Biology subjects

Kirk, M. J.

Publications and source records attributed to Kirk, M. J..

3 recordsLinked to original sources

bioRxiv2026

Evidence that local viscosity and NOX-dependent ROS increases render the tardigrade H. exemplaris resilient to extreme physical force

Biological phase changes provoked by stress, such as vitrification or gel-sol transitions, enable many organisms, including extremotolerant tardigrades, to enter quiescent states and survive extreme environmental conditions. Protein-driven phase transitions are hypothesized to produce large-scale changes in intracellular viscosity, allowing tardigrades to survive extreme stresses such as desiccation. We report that the tardigrade Hypsibius exemplaris undergoes both large-scale and local increases in intracellular viscosity following exposure to anoxic and hyperosmotic stress. Such dramatic shifts in cellular viscosity would be expected to enhance cellular resilience to physical force. Indeed, we found that tardigrades can survive, behave normally, and reproduce after exposure to the highest simulated hypergravity (HG) achievable in an ultracentrifuge (one million times Earths gravity). In contrast, Caenorhabditis elegans, a similarly sized animal, does not survive these extreme forces owing to loss of cellular integrity. Remarkably, tardigrades frozen during exposure to extreme hypergravitational force show minimal disruption of fine cellular ultrastructure and little evidence of stratification of cellular components whose density varies by nearly a factor of two. Further, exposure to anoxia, hyperosmotic stress, and HG all result in a large increase in reactive oxygen species (ROS), which is required for survival under these extreme environments. Inhibition of NADPH oxidase (NOX) suppresses survival both to HG and hyperosmotic stress. Our findings suggest that intracellular viscosity changes in response to multiple extreme stresses may underlie the resilience of these animals to extraordinary physical stress, and that survival in or recovery from these states relies on ROS signaling via NADPH oxidase. Significance StatementTardigrades are renowned for surviving conditions that are lethal to nearly all other life forms. We reveal two mechanisms that support this resilience: intracellular viscosity changes and NADPH oxidase-mediated ROS signaling. Through direct assessment of the effects of altered cellular material properties, found that tardigrades are resilient to forces up to one million times Earths gravity, establishing them as the most hypergravity-resistant animal currently known.

physiology↗

bioRxiv2024

Single-animal, single-tube RNA extraction for quantitative analysis of transcripts in the tardigrade Hypsibius exemplaris

The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes. To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on RNA-sequencing (RNA-seq), which can be performed on populations ranging from large cohorts to individual animals. Reverse Transcription Polymerase Chain Reaction (RT-PCR) and RNA interference (RNAi) are subsequently used to confirm RNA-seq findings and assess the genetic requirements for candidate genes, respectively. Such studies require an efficient, accurate, and affordable method for RNA extraction and measurement of relative transcript levels by quantitative RT-PCR (qRT-PCR). This work presents an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but also reduces the required time and cost for each extraction. This RNA extraction method yields quantities of cDNA that can be used to amplify and detect multiple transcripts by quantitative PCR (qRT-PCR). The method is validated by analyzing dynamic changes in the expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 {beta}2 (HSP70 {beta}2) and Heat-Shock Protein 90 (HSP90), making it possible to assess their relative expression levels in heat-exposed individuals using qRT-PCR. STST effectively complements existing bulk and single tardigrade RNA extraction methods, permitting rapid and affordable examination of individual tardigrade transcriptional levels by qRT-PCR. SUMMARYThis work presents a rapid RNA extraction and transcript level comparison method for analyzing gene expression in the tardigrade Hypsibius exemplaris. Using physical lysis, this high-throughput method requires a single tardigrade as the starting material and results in robust production of cDNA for quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR).

molecular biology↗

bioRxiv2022

Cell-surface targeting of fluorophores in Drosophila for rapid neuroanatomy visualization

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small molecule tethering proteins, HaloTag-CD41 and SNAPf-CD4, which directly label transgene expressing cells with commercially available ligand substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live brain tissue. Pan-neural expression of SNAPf-CD4 enabled registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small molecule probes, drugs, and actuators.

neuroscience↗