|
Qian "Elizabeth" XuClass of 2007Graduated in 2011 Undergraduate Institution: University of Virginia, Charlottesville Major: BS Chemistry, BA Physics, Minor in Mathematics Origin: Chengdu, Sechuan Province, People's Republic of Ch Lab: Jay Groves Location: |
Research |
The intention of my research is to use synthetic lipid membranes, solid-state nanostructures, and high-resolution imaging to study a potentially novel mechanical regulatory mechanism in the EphA2 signaling EphA2 is known to be up-regulated in 40% of human breast cancers and plays an active role in metastasis. The hypothesis, based on both published and preliminary research, is that the multi-scale organization of the EphA2 receptor in the cell membrane regulates its biochemical function. This hypothesis motivates the idea that extracellular mechanical inputs have an important role in intracellular signaling cascades.
EphA2 is a receptor tyrosine kinase that consists of an intracellular kinase domain and an extracellular ligand-binding domain. Through binding to natively membrane-bound ephrin ligands, Eph-ephrin signaling regulates cell adhesion, migration, and vascular development and has been implicated in the development and progression of various types of cancer. Activation of EphA2 occurs after binding to ephrin-A1 presented on an opposing cell membrane. Monomer binding is followed by dimerization of the receptor-ligand complex and then oligomerization.
Clustering mechanisms of EphA2 proteins are not well understood because these signaling molecules function in the cell membrane, which is an environment that is difficult to characterize and manipulate. In order to study the role of multi-scale organization in EphA2/ephrin-A1 signaling, we use a live cell-supported membrane hybrid system. The supported lipid bilayer (SLB), as the model cell membrane, allows us to investigate the behavior of membrane-bound signaling molecules in a physiologically-relevant microenvironment. Along with the creation of chemically modified fluorescent fusion proteins that can stably interact with a subset of capturing lipids within the bilayer, we can control the protein density, precisely image it, and maintain molecular mobility so ligand-induced receptor clustering can occur on the synthetic membrane end.
We will apply our system to well-characterized model human breast cancer cell lines (MDAMB231, MCF10A, etc.) with different EphA2 expression levels and tumorigenicities. The EphA2-expressing cell will be presented to ephrin-A1 functionalized SLBs. The effects of modulations (mechanical, spatial, or chemical) will be characterized in terms of cytoskeleton organization, ADAM10 co-localization, and phosphotyrosine and EphA2 down-regulation. Preliminary data indicates that EphA2-expressing cells respond differently to monomeric, dimeric, and tetrameric ephrin-A1 ligands presented on a synthetic cell membrane.
A more sophisticated level of control over organization on the 1000nm scale will be achieved using patterned membrane technology. This strategy, which is based on patterning structures on supported membrane substrates, has been used in the Groves lab to create sets of chemically identical living cells that differ only in the spatial organization of their signaling machinery. Movement of ephrin-A1/EphA2 complexes will be tracked by Total Internal Reflection Fluorescence (TIRF) imaging in both live-cell and Photoactivatible Localization Microscopy (PALM) modes to resolve receptor organization on multiple length scales.
EphA2 is a receptor tyrosine kinase that consists of an intracellular kinase domain and an extracellular ligand-binding domain. Through binding to natively membrane-bound ephrin ligands, Eph-ephrin signaling regulates cell adhesion, migration, and vascular development and has been implicated in the development and progression of various types of cancer. Activation of EphA2 occurs after binding to ephrin-A1 presented on an opposing cell membrane. Monomer binding is followed by dimerization of the receptor-ligand complex and then oligomerization.
Clustering mechanisms of EphA2 proteins are not well understood because these signaling molecules function in the cell membrane, which is an environment that is difficult to characterize and manipulate. In order to study the role of multi-scale organization in EphA2/ephrin-A1 signaling, we use a live cell-supported membrane hybrid system. The supported lipid bilayer (SLB), as the model cell membrane, allows us to investigate the behavior of membrane-bound signaling molecules in a physiologically-relevant microenvironment. Along with the creation of chemically modified fluorescent fusion proteins that can stably interact with a subset of capturing lipids within the bilayer, we can control the protein density, precisely image it, and maintain molecular mobility so ligand-induced receptor clustering can occur on the synthetic membrane end.
We will apply our system to well-characterized model human breast cancer cell lines (MDAMB231, MCF10A, etc.) with different EphA2 expression levels and tumorigenicities. The EphA2-expressing cell will be presented to ephrin-A1 functionalized SLBs. The effects of modulations (mechanical, spatial, or chemical) will be characterized in terms of cytoskeleton organization, ADAM10 co-localization, and phosphotyrosine and EphA2 down-regulation. Preliminary data indicates that EphA2-expressing cells respond differently to monomeric, dimeric, and tetrameric ephrin-A1 ligands presented on a synthetic cell membrane.
A more sophisticated level of control over organization on the 1000nm scale will be achieved using patterned membrane technology. This strategy, which is based on patterning structures on supported membrane substrates, has been used in the Groves lab to create sets of chemically identical living cells that differ only in the spatial organization of their signaling machinery. Movement of ephrin-A1/EphA2 complexes will be tracked by Total Internal Reflection Fluorescence (TIRF) imaging in both live-cell and Photoactivatible Localization Microscopy (PALM) modes to resolve receptor organization on multiple length scales.
Publications |
- Myosin VIIB from Drosophila is a high duty ratio motor. J. Biol. Chem. 280: 32061-32068, 2005, Yi Yang, Mihaly Kovacs, Qian Xu, John B. Anderson, and James R. Sellers
- Neck Length and Processivity of Myosin V. J. Biol. Chem. 278: 29201-29207, 2003, Takeshi Sakamoto, Fei Wang, Stephan Schmitz, Yuhui Xu, Qian Xu, Justin E. Molloy, Claudia Veigel, and James R. Sellers
