Cell polarization is one of the most fundamental organizational aspects of life, from single-celled organisms such as E. coli and budding yeast up to the wiring of the human brain. Cell polarization is required for the generation of diverse cell types from stem cells, for morphogenesis, directional motility, antigen presentation, and axon guidance;and the loss of cell polarity is a critical step in cancer progression. The wide interest in cell polarity has pushed rapid progress in understanding the scope and complexity of the polarization machinery. However, the fundamental question of how the polarity proteins operate to organize cell structure remains largely unanswered. This proposal builds on progress over the last funding period to address these questions. Our objective is to understand the connections between the PAR polarity proteins and small GTPases. We will focus on MDCK epithelial cell polarization in 3D cultures, and on dendritic spine morphogenesis in hippocampal neurons - two tractable systems in which the PAR proteins have defined but distinct roles.
The specific aims are as follows: 1. Identify the molecular mechanisms through which PAR-3, PAR-6, and aPKC organize the apical domain (which we term a """"""""patch"""""""") during polarized cyst formation of MDCK cells. Using live cell imaging, we will investigate the earliest steps in polarization, which occur during the initial cell division of cells grown in 3D cultures. We will test a new model in which cytokinetic events are proposed to generate the landmark for apical patch assembly. 2. Determine the role of CDC42 in assembly of the apical patch, and identify the CDC42 GEF and GAP that are necessary for cell polarization. RNAi screens are being used to identify the GEF and GAP. Based on preliminary data from a GEF RNAi screen, we propose that a CDC42-GEF called Tuba drives recruitment of PAR-6/aPKC and apicalization. 3. Identify the mechanisms by which PAR-3 controls TIAM1 function in neurons;and determine how PAR- 6, independently of PAR-3, regulates dendritic spine morphogenesis via the Rho GTPase. We will determine the molecular basis for activation of p190 RhoGAP by PAR-6/aPKC. The mechanism by which PAR-6 regulates synaptic activity in hippocampal neurons will also be investigated. Together, these studies will provide new insights into the fundamentally important process of cell polarization, and into the ways by which the PAR polarity proteins exhibit context-specific behavior to control different aspects of cell morphology.

Public Health Relevance

Over 90% of all human cancers originate from epithelial cells. A key property of epithelial cells is that they adhere to one another to form sheets in which the upper surface (which faces the environment) is functionally different from the bottom surface. Cancer progression involves the temporary loss of this cell-cell adhesion, and of up-down polarity. Thus, to understand human cancer, and to identify new chemotherapeutic targets, it is important to find out how epithelial cells polarize. Remarkably, the same process that helps these cells stick to one another is also essential for making the contacts between nerve cells in the brain, which enable us to think and store memories. This grant investigates the detailed molecular mechanisms that enable epithelial cells and nerve cells to polarize and form junctions, and will provide new insights into these mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070902-08
Application #
8101158
Study Section
Intercellular Interactions (ICI)
Program Officer
Deatherage, James F
Project Start
2004-04-01
Project End
2012-09-29
Budget Start
2011-07-01
Budget End
2012-09-29
Support Year
8
Fiscal Year
2011
Total Cost
$311,819
Indirect Cost
Name
University of Virginia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Ahmed, Syed Mukhtar; Macara, Ian G (2017) The Par3 polarity protein is an exocyst receptor essential for mammary cell survival. Nat Commun 8:14867
Ahmed, Syed Mukhtar; Macara, Ian G (2016) Mechanisms of polarity protein expression control. Curr Opin Cell Biol 42:38-45
Iioka, Hidekazu; Macara, Ian G (2015) Detection of RNA-Protein Interactions Using Tethered RNA Affinity Capture. Methods Mol Biol 1316:67-73
Guyer, Richard A; Macara, Ian G (2015) Loss of the polarity protein PAR3 activates STAT3 signaling via an atypical protein kinase C (aPKC)/NF-?B/interleukin-6 (IL-6) axis in mouse mammary cells. J Biol Chem 290:8457-68
Macara, Ian G; Guyer, Richard; Richardson, Graham et al. (2014) Epithelial homeostasis. Curr Biol 24:R815-25
Huo, Yongliang; Macara, Ian G (2014) The Par3-like polarity protein Par3L is essential for mammary stem cell maintenance. Nat Cell Biol 16:529-37
Rodriguez-Boulan, Enrique; Macara, Ian G (2014) Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15:225-42
Andreeva, Anna; Lee, Jianyi; Lohia, Madhura et al. (2014) PTK7-Src signaling at epithelial cell contacts mediates spatial organization of actomyosin and planar cell polarity. Dev Cell 29:20-33
Baschieri, Francesco; Confalonieri, Stefano; Bertalot, Giovanni et al. (2014) Spatial control of Cdc42 signalling by a GM130-RasGRF complex regulates polarity and tumorigenesis. Nat Commun 5:4839
Yasuda, Kyota; Zhang, Huaye; Loiselle, David et al. (2013) The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules. J Cell Biol 203:737-46

Showing the most recent 10 out of 36 publications