Advanced fluorescent probes for imaging distinct processes in the autophagy pathway

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In mammalian cells, two major catabolic processes—the ubiquitin-proteasome system (UPS) and the autophagy pathway—maintain cell health by degrading proteins and organelles that have either reached the end of their life or become compromised. The proteasome system is charged with the degradation of short-lived proteins, whereas the autophagy pathway degrades longer-lived proteins. In addition to protein degradation, autophagy facilitates the removal of superfluous or damaged organelles, as well as invading microbes and protein aggregates. Misregulation of these processes is associated with a number of disease states, from cancers to cardiac myopathies and neurodegenerative disorders. Here we describe a suite of fluorescence-based reagents specifically designed for the study of the proteasome system and autophagy pathway.

Nonradioactive measurements of protein synthesis and degradation with Click-iT® HPG

Both the proteasome system and the autophagy pathway function to degrade cellular proteins and can therefore be studied by labeling proteins in live cells and subsequently monitoring protein lifetimes. Traditionally, these protein measurements required the use of radiolabeled methionine. Although highly sensitive, radioactivity-based approaches do not provide detailed spatial readout, a key feature when studying protein aggregation. Furthermore, they pose a significant hazard to researchers and their surrounding environments.

Life Technologies has developed a click chemistry–based approach that not only circumvents the need for radioactivity but also enables fluorescence imaging of protein aggregates in cells. The Click-iT® HPG Alexa Fluor® Protein Synthesis Assay Kits utilize the methionine analog homopropargylglycine (HPG), which contains an alkyne moiety that can be detected with a fluorescent Alexa Fluor® azide through a click reaction. HPG is fed to cultured cells and incorporates into proteins undergoing translation during the pulse period. Following a chase period, HPG-containing proteins can be detected using either Alexa Fluor® 488 azide or Alexa Fluor® 594 azide, provided in the respective Click-iT® HPG Protein Synthesis Assay Kits [1]. The resulting fluorescent signal is a function of both the protein synthesis occurring in the pulse period and the protein degradation in the chase period.

This approach can be further tailored to study changes in bulk-protein lifetime. For example, Figure 1 shows dose-dependent inhibition of protein clearance—as revealed by increasing Alexa Fluor® 488 fluorescence—in HeLa, A549, and U2OS cells that were pulse-labeled with HPG, treated with 10 different concentrations of the proteasome inhibitor bortezomib during the 6-hour chase period, and then detected with Alexa Fluor® 488 azide.

Proteasome-dependent clearance of short-lived proteins.  
Figure 1. Proteasome-dependent clearance of short-lived proteins. HeLa, U2OS, and A549 cells were incubated with 50 µM homopropargylglycine (HPG) for 1 hr in methionine-free DMEM. Following HPG incorporation, cells were incubated in HPG-free medium for 6 hr (to allow clearance of labeled short-lived proteins) in the presence of different concentrations of the proteasome inhibitor bortezomib. Cells were then fixed, and HPG-containing proteins were detected using green-fluorescent Alexa Fluor® 488 azide provided in the Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit.

Imaging protein aggregates with Click-iT® HPG

In addition to the measurement of protein lifetimes, the Click-iT® HPG Alexa Fluor® Protein Synthesis Assay Kits are useful for visualizing the formation of toxic protein aggregates in cells. In order to gain a more in-depth picture of the mechanisms for clearing these aggregates, click chemistry–based detection can be combined with immunocytochemical analysis using antibodies that recognize key proteins involved in protein clearance. Figure 2 demonstrates the imaging of protein aggregates in HeLa cells. In this example, HPG-containing proteins were detected with Alexa Fluor® 488 azide in cells in which either proteasomal activity (Figure 2, top row) or autophagic activity (Figure 2, bottom row) was blocked; cells were also labeled with an antibody against the autophagosomal resident protein LC3B. Fluorescent protein aggregates can be seen in the cytoplasm in both panels; however, these aggregates colocalized with LC3B only under conditions of autophagy block. 

Protein accumulation as a result of inhibiting the autophagy pathway or the proteasome system.

Figure 2. Protein accumulation as a result of inhibiting the autophagy pathway or the proteasome system. Blockage of the proteasome system (with MG132) or the autophagy pathway (with chloroquine) causes an accumulation of proteins (green), as revealed by the detection of homopropargylglycine (HPG)-containing proteins with the Click-iT® HPG Alexa Fluor® 488 Protein Synthesis Assay Kit. Inhibiting protein clearance through the autophagy block (chloroquine) produces HPG colocalization with the autophagosomal marker LC3B (red), detected with an anti-LC3B antibody; however, under conditions of proteasome block (MG132), HPG-containing protein aggregates do not colocalize with LC3B. Cells were counterstained with Hoechst® 33342 dye (blue).

Imaging autophagosomal clearance of p62 aggregates using Premo™ Autophagy Sensor GFP-p62 and RFP-p62

When proteasomal activity is inhibited (e.g., as shown in Figure 2), unfolded and misfolded proteins can aggregate. Cells have mechanisms to shunt proteins from the proteasome system to the autophagy pathway. A key protein in this crossover corridor between degradation processes is the autophagy receptor p62 (also called SQSTM1), which is able to bind ubiquitinated proteins through a ubiquitin-binding association (UBA) domain [2]. p62 also contains an LC3-interacting region (LIR) that binds autophagosomal proteins in the LC3 family, allowing these protein aggregates to enter the autophagy pathway [3] and ultimately arrive in the lysosome for degradation.

This process can be imaged over time in live cells using either Premo™ GFP-p62 or Premo™ RFP-p62, which are BacMam particles encoding fluorescent protein chimeras of p62. Figure 3A shows the accumulation of p62-positive aggregates in an A549 cell following nutrient deprivation; to enhance the number of protein aggregates seen in this experiment, autophagic flux was also blocked with chloroquine. For these aggregates to interact with the autophagosome, they must be trafficked along microtubules, a process that can be observed by using Premo™ GFP-p62 together with the microtubule marker CellLight® Tubulin-RFP (Figure 3B).

 

p62-positive protein aggregate accumulation during nutrient deprivation and chloroquine-mediated autophagy block

Figure 3. p62-positive protein aggregate accumulation during nutrient deprivation and chloroquine-mediated autophagy block. (A) A549 cells were transduced with Premo™ Autophagy Sensor GFP-p62 (green), cultured for 48 hr, and then incubated in EBSS containing 120 µM chloroquine. Images were acquired every 2 min; selected images show the accumulation of p62-positive protein aggregates over time. (B) A549 cells were transduced with Premo™ GFP-p62 (green) and CellLight® Tubulin-RFP (red) and cultured for 24 hr. Chloroquine was added to a final concentration of 60 µM, and cells were cultured for a further 16 hr before counterstaining with Hoechst® 33342 dye (blue). p62-positive protein aggregates can be seen associated with microtubules.

Imaging autophagosome maturation with the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B

Either underactivity or overactivity of the autophagy pathway can cause cellular defects, and these may arise during autophagosome formation, lysosome fusion, or lysosome/autolysosome acidification. Monitoring this progression is important in studying the flow of molecules through the autophagy pathway.

The autophagosome encompasses cytoplasmic cargo, and its pH is similar to that of the cytosol. Upon fusion of the autophagosome with the highly acidic lysosome, the lumen of the resulting autolysosome becomes more acidic. This change in lumenal pH can be monitored by taking advantage of the differential pH sensitivity of two distinct fluorescent proteins fused to the autophagosomal marker LC3B [3,4].

The Premo™ Autophagy Tandem Sensor provides a pH-sensitive fluorescent protein chimera of LC3B—a fusion of tagRFP, Emerald GFP, and LC3B—encoded in BacMam particles for highly efficient transient expression in live cells. Emerald GFP, an enhanced variant of GFP from Aequorea victoria, has a pKa of >6 and therefore exhibits a fluorescence decrease in acidic environments [5]. The monomeric tagRFP, a variant of RFP from Entacmaea quadricolor, has a pKa of 4, which means its fluorescence is maintained in acidic conditions [6].

Serving as a fluorescent protein–based biosensor, the Premo™ Autophagy Tandem Sensor reports the pH of the environment around LC3B through the various stages of autophagy. In the autophagosome, fluorescence arises from both Emerald GFP and tagRFP, whereas in the acidic autolysosome, only tagRFP fluorescence is observed (Figure 4A). This phenomenon is easily demonstrated through addition of drugs that block autophagy with or without affecting lysosomal pH. For example, chloroquine effectively neutralizes the pH of the lysosome. Under conditions of chloroquine-mediated autophagy block, LC3B-positive structures accumulate that exhibit both Emerald GFP and tagRFP fluorescence (Figure 4B). In contrast, enzyme inhibitors such as leupeptin A inhibit the activity of lysosomal acid hydrolases and block autophagy, but the lysosome remains acidic. Following leupeptin A treatment, LC3B-positive structures accumulate and tagRFP fluorescence is observed (Figure 4B).

The Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B can also be combined with the lysosome-selective LysoTracker® Deep Red dye (see below) to provide an even clearer picture of autophagy within a cell. The combination of these two sensors allows you to follow the formation of autophagosomes, fusion with lysosomes, and acidification of autolysosomes in a single cell (Figure 4C).

 

Visualizing autophagosome maturation.
Figure 4. Visualizing autophagosome maturation. (A) Schematic representation of Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B. BacMam technology uses a modified baculovirus for the transient expression of a gene construct, in this case the RFP-GFP-LC3B fusion. This cytoplasm-localized biosensor is recruited to the autophagosome upon induction of autophagy. Once the autophagosome fuses with the lysosome, lumen acidity increases, quenching fluorescence from GFP but not RFP. (B) Imaging autophagy-inhibited cells. HeLa cells were transduced with Premo™ RFP-GFP-LC3B, and subsequent inhibition of autophagy with either chloroquine or leupeptin A caused the accumulation of LC3B-positive vesicles (relative to vehicle treatment). Chloroquine alkalinizes the lysosome, thus maintaining GFP fluorescence. Leupeptin A does not affect the acidic lysosomal pH, and therefore only RFP fluorescence is observed. (C) Monitoring autophagosome–lysosome fusion and acidification. A549 cells were transduced with Premo™ RFP-GFP-LC3B and subjected to nutrient deprivation with EBSS. Images were captured immediately following nutrient deprivation (at 2 min, early), as well as 20 min (middle), and 1 hr (late) after nutrient deprivation. In cells at rest (early), there are numerous discrete lysosomes (blue) with few autophagosomes (yellow puncta) or autolysosomes (pink puncta); upon nutrient deprivation, there is first an increase in autophagosomes (middle), followed by a decrease in lysosomes and autophagosomes and an increase in autolysosomes (late).

The newest lysosome marker: LysoTracker® Deep Red dye

Lysosomes represent the end point of the autophagy pathway, where cargo is delivered for degradation. In contrast to their oversimplified reputation as cellular garbage cans, lysosomes serve as important signaling hubs, with their number, size, and position regulating a myriad of cellular pathways. Lysosomotropic agents that preferentially accumulate in lysosomes, such as LysoTracker® Green and LysoTracker® Red dyes, have proven extremely useful for imaging these dynamic structures.

To maximize the information a researcher can derive from a single cell, we have developed LysoTracker® Deep Red dye, which exhibits excitation and emission properties that exactly match the Cy®5 fluorescence channel, thereby facilitating multiplex imaging with GFP and RFP markers. The colocalization of lysosome-targeted GFP expression (using CellLight® Lysosomes-GFP) with LysoTracker® Deep Red fluorescence confirms the lysosome selectivity of LysoTracker® Deep Red dye (Figures 5A and 5B). LysoTracker® Deep Red dye is the ideal marker for four-color imaging with GFP, RFP, and a blue-fluorescent counterstain (Figure 5C).

Lysosome-selective staining with LysoTracker® Deep Red dye

Figure 5. Lysosome-selective staining with LysoTracker® Deep Red dye. (A) U2OS cells were transduced with CellLight® Lysosome-GFP (green) and labeled with 50 nM LysoTracker® Deep Red dye (red, Cat. No. L12492). (B) Enlargement of an area of the cell in (A) shows that LysoTracker® Deep Red dye (B1, B3) is located in the lumen of the lysosome, as depicted by the membrane labeling of CellLight® Lysosome-GFP (green; B2, B3). (C) HeLa cells expressing both CellLight® Tubulin-GFP (green) and CellLight® Late Endosomes-RFP (red) were labeled with 50 nM LysoTracker® Deep Red dye (pink) and stained with Hoechst® 33342 dye (purple) Images were acquired using a DAPI/FITC/TRITC/Cy5 optical filter set.

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