Tag: Ken Prehoda

Predicted SH3 Binding Motif in Drosophila aPKC is Required for Proper Localization of aPKC During Asymmetric Cell Division of Neuroblasts

Presenter : Ryan Boileau

Mentor : Ken Prehoda

Major : Biochemistry, Human Physiology, Human Biology

Poster 22

Asymmetric cell division of Drosophila neural stem cells, neuroblasts, require the proper localization of factors that influence the orientation of cell divisions and future fates of mitotic progeny. Errors in the generation of this polarity could cause cells to overproliferate and become cancerous. In neuroblasts, atypical protein kinase C (aPKC) has been previously shown to be a key mediator in the genera- tion of apico-basal polarity by localizing to the apical cortex and restricting fate determinants Numb and Miranda to the basal cortex during cell division. This allows the dividing neuroblast to maintain pluripotency while also generating a daughter cell that differenti- ates into neurons. Although the mechanism of how aPKC restricts basal determinants has become transparent, we seek to evaluate how aPKC itself is apically localized. Using a combination of genetic and biochemical approaches, we have found that a predicted SH3 binding motif within aPKC is necessary for apical polarization. We hypothesize that an SH3 domain containing protein binds to aPKC at this site and plays a role in stabilizing apical localization. Future research will be focused on finding interacting partners of this SH3 binding motif using a candidate gene-based approach.

aPKC Induces Polarization of Numb by Inhibiting Cortical Targeting Sites

Presenter: Lyle McPherson

Mentor: Ken Prehoda

Poster: 23

Major: Biochemistry 

Cell polarity regulates important functions for metazoan cells, including epithelial, neuronal and stem cells. However, little is known about the molecular mechanisms that allow cells to establish cell polarity. aPKC, the kinase of the evolutionarily conserved Par complex, polarizes cellular proteins. In these polarized cells, protein polarization genetically downstream of aPKC maintains tissue integrity and establishes cell identity. For multiple aPKC substrates, phosphorylation induces protein polarization by displacing substrates from aPKCcontaining membrane domains. Despite the clear role of aPKC in establishing cell polarity the molecular mechanism by which aPKC’s kinase activity polarizes its substrates remains unclear. We characterized the polarization mechanism of Numb, an aPKC substrate, using cell biology and biochemistry. We identified lipidbinding sites within Numb that mediate its recruitment to the cellular cortex by binding to negatively charged phospholipids. Additionally, we found that specific amino acids within these sites are phosphorylated by aPKC to inhibit lipid binding. Our findings suggest one mechanism for aPKCmediated cell polarity where aPKC polarizes Numb by phosphorylating it to inhibit cortical association thereby resulting in its polarization. We are currently investigating other domains of Numb containing aPKC phosphorylation sites to further our understanding of the molecular mechanisms behind this process. The mechanism of Numb’s polarization by aPKC illustrates to us a way that a kinase can induce cell polarity by destabilizing a protein’s membrane association in specific regions of the cell.

A Predicted SH3 Binding Motif in Drosophila aPKC is Required for Proper Localization of aPKC During Asymmetric Cell Division of Neuroblasts

Presenter: Ryan Boileau

Mentor: Ken Prehoda

Poster: 5

Major: Human Physiology/Biochemistry/Biology

Asymmetric cell division of Drosophila neural stem cells, neuroblasts, requires the proper localization of factors that influence the orientation of cell divisions and future fates of mitotic progeny. Errors in the generation of this polarity could cause cells to overproliferate and become cancerous. In neuroblasts, atypical protein kinase C (aPKC) has been previously shown to be a key mediator in the generation of apico-basal polarity by localizing to the apical cortex and restricting the fate determinant proteins Numb and Miranda to the basal cortex during cell division. This allows the dividing neuroblast to maintain pluripotency while also generating a daughter cell that differentiates into neurons. Although the mechanism by which aPKC restricts basal determinants is known, how aPKC itself is apically localized is not fully known. Using transgenic fly lines and confocal fluorescence microscopy, we have found that a predicted SH3 binding motif within aPKC is necessary for its apical polarization in neuroblasts. We hypothesize that an SH3 domain containing protein binds to aPKC at this site and plays a role in stabilizing apical localization. Future research will be focused on finding interacting partners of this SH3 binding motif using a candidate gene-based approach and elucidating the mechanism by which this novel interaction may function to polarize aPKC.

Determining Scrib binding partners relevant to its spindle orienting function

Presenter(s): Hussein Al-Zubieri – Biochemistry

Faculty Mentor(s): Ken Prehoda, Nicole Paterson

Poster 82

Research Area: Molecular Biology

Asymmetric stem cell division requires a mitotic spindle oriented relative to its axis of polarity. Spindle orientation determines where the cleavage furrow is positioned, thereby determining the location of cell division. Without a correctly oriented spindle, tumorigenesis can occur. Indeed, many of the proteins in the pathway are tumor suppressors. Two pathways have been found to position the spindle, Mud pathway and Dlg pathway. Both are required for spindle orientation. Our research focuses on a Dlg pathway protein member called Scrib which is a tumor suppressor protein that has been shown to be required for spindle orientation. The function of Scrib is not yet fully understood, and our research is focused on discovering the mechanism of its function in this process. A starting point for identifying function is to test the requirement of the functional domains of Scrib in vivo, and determine the specific function of these domains in vitro. My project is to determine the Scrib binding partners relevant to its spindle orienting function.