Lysosomes are ubiquitous, dynamic, membrane-bound organelles responsible for macromolecule degradation and catabolite recycling in eukaryotic cells. Lysosomes show highly dynamic behaviors, involving self-budding, extension, and scission. Lysosomes not only receive material via the endocytic pathway, but also through the secretory pathway that directly from the cytoplasm. The material received from endocytic and secretory, such as nucleic acids, lipids, proteins and oligosaccharides, are degraded within lysosomes. Breakdown products are delivered to the processes of cellular anabolism or catabolism. Besides, lysosomes also act as a signaling hub to assemble coordinated cellular responses and related factor information via key regulatory modules docked on the lysosomal surface. In addition, lysosomal function is associated with diverse fundamental processes, including cell surface receptor turnover, antigen processing, plasma membrane repair, ion storage, autophagy, and aging. By serving as a center of degradation, recycling and signaling, lysosomes play vital roles to maintain cell and tissue homeostasis.
Lysosome-related organelles (LROs) are specialized, membrane-bound cellular compartments found in organisms from worms to humans, and share many common features of canonical lysosomes. For Caenorhabditis elegans, the LROs known as gut granules, are the site of microscopic autofluorescence and can be accumulated with ages. Due to the characteristics of autofluorescence and birefringence, the LROs of worms are easily recognized under polarized microscopy. Previous studies uncovered that mutations can disrupt LRO function as well as age-dependent accumulation of autofluorescence. Additionally, C. elegans LROs also act as a cellular reservoir for zinc, in order to prevent toxicity of high dietary zinc.
We offer services to exam adult C. elegans' lysosome morphology. Lysosomal tubule length, number, and volume are the main considerations. The parameters of lysosomes are measured using NUC-1::CHERRY reporter with laser scanning confocal and spinning-disk microscopy. Additionally, the high voltage electron microscopy (HVEM) is adopted for further testing.
Due to the highly dynamic behaviors of lysosomes, we employ spinning-disk microscopy to follow lysosomal dynamics in nematodes expressing NUC-1::CHERRY. The e time-lapse images of C. elegans L4-stage larvae and adults are captured every 1 s for 1-2 min and then analyzed using Volocity software.
Cathepsin L (CPL-1) is an inactive pro-enzyme and converted to the active mature form through proteolytic removal of the pro-domain in lysosomes. The processing of endogenous CPL-1 can be examined to indicate the degradation activity of lysosomes. Besides, the NUC-1::CHERRY fusion protein is also adopted to examine the lysosomal degradation activity. Since CHERRY is cleaved from the fusion protein by cathepsins when delivered to lysosomes, the degree of cleavage can be quantified to indicate the degradation activity of lysosomes by western blot.
|C. elegans Lysosomal Detection Services|
|Items||Content||Experimental subject||Method of measurement|
|Examination of lysosome morphology||Lysosomal tubule length||Adult C. elegans||NUC-1::CHERRY reporter with laser scanning confocal microscopy|
|Lysosome number and volume||Adult C. elegans||NUC-1::CHERRY reporter with spinning-disk microscopy|
|Quantification of lysosome dynamics||Quantification of lysosome dynamics||C. elegans L4-stage larvae and adults||NUC-1::CHERRY reporter with spinning-disk microscopy|
|Lysosome degradation activity assay||Examination and quantification of CPL-1 processing||Adult C. elegans||Western blot|
|Quantification of NUC-1::CHERRY cleavage||Adult C. elegans||Western blot|
Creative Biogene is a professional C. elegans model service provider. Since intestinal cells of Adult C. elegans are large and have extensive endocytic networks. There is a good system for deciphering the endocytosis and other related pathways by detecting lysosomal combining with live imaging techniques. Here we provide lysosome measurement service to assist customers in relevant areas. If you are interested in our services, please contact us for more details.
* For research use only.