A09 - Scaffolding of mechanotransduction domains in migratinfg and non-migrating cells
The mechanosensitive ion channels, PIEZO1 and PIEZO2, are involved in multiple cellular processes, ranging from vessel formation to touch sensation (Ranade et al., 2014). We have previously shown that STOML3 modulates PIEZO channels and is present in oligomeric form in membrane micro domains (Wetzel et al., 2017, Poole et al., 2014). We also identified a range of stomatin‐domain modulating small molecules that either decrease or increase STOML3 domain‐size in the plasma membrane as measured with dSTORM super‐resolution microscopy (Wetzel et al., 2017). Recently, we made the surprising observation that >90% of PIEZO channels cannot be opened by mechanical stimuli under physiological membrane potentials (Moroni et al., 2018). However, very positive voltage steps or disease-causing mutations near the channel pore can release channels for efficient opening (Moroni et al., 2018). The same disease-causing mutations that change PIEZO2 voltage sensitivity are associated with craniofacial abnormalities in humans.
There is increasing evidence that mechanosensitive PIEZO proteins play a role in cell migration during development by sensing the mechanical environment during cell movement. We recently found that there is a very early migratory population of cells that express PIEZO2 channels that later in development contribute to the endothelial cell population in vessels throughout the mouse embryo. We will use transgenic mouse models to manipulate PIEZO sensitivity in these migrating cells for instance by miss‐expressing STOML3 in these cells. This will be done by using a Cre‐inducible overexpression of an epitope‐tagged STOML3 from the ROSA locus; STOML3 overexpression can initiated using a PIEZO2‐Cre mouse available to us (Ranade et al., 2014). We hypothesize that the scaffolding proteins like STOML3 may control the localization of mechanotransduction domains in migrating cells to sensitize mechanosensing regions of the cells, like filopodia, to mechanical perturbation. We also wish to label PIEZO2 expressing endothelial precursors to study the fine architecture of PIEZO protein localization in migrating cells.
We recently showed that both PIEZO1 and TRPV4 channels are required for fast mechanotransduction by chondrocytes in the joint; a stationary mechanosensitive cell type (Servin‐Vences et al., 2017). TRPV4 appears to be unique amongst TRP channels in that it is gated extremely efficiently in heterologous cells by substrate deflection (Servin‐Vences et al., 2017). We will use a TRPV1/TRPV4 chimeric channel approach to map the domain of TRPV4 that is required for substrate gating. We will use pull‐down approaches with proteomics to try and identify TRPV4‐interacting proteins that provide the mechanical link with the extracellular matrix. Such interacting proteins may represent novel scaffolding proteins with essential roles in mechanotransduction.
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