Cell polarization and directed migration are fundamental processes required for human cells to mediate immune responses, wound healing, angiogenesis, development, and cancer metastasis. While much work has been done to identify and characterize regulatory proteins involved in these processes, the large number of critical proteins and second messengers has left the questions open of how cells induce polarization, drive migration and trigger chemotaxis towards a source of chemoattractant. The goal of the proposed work is to answer these questions by employing a microscopy based quantitative analysis of the regulatory control systems in endothelial cells and neutrophils. Understanding how these regulatory systems are build will likely reveal critical control points that have potentil as therapeutic targets. Our specific strategy is based on cell lines that we developed expressing FRET- and translocation- based fluorescent biosensors for three different core second messengers, PIP3, Ca2+ and diacylgycerol as well as for four core small GTPases, Ras, RhoA, Rac and Cdc42. We have also developed rapid perturbation strategies, based on synthetic expressed DNA constructs that allow us to use small molecules to rapidly activate each of these core regulators of polarization and identify and characterize the relevant local feedback loops in the two cell models. This already led to the identification of important proteins that connect these core regulators to each other. Furthermore, by being able to track cells and monitor local signaling activities as they migrate and turn towards sources of chemoattractant, we have been able to identify an unexpected central role of Cdc42 in the polarization and directed migration of both cells. The fundamental nature of our experiments on polarization and directed cell migration will make our studies relevant to understand these processes in a wide range of cell types and organisms. Our studies will also provide important tools for other researchers to use for studies of polarization and migration.

Public Health Relevance

Cell polarization and directed migration play critical roles in human immune responses, wound healing, angiogenesis, development, and cancer metastasis. Our cell-based fluorescence microscopy approach will provide systems level molecular and mechanistic insights into the internal control networks that ultimately allow human endothelial cells to form new vasculature and enable human neutrophils to chemotax. The fundamental nature of our experiments on polarization and directed cell migration will make our studies relevant to understand these processes in a wide range of cell types and organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM063702-13
Application #
8762335
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Nie, Zhongzhen
Project Start
2001-07-01
Project End
2018-07-31
Budget Start
2014-09-16
Budget End
2015-07-31
Support Year
13
Fiscal Year
2014
Total Cost
$356,997
Indirect Cost
$134,569
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Yang, Hee Won; Collins, Sean R; Meyer, Tobias (2016) Locally excitable Cdc42 signals steer cells during chemotaxis. Nat Cell Biol 18:191-201
Hayer, Arnold; Shao, Lin; Chung, Mingyu et al. (2016) Engulfed cadherin fingers are polarized junctional structures between collectively migrating endothelial cells. Nat Cell Biol 18:1311-1323
Miyazaki, Yusuke; Mizumoto, Kota; Dey, Gautam et al. (2016) A method to rapidly create protein aggregates in living cells. Nat Commun 7:11689
Winans, Amy M; Collins, Sean R; Meyer, Tobias (2016) Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation. Elife 5:e12387
Ge, Xuecai; Milenkovic, Ljiljana; Suyama, Kaye et al. (2015) Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma. Elife 4:
Collins, Sean R; Yang, Hee Won; Bonger, Kimberly M et al. (2015) Using light to shape chemical gradients for parallel and automated analysis of chemotaxis. Mol Syst Biol 11:804
Dey, Gautam; Meyer, Tobias (2015) Phylogenetic Profiling for Probing the Modular Architecture of the Human Genome. Cell Syst 1:106-15
Dey, Gautam; Jaimovich, Ariel; Collins, Sean R et al. (2015) Systematic Discovery of Human Gene Function and Principles of Modular Organization through Phylogenetic Profiling. Cell Rep :
Galic, Milos; Tsai, Feng-Chiao; Collins, Sean R et al. (2014) Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons. Elife 3:e03116
Tsai, Feng-Chiao; Seki, Akiko; Yang, Hee Won et al. (2014) A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration. Nat Cell Biol 16:133-44

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