Developing a long-term microfluidic system to study the mesoscopic dynamics of Piezo1 activity
File(s)
Author(s)
Tang, See Swee
Type
Thesis
Abstract
The cellular microenvironment (or cell niche) is a complex and specialised environment, consisting of physical, chemical, and biological factors that collectively influence the cells residing within. Interactions within this dynamic microenvironment play a critical role in regulating and maintaining cellular activities and function. Within the cellular microenvironment, resident cells constantly experience various mechanical force stimuli, which previous in vitro studies cannot fully recapitulate. Hence, the inclusion of mechanical forces (for example fluid shear stress) in the cell culturing system would yield a more physiologically relevant investigation of cellular behaviour.
Cells commonly express mechanosensitive proteins and receptors to sense and transduce external mechanical stimuli to respond appropriately. A widely expressed mechanosensitive ion channel, Piezo1, has been shown to play an important role in cellular mechanotransduction. Our lab previously generated a genetically-encoded Piezo1 sensor (GenEPi), which allows precise detection of Piezo1-specific activities. To investigate the activities of Piezo1 in near-physiological conditions, I developed a microfluidic-based system to study the dynamics of Piezo1 in mammalian cells using our GenEPi sensor.
Using my developed system, I discovered that Piezo1 clusters exhibit more directed diffusive motion after fluid shear stress. By studying the dynamics of the protein, I revealed differential Piezo1 responses across fluid shear stress magnitudes. I also reported a mechanical threshold to HEK-Piezo1 activation during constant fluid shear stress. Through the GenEPi dynamic responses, I observed that cellular Piezo1 activations are synchronised to mechanical shear triggers but become increasingly asynchronous under sustained shear stress. Crucially, the characteristics of Piezo1’s dynamic within cells reported here will significantly advance our understanding of the interplay between mechanical forces and cellular behaviour within the cellular microenvironment. This system can also be a model for others to adopt in investigating the dynamics of other mechanosensitive proteins expressed in the cells.
Cells commonly express mechanosensitive proteins and receptors to sense and transduce external mechanical stimuli to respond appropriately. A widely expressed mechanosensitive ion channel, Piezo1, has been shown to play an important role in cellular mechanotransduction. Our lab previously generated a genetically-encoded Piezo1 sensor (GenEPi), which allows precise detection of Piezo1-specific activities. To investigate the activities of Piezo1 in near-physiological conditions, I developed a microfluidic-based system to study the dynamics of Piezo1 in mammalian cells using our GenEPi sensor.
Using my developed system, I discovered that Piezo1 clusters exhibit more directed diffusive motion after fluid shear stress. By studying the dynamics of the protein, I revealed differential Piezo1 responses across fluid shear stress magnitudes. I also reported a mechanical threshold to HEK-Piezo1 activation during constant fluid shear stress. Through the GenEPi dynamic responses, I observed that cellular Piezo1 activations are synchronised to mechanical shear triggers but become increasingly asynchronous under sustained shear stress. Crucially, the characteristics of Piezo1’s dynamic within cells reported here will significantly advance our understanding of the interplay between mechanical forces and cellular behaviour within the cellular microenvironment. This system can also be a model for others to adopt in investigating the dynamics of other mechanosensitive proteins expressed in the cells.
Version
Open Access
Date Issued
2023-12-31
Date Awarded
01/06/2024
License URL
Advisor
Pantazis, Periklis
Publisher Department
Bioengineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)