Text

theses

To study the functional role of keratin filaments during early embryological development I developed a novel biomolecular tool for specifically disrupting keratin filaments in living cells. Conventional genetic approaches such as, knockdown, knockout and transgenic overexpression have attempted to study the role of keratin IFs but fall short of identifying the functional significance of keratin filamentous network during early development. The utility of small molecular inhibitors withaferin A and acrylamide to experimentally interfere with keratin network organization were also evaluated in this study. The comparative analysis done here emphasizes that the effect of these inhibitors is dependent on many factors, such as cell type, cell density, dose of the drug and time of exposure. Furthermore, correlative evidence suggests that the keratin network in the vicinity of the cell-cell contacts was resistant to both the small molecular inhibitors. It was also observed that both keratin filaments and keratin precursor particles have spatially defined subcellular localization in tissues, such as presumptive ectoderm and mesendoderm. In order to specifically study the functional role of keratin filamentous network, a genetically encoded PhotoActivatable disruptor of keratin filaments was developed, taking advantage of Xenopus keratin 8 mimetic peptide and a light sensitive LOV domain of the Avena sativa phototropin. The inhibitory effects of this peptide were validated in multiple cell lines and tissues. Upon photoactivation, PA-2B2 induces localized rapid subcellular disruption of the keratin filaments and thus destabilization of the network leading to cell shape changes. Specific disruption of keratin filaments by light-based activation of PA-2B2 leads to a dramatic disruption of early embryological development in Xenopus. Additionally, disruption of keratin filaments leads to failure of fibronectin assembly along the blastocoel roof. Collectively, the data shows the critical importance of keratin filaments in morphogenetically active tissues and early embryogenesis. The optogenetic novel tools developed here to disrupt keratin filaments with spatiotemporal precision are a powerful means to probe keratin filament function within living cells and direct future investigations.

ETD_10364

Ph.D.

Includes bibliographical references

doi:10.7282/t3-b5w5-ec75

ETD doctoral

The author owns the copyright to this work.