We successfully engineered mechano-responsive cells by expressing mechanosensitive ion channels MscS and P3:FAStm:TRPC1 in mammalian cells.
Localization of mechanosensitive channel TRPC1 on the plasma membrane was demonstrated and improved by fusing it with segments of FAS receptor, including the transmembrane domain.
Addition of gas-filled lipid microbubbles increased the sensitivity of mammalian cells to ultrasound.
We demonstrated for the first time that gas vesicle-forming proteins are expressed in a human cell line, are not toxic and improve the sensitivity of cells to mechanical stimuli.
A custom-made ultrasound stimulation device (Moduson), suitable for use in different experimental setups that require ultrasound stimulation of cells was developed.
New graphical analysis software that enables fast analysis of fluorescent microscopy data was also developed to quantify the response to ultrasound stimulation.
A new split calcium sensing/reporting system was designed that is able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation by emitted light.
Cells interact with other cells and environment in various ways in order to appropriately respond to the microenvironmental changes. Some of the important extracellular physical
signals are the mechanical forces and adaptation upon mechanical stimuli is crucial for regulating the cell volume, signalization, growth, cell to cell interactions and
overall survival.
Mechanical forces such as changes in osmolality, fluid flow, gravity or direct pressure result in changes in tension of the phospholipid bilayer and arrangement of
the cellular cytoskeleton. The detailed mechanism of mechanosensing is not known, however most mechanosensitive receptors respond to mechanical stimuli through opening
of the channel pore and allowing calcium ions to enter the cell Zheng2013. Role of the membrane composition has been shown for bacterial channels, however
cytoskeleton apparently plays an important role as several mechanosensitive channels comprise domains that can interact with cytoskeleton.
INSERT!!!!!
This mechanism, serves as a force-sensing system Zheng2013, Haswell2011. Furthermore, it has already been shown that living organisms can detect
and respond to mechanical stress generated by ultrasound, which represents an external stimulus with many potential applications Ibsen2015. Ultrasound
offers remarkable advantages over other external stimuli used for targeted cell stimulation. In our project we aimed to explore the potential of mechanosensors and to
improve the sensitivity of cells to mechanical stimulation with the idea of designing ultrasound-responsive devices.
