CAREER: Single-molecule dissection of the Partitioning Defective cell polarity machinery

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The goal of this project is to better understand how cells establish and maintain an internal “sense of direction” called cell polarity. The cells in our bodies rely on this intrinsic polarity to orient themselves within a tissue. For example, to function properly, cells in our intestines need to know which side of the cell is facing the luminal space, where food is being digested, and which side is facing the underlying blood vessels, where nutrients need to be absorbed. This project uses new, highly sensitive techniques to study how molecules inside cells can interact to produce spatial patterns that polarize a cell. To ensure that the results are generalizable, polarizing cell types from both worms and mice will be studied in parallel. The results are expected to shed light on how animal cells orient themselves within a developing embryo and in the adult body. Much of the work required for this project will be carried out by undergraduate researchers recruited from the diverse student body at the University of Texas at Austin. Freshman undergraduate researchers will learn genome editing technologies while conducting a large-scale search for new polarity genes, as part of a course-based undergraduate research experience. A subset of these students will participate more deeply in the research as upperclassmen, by taking on independent projects within the lead scientist’s lab. As a result, this project will contribute to training and developing a diverse STEM workforce.Cell polarity is the asymmetric localization of molecules and activities within a cell, and is essential for the development and homeostasis of animal tissues. The scaffold protein PAR-3 is a key regulator of polarity across animals and in many different polarized cell types. This research group has previously shown that in Caenorhabditis elegans zygotes, PAR-3 forms oligomeric complexes containing the central polarity kinase aPKC, and these complexes are carried to the anterior of the zygote by actomyosin cortical flows. The goals of the present project are to determine 1) how PAR-3 complexes are assembled and disassembled in C. elegans zygotes, and 2) whether these mechanisms are conserved in mammalian cells. To address these questions, the group has developed and will apply an innovative single-cell, single-molecule biochemistry approach that allows direct extraction and quantification of protein complexes from polarizing cells. By measuring the composition and dynamics of PAR-3 complexes in polarizing C. elegans zygotes, the project will shed light on the biochemical basis for PAR-3 complex assembly. These experiments will incorporate both known players and new molecules identified from proteomic screens. This approach will then be extended to mouse embryonic stem cells, a mammalian epithelial polarity model that will enable the findings to be generalized across animal systems. Together, this work will elucidate how oligomeric PAR-3 complexes are assembled and controlled to enable proper cell polarization in vivo.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.