Archaea as a microbial model for developmental mechanoplasticity - PROJECT SUMMARY Cells with identical genomes can follow different developmental paths depending on their mechanical interactions with the environment. Archaea impact human health by playing critical roles in environmental cycles and animal microbiomes. Yet little is known about their cell biology. In contrast to most microbes, archaea are as plastic as mammalian cells, have low turgor pressure, and lack a rigid cell wall. Hence, to advance the mechanistic understanding of how microbiome complexity influences human physiology, it is crucial to understand how archaea respond to mechanical stresses under intermittent crowding changes that mimic the human microbiome. Under this light, our group studies the fundamental principles of how archaeal cells convert mechanical signals into biological outcomes in the face of shear, confinement, osmoshock, and crowding. This proposal furthers our vision by using (1) a top-down approach in which archaeal mechanoresponses are quantitatively and systematically described across spatial-temporal scales from populations to cells and cellular processes; (2) a bottom-up approach to uncover the specific activity-dependent molecules and molecular functions orchestrating cellular and populational responses. To address the first thrust, multi-layer microfluidic devices will be integrated with imaging-based analysis of cellular organization, behavior, biomechanics, sub- cellular dynamics, and gene expression profiles under different shear, compression, and crowding levels. Preliminary data suggests that shear and confinement trigger dramatic but distinct developmental programs in the haloarchaeon model Haloferax volcanii. Closing the gap toward the venture, the second direction will provide mechanistic insights into how different mechanoresponsive factors contribute to the described phenomena above. We will focus on mechanosensor candidates identified by us and other groups, including cytoskeletal polymers (volactin, halofilins, CetZs), mechanochannels, and immunoglobulin glycoproteins. In parallel, we will also leverage the power of protein pulldowns and in vitro reconstitution, combined with the whole-genome CRISPRi library recently built in our group, to expand the interactome and identify new molecular and biophysical pathways underlying mechanosensation. Our past studies and preliminary results demonstrate i) technical skills in mastering microfluidics and advanced archaeal live-cell imaging to probe molecular and biophysical mechanisms, ii) cultivation and reverse genetics of dozens of archaeal species, and iii) proof of concept that some of our dashing ideas will yield valuable insights in laboratory model systems. Our research program envisions a better understanding of how microbes adapt in reconstituted environments akin to human physiological conditions, supporting future mechanistic in vitro and in vivo multi-species microbiome studies.
