Development of a molecular tension sensor to visualize force production by nonmuscle myosin IIB in living cells
Abstract/Contents
- Abstract
- Mechanical forces exert a strong influence over biological form and function. For example, during the transition of a fertilized egg cell into a complete multicellular organism, an embryo experiences growth, but also movements that deform and rearrange its cells. These movements ultimately shape tissues and sculpt functional organs. Cellular movement originates in molecular events that generate, resist, and transduce mechanical force, and work with chemical signaling to direct individual and collective cellular functions. As such, improved insight into the fundamental aspects of cellular biophysics would dramatically enhance our understanding of cell motility, cell division, and embryonic development, with potentially transformative consequences for the fields of tissue engineering and medicine. In my thesis research I designed tools to explore the mechanical state of molecular motors and crosslinkers, and investigated the role that mechanical forces play in cellular motility and mechanotransduction. I first describe the development of a FRET-based molecular tension sensor specific for nonmuscle myosin IIB, and show that the sensor localizes similarly to endogenous myosin IIB, responds to strain and relaxation, and provides the first force measurement of myosin II in a living cell. Next, I examine the role of ion gradients and nonmuscle myosin II dynamics in the formation of membrane blebs, a cellular protrusion required for some forms of three-dimensional cell motility. Our results suggest that that blebbing M2 cells exhibit a constitutive alkaline pH beneath the membrane, and that distinct organizational patterns for nonmuscle myosin IIA and IIB occur during bleb retraction. Finally, I test the hypothesis that the crosslinker ezrin transduces tension between the cytoskeleton and the sodium proton exchanger 1 (NHE1). pH measurements to monitor NHE1 activity reveal that epithelial cells do not show a substantial proton flux response to applied touch, and FRET measurements on an ezrin tension sensor do not support its role as a tension-sensing cytoskeletal element.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Ramallo Pardo, Diego Fernando |
|---|---|
| Associated with | Stanford University, Biophysics Program. |
| Primary advisor | Dunn, Alexander Robert |
| Thesis advisor | Dunn, Alexander Robert |
| Thesis advisor | Goodman, Miriam Beth |
| Thesis advisor | Theriot, Julie |
| Advisor | Goodman, Miriam Beth |
| Advisor | Theriot, Julie |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Diego Fernando Ramallo Pardo. |
|---|---|
| Note | Submitted to the Program in Biophysics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Diego Fernando Ramallo Pardo
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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