Fluorescence-based Tools For Investigating Autophagy, Cells Under Stress

When it comes to managing stress, our cells function like efficient machines, quickly assessing the situation and finding ways to adjust. Autophagy—literally, “eating oneself”—is one way that cells deal with typical day-to-day stress (misfolded proteins, aged or defective organelles) as well as unexpected disasters (hypoxic conditions, serum starvation, viral infections). The cellular machinery either digests the damaged or defective molecules, effectively eliminating the stressors that threaten cell health, or, in the case of hypoxia and serum starvation, devours existing proteins and organelles to generate key nutrients for survival. Much about the autophagic process is still not well understood, including its role in diseases such as cancer and neurodegeneration. Here we describe new fluorescence-based imaging tools to help visualize (Figure 1) this important intracellular process.

Figure 1. Imaging autophagy with the Premo™ Autophagy Sensor LC3B-GFP and CellLight™ Lysosomes-RFP. HeLa cells were cotransduced with the Premo™ Autophagy Sensor LC3B-GFP (green) and CellLight™ Lysosomes-RFP. (red). The following day, cells were treated with 50 μM chloroquine diphosphate. Twenty-four hours later, cells were incubated with 1 μg/mL Hoechst 33342 (blue) and 20 μg/mL Alexa Fluor® 647 70,000 MW dextran conjugate (purple) for 40 min at 37°C before imaging.

Targeting Proteins and Organelles for Degradation

The process of autophagy begins with the formation and elongation of isolation membranes, or phagophores (Figure 2). The cytoplasmic cargo is then sequestered, and the double-membrane autophagosome fuses with a lysosome to generate the autolysosome. Finally, degradation is achieved through the action of hydrolytic enzymes within the autolysosome. Autophagy was first described in 1963; however, only in the past decade has this pathway become the subject of intense study. Researchers have sought to gain further insight into the role basal autophagy plays in cell homeostasis and development, and to further elucidate the role of induced autophagy in the cell’s response to stress, microbial infection, and disease processes such as neurodegeneration, cancer, and others [1–3].

Click image to enlarge

Figure 2. Schematic depiction of the multistep autophagy pathway in a eukaryotic cell. he first step involves the formation and elongation of isolation membranes, or phagophores. In the second step, which involves the LC3B protein, the cytoplasmic cargo is sequestered, and the double-membrane autophagosome is formed. Fusion of a lysosome with the autophagosome to generate the autolysosome is the penultimate step. In the fourth and final phase, the cargo is degraded.

Observe LC3B Temporally and Spatially

The LC3B protein plays a critical role in autophagy. Normally this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the phagophore and can be used as a general marker for autophagic membranes. The new Premo™ Autophagy Sensor combines the selectivity of an LC3B–fluorescent protein (LC3B-FP) chimera with the transduction efficiency of BacMam technology, enabling unambiguous visualization of this protein in live cells (Figure 3). The reagent also tolerates fixation with formaldehyde and thus is compatible with fixed-cell analysis, which is required for multiplex experiments using antibodies, and is preferred for HCS analysis workflows.

BacMam reagents (insect baculoviruses with a mammalian promoter) are nonreplicating in mammalian cells and thus are safe to handle (biosafety level 1). They are also noncytotoxic and ready to use. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high cotransduction efficiency; therefore, multiple BacMam reagents can readily be used in the same cell.

Recent improvements to the BacMam system enable efficient transduction in a wider variety of cells, including neurons and neural stem cells, with an easy one-step protocol. Now, to image autophagy, simply add BacMam LC3B-FP to cells and incubate overnight for protein expression. Each Premo™ Autophagy Sensor Kit includes BacMam LC3B-FP, a control BacMam LC3B (G120A)-FP, and chloroquine diphosphate to artificially induce phagosome formation. Following treatment with chloroquine diphosphate, normal autophagic flux is disrupted, and autophagosomes accumulate as a result of the increase in lysosomal pH. The mutation in the control BacMam LC3B (G120A)-FP prevents cleavage and subsequent lipidation during normal autophagy, and thus protein localization should remain cytosolic and diffuse.

Available separately is the LC3B Antibody Kit for Autophagy which includes a rabbit polyclonal anti-LC3B antibody and chloroquine diphosphate, for imaging autophagy via LC3B localization in fixed samples (Figure 4).

Figure 3. Detecting autophagy with the Premo™ Autophagy Sensor and fluorescence microscopy (left) or high-content imaging and analysis (right). (Left) U2OS cells were cotransduced with the Premo™ Autophagy Sensor LC3B-RFP (red) and CellLight™ MAP4-GFP (green). The following day, cells were treated with 50 μM chloroquine. The following day, cells were incubated with 1 μg/mL Hoechst 33342 (blue) before imaging. (Right) HeLa cells were plated at 5,000 cells per well and left to adhere overnight. Cells were then transduced with the Premo™ Autophagy Sensor LC3B-GFP. The following day, cells were incubated with 50 μM chloroquine or left untreated (control) for 16 hr. Quantitative analysis was performed by quantifying fluorescence from vesicular structures in the perinuclear region using the Thermo Scientific Cellomics® ArrayScan® VTI platform.

Localization of LC3B visualized with the LC3B Antibody Kit for Autophagy.

Figure 4. Localization of LC3B visualized with the LC3B Antibody Kit for Autophagy. (A) Cells were transduced with the Premo™ Autophagy Sensor LC3B-RFP. (B, C) Cells were treated with chloroquine to induce phagosome accumulation. Panel C includes the LC3B-antibody from the LC3B antibody Kit for Autophagy visualized with Alexa Fluor® 647 goat anti–rabbit IgG, demonstrating colocalization of LC3B-RFP and the LC3B antibody. (D) HeLa, A549, and HepG2 cells were treated with 50 μM chloroquine for 16 hr at 37°C. Following fixation and permeabilization, autophagosomes were stained with anti-LC3B rabbit polyclonal antibody and visualized with Alexa Fluor® 647 goat anti–rabbit IgG. The fluorescence intensity of the autophagosomes and cytosol was quantified using Slidebook™ digital microscopy software.

Image Autophagic Organelles

Two organelles that play a crucial role in autophagy are the mitochondria and lysosomes. Old, damaged, or surplus mitochondria are major targets for autophagy, which in this case is sometimes referred to as “mitophagy”. Degradation of mitochondria through this process can be used to recover their amino acids and other nutrients as well as remove damaged mitochondria from the cell [4,5]. Fusion of a lysosome with the phagophore to form the autolysosome is the penultimate step of the autophagic pathway (Figure 2). A variety of reagents including fluorescent dyes, fluorescent protein chimeras, and antibodies can be used for imaging autophagic organelles, like the lysosomes and mitochondria, during basal and induced autophagy (Table 1, Figure 5).

Figure 5. Imaging autophagy in live HeLa cells with CellLight™ reagents for mitochondria and lysosomes. Cells were transduced with CellLight™ Mito-RFP and CellLight™ Lysosomes-GFP. Following treatment with 200 μM chloroquine, nuclei were stained with Hoechst 33342, and live-cell imaging was performed using a DeltaVision® Core microscope and standard DAPI/FITC/TRITC filter sets.

Table 1. Fluorescent Detection Reagents for Imaging Mitochondria and Lysosomes.

	Organic Dyes	BacMam-based Fluorescent Proteins	Primary Antibodies
	(e.g., MitoTracker® and LysoTracker® dyes)	(e.g., CellLight™ reagents)
How They Work	Positively charged MitoTracker® dyes localize to actively respiring mitochondria; weakly basic LysoTracker® dyes accumulate in compartments with low pH.	Combine targeting sequence–fluorescent protein fusion with the transduction efficiency of BacMam to label organelles independently of function (i.e., pH, mitochondrial membrane potential).	Recognize specific target of interest (e.g., LAMP1, a lysosomal protein).
Applications	Live-cell imaging applications; fixable,* thus compatible with antibody-based imaging applications.	Live-cell imaging applications; fixable,*† thus compatible with antibody-based imaging applications.	Imaging fixed cells or tissue. Compatible with labeling with MitoTracker®, LysoTracker®, and CellLight™ reagents.
Typical Workflow	Incubate cells with the MitoTracker® or LysoTracker® reagent for approximately 5–30 min.	The ready-to-use CellLight™ reagent is added to live cells, followed by an overnight incubation to allow for protein expression.	Cells are fixed and permeabilized, then incubated with the antibody for labeling, and visualized with a fluorescently labeled secondary antibody.**
* Please consult the product manual or contact technical service for additional information on the fixability of these reagents. † With fluorescent protein constructs, anti-GFP or anti-RFP antibodies can be used to amplify or change any fluorescence. **Secondary antibody is required if the primary antibody is not directly labeled.

Monitor Degradation of Long-lived Proteins

Autophagy helps to eliminate long-lived proteins from cells. The current method of monitoring this degradation recommends the use of a radiolabeled amino acid to first label cellular proteins, followed by a long cold chase to allow for degradation of any short-lived proteins. Degradation of the radiolabeled long-lived proteins can then be calculated by measuring the amount of radioactivity in the medium relative to the cells [6–8].

A potential alternative to this radioactive method is the use of click chemistry, whereby an azide- or alkyne-modified amino acid (Click-iT® AHA or Click-iT® HPG, respectively) is substituted for the radiolabeled amino acid and detection uses the corresponding fluorescently-labeled alkyne or azide. The Click-iT® AHA assay provides both the labeled amino acid and a fluorescent detection reagent based on the bright and photostable Alexa Fluor® 488 dye.

Visualize Autolysosome Formation

The compound DQ™ BSA, when used in conjunction with an autophagic marker like LC3B, can be used to accurately visualize the formation of the autolysosome in live cells [9]. DQ™ Green BSA and DQ™ Red BSA are bovine serum albumin (BSA) conjugates that have been labeled to such a high degree that the fluorescence is self-quenched. To visualize autolysosome formation, cells that express GFP-LC3B or RFP-LC3B are incubated with the contrasting color of DQ™ BSA. The convergence of the lysosome with the autophagosome results in dequenching, and the release of brightly fluorescent fragments and the autolysosomes are identified by colocalization of green and red fluorescence.

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