Sensitivity to Strain and Shear Stress of Isolated Mechanosensitive Enteric Neurons.

Within the enteric nervous system, the neurons in charge to control motility of the gastrointestinal tract reside in a particular location nestled between two perpendicular muscle layers which contract and relax. We used primary cultured myenteric neurons of male guinea pigs to study mechanosensitivity of enteric neurons in isolation. Ultrafast Neuroimaging with a voltage-sensitive dye technique was used to record neuronal activity in response to shear stress and strain. Strain was induced by locally deforming the elastic cell culture substrate next to a neuron. Measurements showed that substrate strain was mostly elongating cells. Shear stress was exerted by hydrodynamic forces in a microchannel. Both stimuli induced excitatory responses. Strain activated 14% of the stimulated myenteric neurons that responded with a spike frequency of 1.9 (0.7/3.2) Hz, whereas shear stress excited only a few neurons (5.6%) with a very low spike frequency of 0 (0/0.6) Hz. Thus, shear stress does not seem to be an adequate stimulus for mechanosensitive enteric neurons (MEN) while strain activates enteric neurons in a relevant manner. Analyzing the adaptation behavior of MEN showed that shear stress activated rapidly/slowly/ultraslowly adapting MEN (2/62/36%) whereas strain only slowly (46%) and ultraslowly (54%) MEN. Paired experiments with strain and normal stress revealed three mechanosensitive enteric neuronal populations: one strain-sensitive (37%), one normal stress-sensitive (17%) and one strain- and stress-sensitive (46%). These results indicate that shear stress does not play a role in the neuronal control of motility but normal stress and strain.

Substrate, focal adhesions, and actin filaments: a mechanical unit with a weak spot for mechanosensitive proteins.

Mechanosensing is a vital prerequisite for dynamic remodeling of focal adhesions and cytoskeletal structures upon substrate deformation. For example, tissue formation, directed cell orientation or cell differentiation are regulated by such mechanosensing processes. Focal adhesions and the actin cytoskeleton are believed to be involved in these processes, but where mechanosensing molecules are located and how elastic substrate, focal adhesions and the cytoskeleton couple with each other upon substrate deformation still remains obscure. To approach these questions we have developed a sensitive method to apply defined spatially decaying deformation fields to cells cultivated on ultrasoft elastic substrates and to accurately quantify the resulting displacements of the actin cytoskeleton, focal adhesions, as well as the substrate. Displacement fields were recorded in live cell microscopy by tracking either signals from fluorescent proteins or marker particles in the substrate. As model cell type we used myofibroblasts. These cells are characterized by highly stable adhesion and force generating structures but are still able to detect mechanical signals with high sensitivity. We found a rigid connection between substrate and focal adhesions. Furthermore, stress fibers were found to be barely extendable almost over their whole lengths. Plastic deformation took place only at the very ends of actin filaments close to focal adhesions. As a result, this area became elongated without extension of existing actin filaments by polymerization. Both ends of the stress fibers were mechanically coupled with detectable plastic deformations on either site. Interestingly, traction force dependent substrate deformation fields remained mostly unaffected even when stress fiber elongations were released. These data argue for a location of mechanosensing proteins at the ends of actin stress fibers and describe, except for these domains, the whole system to be relatively rigid for tensile strain with a mechanical coupling between the front and rear end of a cell.