Waddellmccarty4256

Endosomal microautophagy (eMI) is a type of autophagy that allows for the selective uptake and degradation of cytosolic proteins in late endosome/multi-vesicular bodies (LE/MVB). This process starts with the recognition of a pentapeptide amino acid KFERQ-like targeting motif in the substrate protein by the hsc70 chaperone, which then enables binding and subsequent uptake of the protein into the LE/MVB compartment. The recognition of a KFERQ-like motif by hsc70 is the same initial step in chaperone-mediated autophagy (CMA), a form of selective autophagy that degrades the hsc70-targeted proteins in lysosomes in a LAMP-2A dependent manner. The shared step of substrate recognition by hsc70, originally identified for CMA, makes it now necessary to differentiate between the two pathways. Here, we detail biochemical and imaging-based methods to track eMI activity in vitro with isolated LE/MVBs and in cells in culture using fluorescent reporters and highlight approaches to distinguish whether a protein is a substrate of eMI or CMA.Macroautophagy is an intracellular degradation system in which autophagosomes and autolysosomes degrade the contents they contain in order to realize cell homeostasis and organelle renewal. Measuring autophagy activity and autophagic flux is very important for studying the role of autophagy, but accurate measurement of autophagic flux is quite complicated. Here, we use the GFP-mRFP-LC3 tandem probe to evaluate the cell autophagic flux. GFP is more sensitive to acidic environment and can be degraded in autolysosome due to the acidic environment. On the contrary, mRFP can be stably present in autolysosome due to its better tolerance to PH reduction. Hence, autophagic flux can be evaluated by calculating the ratio of GFP/RFP signal values. In addition, using this probe, we can more accurately measure the basal autophagic flux and induced autophagic flux in cells or animals. Summarily, the GFP-mRFP-LC3 tandem probe is a simple quantitative method to evaluate autophagic flux of cells and even whole organism.Fasting induces vast metabolic adaptations on the cellular level and leads to an organism-wide induction of autophagy. Autophagic degradation subserves resource recycling and facilitates the maintenance of energetic homeostasis. Mass spectrometry offers the possibility to assess changes in the metabolome that occur in conditions of nutrient deprivation and to profile such adaptations. Here we describe a detailed workflow for the targeted quantitation and untargeted profiling of metabolites that can be used to assess the intracellular metabolome of starving cells. Moreover, we outline a workflow for the use of non-radioactive isotope labeled metabolites. Altogether, we show that mass spectrometry is a powerful tool for monitoring metabolic changes in conditions of fasting.Neutralization of pathogens by phagocytic immune cells requires the biogenesis of a compartmentalized hotspot of reactive species called the phagosome. One of these reactive species is hypochlorous acid (HOCl), produced by the enzyme myeloperoxidase (MPO) after the phagosome fuses with the lysosome. Mapping HOCl during phagosome maturation can report on pathogen killing and offer insights into regulation of MPO activity, mechanisms of resistance and host-pathogen interactions. However, this has been difficult because of a lack of a suitable method to chemically map a transient organelle with pH fluctuations like the phagosome. Here, we detail a protocol for quantifying HOCl dynamics in phagosomes using a fluorescent DNA-based reporter. Cyclopamine supplier Compared to traditional methods of visualizing HOCl or measuring MPO activity, this method offers sub-cellular spatial resolution and the capacity to assay HOCl production with single cell resolution.Mitophagy is an evolutionally conserved cellular process that eliminates dysfunctional and excess mitochondria, thereby facilitating mitochondrial quality control and metabolic recycling. In addition, mitophagy is essential for cellular homeostasis and tissue development, and mitophagic dysfunction is related to various pathologies including neurodegenerative diseases and cancer. Thus, accurate quantitative measurement of mitophagy is one of the hot topics in the field of mitochondrial research. Fluorescence microscopical technology, one of the most widely used technologies at present, can well explain the occurrence and activity of mitophagy. Here, we introduce in detail an experimental method for the immunofluorescence-based quantitativ determination of mitophagy, which not only servers the in-depth study of mitochondrial homeostasis regulation, but also allows for the analyzing mitochondrial autophagy pathologies such as aging, neurodegenerative diseases and cancer.Mechanical stress has been shown to induce the degradation of lipid droplets in kidney epithelial cells. Here, we illustrate the technical equipment and devices that are currently used in our laboratory to apply shear stress on cells. We provide a detailed protocol to monitor lipophagy in response to shear stress. The aim of this review is to guide and help people understand the challenges in studying acidic lipolysis in cells subjected to fluid flow.The transcription factor EB (TFEB) plays a critical role in autophagy induction and lysosomal biogenesis by orchestrating the expression of autophagy- and lysosome-related genes. In response to a series of stresses such as nutrient starvation, TFEB translocates from the cytoplasm to the nucleus, where it exerts its regulatory function. The activity of TFEB is tightly regulated by multiple phosphorylation and acetylation sites. Methods that rely on the analysis of posttranslational modification as a proxy for TFEB activation are often misleading. Here, we elaborate on protocols for monitoring nuclear translocation of TFEB by fluorescence microscopy.Food allergy evaluation for dermatologic disorders is warranted when Type 1 allergy is suspected, and includes skin prick testing (SPT) or measurement of specific immunoglobulin E (IgE) levels. The utility of these tests for identifying triggers is improved with clinical correlation, especially for contact urticaria, and protein contact dermatitis, which are mixed mechanism diseases. In atopic dermatitis (AD), patients are at risk for development of food allergy, and screening with SPT or IgE may be considered in severe AD, especially to guide early food introduction. Management of food-related AD exacerbations should focus on modifications in skincare before evaluating for allergy.