Analysis of mechanical induction of bioelectric activity in cells
Abstract
This research proposal seeks to apply novel optical methods for visualizing mechanical stresses in live cells to analyze the induction of bioelectric activity by mechanical stimuli. It is known that cells react to mechanical forces through a variety of mechanisms. One of the key features of cell regulation is the use of actively controlled ion exchange with the environment via channels and pumps, instead of passive exchange via diffusion. However, there is still a very limited knowledge of how mechanical and electrical stimuli are transduced to initiate changes in the bioelectric state of a cell. We now seek to leverage advances made in our laboratories to better understand these relationships. The nanoscale sensitivity of quantitative phase imaging (QPI) can be used to reveal mechanical properties of cells. In this modality, an external shear stress is applied and sub-cellular changes in mass distribution are observed with QPI. A significant advantage of this assay is that the cells are not displaced during measurement. Recent efforts have incorporated FRET (Fšrster resonance energy transfer)-based sensors into the QPI platform. FRET technology reports the nanoscale separation of donor and acceptor molecules. In this approach, FRET is used to report local cell conditions such as the tension experienced by load-bearing proteins or the concentration of calcium ions. The combination of FRET-based sensors with QPI permits the study of dynamic loading of cells. Traditionally, such dynamic loading has required indirect measures and complex devices but with this platform, cell stresses can be directly visualized without mechanical displacement. This project seeks to use the combined imaging platform to examine how external stimuli are transduced by cellular structures to produce changes in internal electrical state via modulation of ion channel transmission. To meet this goal, we propose the following specific tasks: Task 1) Use measurements of FRET-based sensors and QPM to characterize signal transduction in cells under environmental challenges in ion concentration. In this task, methodology will be developed to analyze the mechanical signal transduction pathways which produce a change in cellsÕ internal state. Cells will be engineered to express FRET-based voltage and tension sensors. As a test system, exposure to heavy metal ions, which are known to both block certain ion channels and modulate cell mechanical properties, will be used to induce changes in cell behavior. Task 2) Use measurements of FRET-based sensors and QPM to characterize signal transduction in cells under environmental challenges in mechanical load. In this aim, cell behavior will be examined in response to atypical mechanical loading such as that found as metastatic cells migrate through narrow openings. The integrated optical platform will be applied to image cells as they move through a small capillary, using FRET based sensors to examine the interaction of ion concentration, alteration in sub-cellular structures, and loading of mechanosensitive proteins. The expected outcome of this project will be to improve fundamental understanding of the coupling between ion flow, mechanics, and mass distribution in response to a mechanical stimulus. Furthermore, the experiments proposed here will enable comparison of the pathways for ionic and mechanical stimuli and determine if they induce similar or distinct types of coupling between key cellular processes.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jun 17, 2019
- Source ID
- W911NF1910306
Entities
People
- Adam Wax
Organizations
- Army Contracting Command
- Duke University
- United States Army