Bacterial Mechanosensitive Channel of Large Conductance (MscL) in Mammalian Cells for Novel Mechanobiology Applications

Abstract

Mechanobiology, a relatively young field that centers on how external physical forces on cells or tissues and their intrinsic mechanical properties can influence physiology and disease, has become a pillar in cell biology. Indeed, cells experience a myriad of external, mechanical stimuli such as shear stress, stretch, substrate and matrix rigidity, surface topography, compression, and inter-cellular junction forces. Mechanosensors on the cell surface interfacing with the external environment (e.g., receptors, mechanosensitive ion channels, focal adhesions) and within the cell (e.g. the cytoskeleton) can sense, transmit, and amplify these inputs. This results in a cascade of intracellular biochemical signaling that leads to altered gene expression, protein expression, and finally, altered cell behavior and function. This process is known as mechanotransduction. Mechanotransduction at the cellular-scale has perceptible, large-scale implications such as proper organism development, our ability to sense sound and touch, function and homeostasis of organ systems, and disease progression. Previous work in mechanobiology has focused on investigating or capitalizing on native, endogenous mechanotransduction. This dissertation work proposes a relatively unexplored frontier in mechanobiology: exogenous mechanotransduction, by demonstrating a novel approach towards achieving signal transduction and mechanically driven behavior in cells through the introduction of exogenous mechanosensory components. Here we demonstrate how the functional expression of the E. coli membrane tension gated mechanosensitive channel of large conductance (MscL) in mammalian cells endows the cells with new mechano-sensing capabilities such as the activation of MscL in the plasma membrane through membrane tension resulting from (1) osmotic down-shock and (2) new interactions with native mechano-sensory components, as well as altered cell function such as (3) impairment of cell migration in metastasis in vivo and narrow, 3D confinement in vitro. The first major contribution in this thesis was to show that MscL can be expressed in mammalian cells, localize to cellular membranes, and responds to membrane tension via osmotic down-shock. The second contribution was demonstrating that the activation of the bacterial MS channel expressed in mammalian cells can be mediated through localized membrane stress that is dependent on the native actin-cytoskeleton. This was done by using acoustic tweezing cytometry (ATC) where acoustic excitation of microbubbles targeted to surface integrin receptors generated localized forces that robustly gated MscL. Impermeable, fluorescent dye uptake was used to report MscL activation; also showing that activated MscL can deliver large molecules into the cell. Lastly, we investigated the effect of MscL mechanotransduction on the cell function of migration in cancer metastasis and then more specifically, 3D-confinements. Our findings in our in vivo mouse model showed that there was a marked reduction in metastasis to the lung for MscL expressing cancer cells compared to controls. In vitro migration experiments using a biomimetic microfluidic device revealed that MscL activation due to 3D-confined migration could be responsible for the observed reduction in metastasis. We found that ~46% of MscL-expressing cancer cells that entered extremely narrow confinements of 30 µm2 cross-section had activated MscL and only 11% of these cells were able to fully enter the channel and migrate. Implications of this thesis are that MscL: (1) can be used as a molecular delivery tool for live-cells via mechanical stimulus; (2) can provide insight into the metastatic cascade and mechanobiology focused therapies; and (3) in mammalian cells can serve to study existing mechanotransduction or potentially engineer new mechanical properties and signaling in cells.