C. elegans Osmotic Stress Model Service
Keeping the balance of cellular osmotic is an essential prerequisite for cellular life. Cellular osmotic homeostasis is determined by the accumulation and loss of inorganic ions and organic osmolytes. When an organism or tissue is exposed to the damaging effects of high osmolarity, intracellular water rapidly diffuses across cell membranes, leading to an intracellular ionic imbalance, and following protein damage by aggregation. C. elegans normally lives in the soil, where it can be exposed to constant and extreme osmotic stress conditions. Additionally, C. elegans has numerous experimental advantages. Therefore, the worm provides a powerful model system to characterize the genetic basis of animal cell osmoregulation.
Osmotic Stress in C. elegans
The natural soil environment is full of challenges of wide and rapid change of osmolarity. Due to a high surface-to-volume ratio, low gut and cuticle water permeability, C. elegans are particularly vulnerable to osmotic stress and have the ability to survive extreme conditions. Previous studies indicated that C. elegans can readily adapt to growth media containing 21~500 mM NaCl, and there exist two independent mechanisms that depend on different stress conditions. It has been reported that GPDH-1 (a glycerol-3-phosphate dehydrogenase) induced glycerol synthesis rapidly increases to attenuate the damaging effects of high osmolarity, which is a typical effect of hyperosmotic stress in cells. The response is mediated by GPDH-1, and its activation occurs rapidly and at relatively low levels of salt (200 mM NaCl). Another mechanism is mediated with the osmotically induced accumulation of damaged proteins, which occurs only at high salt concentrations (>500 mM NaCl) and takes approximately 1 h. The accumulation of damaged proteins is an important feature of diseases such as Alzheimer's and Parkinson's. Insight into the molecular mechanisms in C. elegans may help to unravel these complex phenotypes genetically and screen for potential Alzheimer's and Parkinson's therapeutics.
Osmotic Stress Model Services in CD BioSciences
- Fluorescent protein aggregate measurement
Blinded experiments using a Zeiss Stemi SV11 microscope to assess the proteostasis capacity in response to hyperosmotic stress.
- Identification and quantification of organic osmolytes
Organic solutes and protein content are extracted in perchloric acid (PCA). The precipitate proteins are quantified by bicinchoninic acid (BCA) assay. Organic solutes are determined by high performance liquid chromatography (HPLC) analysis. After removing protein, glycerol content can be measured.
- C. elegans analysis
- Survival analysis
After 24 h dealing with hyperosmotic stress, survival is determined. Worms are scored as dead if they don't respond to repeated prodding with a platinum wire.
- Volume measurements
Assuming that the worm body shape approximates a cylinder, the total body volume is calculated by measuring the length and width at the widest point of the animal, combining with Zeiss Stemi SV11 dissecting microscope, a video camera and imaging software.
-Measurement of cuticle disintegration
- Lifespan analysis
- RNA-sequencing and gene expression analysis
RNA-sequencing and gene expression analysis are used to study molecular mechanisms of osmotically induced protein alternation. It is worth mentioning that in order to avoid gene expression changes that might be associated with the L4/adult molt, plates are visually inspected. Before they entered the molting period, and no evidence of molting or development of worms into gravid adults is detected, isolation of total RNA is carried out. In addition, functional annotation for genes is obtained using WormBase and Ensembl BioMart.
Workflow of Osmotic Stress Model Services
CD BioSciences focuses on the stress model system to help our customers to insight into the genetic and molecular mechanisms underlying osmotic stress-associated human diseases, including Alzheimer' and Parkinson' diseases. Additionally, our customers can consult with our experts to design the best solution according to your specific requirements. Please feel free to contact us if you would like to know more about C. elegans osmotic stress service.
Reference
- Lee EC, et al. (2016). "Strange K. Abnormal Osmotic Avoidance Behavior in C. elegans Is Associated with Increased Hypertonic Stress Resistance and Improved Proteostasis". PLoS One. 11(4):e0154156.
* For research use only.
