Periodic Reporting for period 4 - MechanoSystems (How to build a brain? Engineering molecular systems for mechanosensation and -protection in neurons)
Período documentado: 2021-11-01 hasta 2023-10-31
whose nervous system is mapped and beginning to be well characterized, 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 mechanoelectrical 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. We showed that a protein called MEC-2 forms biomolecular condensates that ensue from liquid-liquid phase separation and mature into viscoelastic gels upon an interaction with a protein that was thought to be restricted to muscles. We showed that binding of MEC-2 to Titin releases a heterotypic interaction and allows these condensates to rigiditify and transmit forces delivered during body touch. These results suggest a strong mechanical compartmenatilization at distinct mechanoreceptor sites. To understand the general principles for information processing in mechanoreceptor neurons we turned to a second mechanosensory modalities, namely to the sense of the self. We identified a mutant in the spectrin cytoskeleton that displays severe defects in body posture regulation. Using a combination of molecular genetics, in vitro optical trapping and elastic substrate deformations, together with in vivo calcium imaging of neuronal activity and thorough behavioral analyses we found that a major proprioceptive neuron selectively responds to compressive and tensile stresses through opening of two distinct mechanoelectrical transduction channels. These ion channels of the TRP and K2P superfamily transduce mechanical information into depolarization or hyperpolarization, respectively, depending on whether the axon is compressed or extended. Because the long axons are subjected to compressive and extensive mechanical stresses at the same time, such a mechanism leads to a dynamic mechanical compartmentalization that ensure local mechanosensing and coordination of locomotion. In future, we will be investigating how mechanosensation changes during disease with a special focus on how proprioceptive coordination is lost in older animals. These results and the future research will have profound consequences on our understanding how mechanical information is processed by neuronal systems and it will shed further light into the pysiological and pathological transformations that results from changes in mechanical properties of neurons.
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.
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