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How to build a brain? Engineering molecular systems for mechanosensation and -protection in neurons

Periodic Reporting for period 2 - MechanoSystems (How to build a brain? Engineering molecular systems for mechanosensation and -protection in neurons)

Reporting period: 2018-11-01 to 2020-04-30

Summary of the context and overall objectives of the project
The major focus of our lab is to understand the impact of mechanical stresses and material properties on the physiological functions and pathological transformations in neurons. We perform our investigations in the non-vertebrate model organism Caenorhabditis elegans, whose neuronal output is well stereotyped, thus, mechanical manipulations can be readily assessed on the physiological level. We began our quest by analysing the force transmission pathway that leads to the activation of the mechanoelectral transduction channel upon a deformation of the skin during the sense of touch. Whereas the molecular players are known for a long time, their interplay and mechanical interactions are less understood. Preliminary data shows that a yet to be identified connection to the cytoskeleton is critical to activate the mechanosensitive ion channel during touch, either delivered by an eyebrow hair or using novel microfluidic devices. In future, we will extend these analyses to other mechanosensitive systems, such as proprioception.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
To understand force transmission at the molecular scale, we produced mutant alleles of an ion channel adaptor subunit using CRISPR/Cas9. Importantly, these mutants almost completely eliminates the sense to external touch, without perturbing localization of the subunit to the ion channel. We next setup a theoretical model that integrates a two component system under the action of a impinging force to simulate the evolution of the open state of the mechanoelectrical transduction channel. The results are consistent with a molecular tether that transfers the force to the channel. To proof this prediction, we inserted a tension-sensitive FRET cassette into the adaptor protein and visualized the transfer of mechanical stress during touch. In order to simultaneously perform imaging and force delivery to the animals, we immobilized individual animals in microfluidic chips with integrated pneumatic actuators. Importantly, under static condition, we find that the adaptor protein is not under mechanical tension, but efficiently is set under stress when the animal is indented using the pneumatic channels. We are now currently investigating the origin of the force transmitting component, e.g. possible cytoskeletal components.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Whereas the molecular components of the force transduction apparatus have been known for a long time in C elegans and are now also emerging in humans, the interplay and mechanism of force transduction is still elusive. Much effort has been put on deciphering the influence of membrane properties on ion channel activity. Our results, however, argue against this mechanism, as artificially increasing membrane tension by a factor of 20x using optical tweezers does not activate the mechanoreceptor neurons. Rather, force becomes transmitted through the cytoskeleton – a prosthetic, optogenetic interaction of the mutant channel adaptor to the actomyosin cortex is able to rescue the touch insensitivity phenotype. Thus, our combined results strongly favor a scenario during which force is transmitted from the cytoskeleton during touch. Further work needs to perfomed if this is a unifying principle for other mechanosensitive modalities, e.g. nociception and proprioception.