Utilizing the auxin-inducible degron to study cell polarity dynamics in C. elegans and mouse embryonic stem cells

dc.contributor.advisorDickinson, Daniel J.
dc.creatorde Jesus, Bailey
dc.description.abstractDuring embryogenesis, early developmental events proceed on a tightly coordinated timescale. This is especially true for cell polarization, whereby cells establish molecularly distinct plasma membrane domains that orient asymmetric cell divisions and enable differentiation. Traditional methods of protein perturbation, such as genetic knockouts or RNA interference (RNAi), typically lack the flexibility to target proteins at multiple specific times during development. Therefore, we sought to implement a method that would allow for temporal treatment in order to examine the regulation of cell polarity. The auxin-inducible degron (AID) system enables proteins of interest to be tagged with a short degron sequence, which is recognized and polyubiquitinated by an E3 ligase specifically in the presence of the plant-derived molecule, auxin. The polyubiquitinated protein is then degraded by the proteasome. We first used this system in the C. elegans embryo to degrade the mitotic kinase Aurora A at different times in the cell cycle, which demonstrated that Aurora A temporally regulates polarity establishment through different mechanisms. Next, we were interested in applying the AID system to mouse embryonic stem cells (mESCs) to study the role of the tight junction protein ZO-1 during epithelial polarization. To do this, we used a CRISPR-Cas9-based universal donor system to endogenously tag the ZO-1 locus with the degron, thereby eliminating the previous need to use transgenes and knockdown endogenous loci when working with mammalian cells.We found that depleting ZO-1 increased the proportion of unpolarized clusters, which suggests that ZO-1 is important for polarization. This approach can be applied to other proteins in order to further elucidate the dynamics of epithelial polarity as well as other developmentally important processes.
dc.description.departmentMolecular Biosciences
dc.relation.ispartofHonors Thesesen
dc.subjectCell polarity
dc.subjectC. elegans
dc.subjectmouse embryonic stem cells
dc.subjectauxin-inducible degron
dc.titleUtilizing the auxin-inducible degron to study cell polarity dynamics in C. elegans and mouse embryonic stem cells

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