from these models are being proposed to predict the mechanical properties and functionality of the constructs. This modeling scheme may be useful in elucidating more specific mechanisms governing ECM accumulation. Given their potential predictive power, these models may also reduce the cost of performing long-term culture experiments.