Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

We want to understand the role of lipophagy on lipid droplet degradation and the function relationships between lipophagy and cancer. Cancer cell exhibit metabolic flexibility with lipid droplet accumulation serving as a hormone. The droplet is stored and supply lipids supporting lipid synthesis and energy production under nutrient scarcity. BODIPY-based fluorescent proofs enable imaging of lipid droplet dynamic for following lipophagy and interaction with organelle like the nucleus, lysosome, and mitochondria, advanced lipid droplet research.

Studying lipophagy in cancer requires advanced microscopy and tumor mimicking models. To quantify lipid droplets' autophagy interaction, crucial for understanding lipophagy's roles in cancer prevention. KDEL receptor signaling coordinate lysosome, autophagy, and lipid-droplet turnover via tethers, linking nutrients and membrane sensing to sustain Golgi-dependent protein secretion.

[Instructor] To begin, take epithelial adenocarcinoma HeLa cells expressing wild-type and two mutants of the autophagy receptor p62/SQSTM. For organelle labeling, wash the cells in a glass bottom dish twice with PBS. Incubate the cells with diluted BODIPY dye and keep them for 30 minutes at 37 degrees Celsius with 5% carbon dioxide. Wash the cells with PBS at room temperature, then maintain them in red-phenol free DMEM, supplemented with 10 millimolar ES-qualified HEPES buffer for live cell imaging. For confocal image acquisition, adjust the Multi-Line Argon gas laser to 10% working power with the 488-nanometer laser line at 1% to 2% potency, resulting in an overall laser power of 0.1% to 0.2%. To minimize cell damage in photobleaching of the probe, adjust the 568-nanometer laser to 3% to 5% potency. Then set the image acquisition resolution in the software to 1024 by 1024 pixels. Adjust the spectral ranges for image capture to 478 to 494 nanometers for green lipid droplet emission and 600 to 625 nanometers for red mCherry emission. Then, set the pinhole size to one area unit for the 488-nanometer wavelength. Incubate the cells with 8-Br-cAMP at a concentration of 100 millimolar to activate protein kinase A kinase, promoting lipid droplet degradation. Finally, capture fluorescent images every five minutes at 37 degrees Celsius for 60 minutes. To begin, take BODIPY 493/503-stained adenocarcinoma HeLa cells. Capture multispectral images every second for five minutes at 37 degrees Celsius using a Multi-Line Argon laser at 10% power, with the 488-nanometer line set between 0.1 and 0.5% to reduce cell damage and photobleaching. In the software, select Plugins, ComDet v0.5.3, and Detect Particles. Define the minimum distance points that must be considered colocalized. Choose particle parameters, including size in millimeters or pixels and the threshold for each channel independently. Define regions of interest for beads and cells by clicking on Tools, followed by ROI Manager. Use the freehand circle to draw the ROI and save them. Then, go to Plugins, Tracking, and TrackMate. Open TrackMate. To confirm the time intervals without making changes, click on Next. Now, select the LoG detector, then click Next. In the LoG detector configuration, filter particles by diameter from 0.8 to 1.0 micrometers and set an appropriate threshold. Perform a preview to confirm the parameters, and click Next. Then, set the initial quality threshold to limit the number of spots for analysis. Press Next without changing any parameters. Select a view by choosing HyperStack Displayer, and then click on Next. Next, choose Linear motion LAP tracker for particles with constant speed in the plane. Then, set the maximum distance from a predicted position for candidate spots in the analysis. Set the maximum frame gap to the maximum time allowed for following a spot that may temporarily disappear from the focal plane. Select display options and then choose plot features. To represent results visually, go to Tracks. Choose an action to save videos that show the movement of spots. p62/SQSTM1-S182A expression resulted in a higher number and intensity of lipid droplets compared to control and wild-type cells, while p62/SQSTM1-S182E expression led to a significant reduction in both number and fluorescence intensity of lipid droplets. Cells expressing p62/SQSTM1-S182A showed fewer lipid droplet autophagosome interactions, averaging 59 interactions per cell with lipid droplet speed at 0.25 micrometers per second. Cells expressing p62/SQSTM1-S182A showed fewer lipid droplet autophagosome interactions, averaging 59 interactions per cell with lipid droplet speed at 0.25 micrometers per second. Cells expressing p62/SQSTM1-S182E showed an increased number of interactions between lipid droplets and autophagosomes, with lipid droplet speed significantly higher at 0.61 micrometers per second.