Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Autophagy is the cell’s way of taking out its own garbage and recycling. But while failure to take out the trash can make a house malodorous, dysregulation of autophagy has been implicated in disease, including infection, cancer, diabetes and more.
Autophagy is a complicated sequence of only partially understood events by which cells degrade their own intracellular parts, including misfolded proteins, damaged mitochondria and pathogen-infected intracellular constituents. Autophagy can be selective, too, and researchers are actively investigating the mechanisms of regulation and selection. Here we highlight some of the tools researchers are using to do just that.
Antibodies
Using antibodies specific to one target molecule is a powerful way to pinpoint proteins, kinases and enzyme substrates involved in biological processes such as autophagy. Novus Biologicals offers a broad range of monoclonal and polyclonal antibodies designed to target all steps in the autophagy process, and researchers can apply these to get both quantitative and qualitative information on specific molecular targets or signaling pathways, says Mike Fan, product manager at Novus Biologicals.
Cell Signaling Technology (CST) also markets antibodies for autophagy research. “We offer not only antibodies against some of the key markers in autophagy, like LC3, but we also offer a lot of signaling antibodies,” says Gary Kasof, senior group leader in development at CST. For example, the company has research antibodies against key sites phosphorylated by kinases known to be involved in autophagy signaling pathways, such as mTOR, AMPK and ULK1.
Kasof says most of CST’s autophagy antibodies are monoclonals, which offer higher performance and better reliability from lot to lot than polyclonal antibodies. “In the case of our LC3 antibody, we spent a lot of time trying to develop monoclonal antibodies that would perform well for immunofluorescence, which is really key for LC3, because investigators are looking for punctate staining indicative of autophagosomes that are being triggered by autophagy,” he explains.
‘Do not disturb’ using dyes
Another approach to autophagy research is to use fluorescent dyes. For instance, Thermo Fisher Scientific’s Premo™ Autophagy sensors and CellLight® reagents “work well in cells that are typically recalcitrant to gene delivery, for example primary human cells and neurons,” says Stephen Oldfield, the company’s senior market development manager. The Premo Autophagy Sensor detects the recruitment of LC3B protein from cytoplasm to membrane in early autophagy, and the Premo Autophagy Sensor Tandem LC3B indicates whether LC3B-positive vesicles have an acidic or neutral pH.
Thermo Scientific also offers “fluorescent proteins to detect the recruitment of autophagy markers, and a tandem sensor to report on maturation of the autophagosome,” says Oldfield. “Single-color LC3B chimeras can be combined with organelle dyes such as MitoTracker and LysoTracker or the CellLights® fluorescent proteins to provide more biological information.”
Oldfield says the company’s tools typically are used with fluorescence microscopes, high-content imaging systems or microplate readers. “Depending on the application, researchers can monitor the progress of autophagy through wavelength shifts, intensity measurements or by counting cellular features,” he says. Thermo Fisher also offers new, high-throughput screening autophagy assays that use time-resolved FRET (fluorescence resonance energy transfer) to detect autophagy induction.
Enzo Life Sciences also offers a range of autophagy tools; its flagship product is the Cyto-ID® Autophagy Detection Kit. A cell-based assay, Cyto-ID uses a fluorescent, cationic, amphiphilic tracer “to highlight the various vacuolar components of the autophagy pathway,” says Courtney Noah, senior marketing manager at Enzo Life Sciences. “The assay is compatible with flow cytometry, allowing, for the first time, easy quantitation of autophagy without the need for transfection.”
The assay lets users track autophagy in live cells using fluorescence microscopy and in flow cytometry for high-throughput screening, and it even labels primary cells. It also “enables kinetic analysis of the autophagy pathway and is able to distinguish between increases in autophagic flux”—the equilibrium between the formation and elimination of autophagosomes—“vs. autophagic vacuole accumulation,” says Noah. The assay also can be multiplexed with probes specific for other organelles, such as mitochondria and lysosomes. For example, combining the Cyto-ID® Green Autophagy Dye with Mito-ID® Red enables users to differentiate autophagy from mitophagy, the process by which cells selectively degrade damaged (or otherwise unwanted, as in developing erythrocytes) mitochondria.
A lentiviral biosensor
EMD Millipore offers a range of tools for autophagy analysis, including antibodies to autophagy targets, autophagy inducers and autophagy inhibitors. Generally, the inducers work by blocking pathways that normally inhibit autophagy. The inhibitors work by blocking the formation of autophagosomes.
In addition, EMD Millipore has created so-called biosensors, or lentiviral-based GFP-LC3 reporter cell lines, for visualizing an initial step in the autophagy pathway: LC3 localization to autophagosomes. Using the reporter cell lines, the translocation event can be observed by microscopy in live cells or with flow cytometry. “GFP- or RFP-protein [LC3] localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype,” says Kevin Long, neuroscience lead and manager of technical content marketing at EMD Millipore. This gives researchers the opportunity to study the regulation of this segment of the autophagy pathway—for example, by using the autophagy inducers and inhibitors described above.
EMD Millipore’s FlowCellect™ GFP-LC3 Reporter Autophagy Assay Kits let users monitor autophagy with flow cytometry. The kits include a GFP-LC3 reporter cell line for tracking cellular LC3 levels. They can even distinguish between cytosolic and autophagic (i.e., membrane-associated) LC3 using selective cell permeabilization.
Another tool for studying autophagy is EMD Millipore’s microfluidic CellASIC® ONIX Platform, which enables control of the cells’ environment during live-cell imaging. ONIX uses microfluidics-based cell-culture plates and a standard inverted microscope. By precisely controlling parameters such as temperature, gas levels and solution exchange, users can conduct time-lapse experiments with dynamic environmental conditions while sampling data over long time periods. “Remember that autophagy is often a microenvironment-induced cellular event, and so the CellASIC system is a fantastic tool for studying induction since it completely controls the cellular environment in the culture,” Long says.
With such a broad tool set available, users should have no problem detecting autophagy. But autophagy is just one of many cellular responses to changing environmental conditions, Long says. “There is increasing need to study autophagy in the context of epigenetic regulation, oxidative stress and programmed cell death, or apoptosis,” he explains. Looking forward, such work could benefit from imaging flow cytometry, which images cells during flow cytometry, he says. “In situ and culture-based single-cell analysis, but using sizable populations, may turn out to be valuable here.”
Image: Punctate LC3A staining (green) in HeLa cells induced by chloroquine treatment. (Red, actin; blue, nuclei). Courtesy of Cell Signaling Technology. (PDF)