![]() ![]() Using these two markers we are able to visualize by fluorescent microscopy how both the lysosomal and the endosomal pathway are responding to different experimental conditions and to assess sensor puncta formation in various genetic backgrounds. Typically, we simultaneously express UAS-GFP-HsLAMP1 to mark (endo)lysosomes in the larval fat body ( Figure 1). its localization to endo/lysosomes) under various experimental conditions and tissues in pulse-chase type of experiments. More specifically, the sensor consists of a KFERQ targeting motif of RNAseA fused to a photoactivatable mCherry and is expressed tissue-specifically under the control of Gal4-UAS system which allows us to visualize the formation of sensor puncta (i.e. Upon eMI activation, this biosensor localizes to late endosomes/lysosomes in an ESCRT machinery-dependent manner. For example, using a KFERQ-tagged fluorescent biosensor, we have identified an eMI-like pathway in Drosophila. To date, quantification of punctate structures such as autophagosomes, endosomes, or lysosomes using Fiji/ImageJ requires repetitive tedious manual step by step processing of images of genotypes or samples of interest treated by different means, which makes the analyses of a high number of cells very time consuming. CMA and eMI can specifically degrade proteins containing KFERQ-related targeting motifs. MA can selectively degrade organelles or aggregated proteins, but selective degradation of single proteins has mostly been described for CMA and eMI. ![]() Three types of autophagy have been identified in mammalian cells: Macroautophagy (MA), chaperone-mediated autophagy (CMA), and endosomal microautophagy (eMI). Methods to measure autophagy or vesicular transport in general are very sought after and are frequently based on the analysis of punctate structures in fluorescent micrographs. ![]() This is reflected by its association with a large array of pathologies and dysfunctions, including cardiovascular, neurodegenerative, cancer as well as aging associated diseases. Autophagy is a catabolic pathway that occurs in all eukaryotic organisms analyzed to date and is critical for the maintenance of cellular homeostasis. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |