Utilizing flow cytometry to monitor autophagy in living mammalian cells

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Nov 20, 2007 - to monitor this process in mammalian cells are limited due to lack ..... measured using flow cytometry and normalized to cells incubated in serum-free, .... (control) media in the presence of rapamycin (200 nM) or tunicamycin (5.
[Autophagy 4:5, 621-628; 1 July 2008]; ©2008 Landes Bioscience

Research Paper

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Utilizing flow cytometry to monitor autophagy in living mammalian cells

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Elena Shvets, Ephraim Fass† and Zvulun Elazar* Department of Biological Chemistry; The Weizmann Institute of Science; Rehovot, Israel †Present

address: Department of Molecular Microbiology; Washington University School of Medicine; St. Louis, Missouri USA

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with cancer, muscular and neurodegenerative diseases.3-5 However, methods to monitor and quantify autophagy have been limited because of lack in autophagic markers. In recent years, the molecular mechanism underlying autophagy has been extensively researched in yeast,6 and two evolutionarily conserved ubiquitin-like (UBL) conjugation systems, of Atg12 and Atg8, were found to play a pivotal role in early stages of autophagosome biogenesis.7 Atg8 and its mammalian homologues, including MAP1-LC3 (LC3), undergo conjugation to a lipid (phosphatidylethanolamine) at their C-terminus and the resultant lipidated form remains associated with autophagosomal membrane until fusion with lysosome, serving as the first bone fide marker for autophagosomes.8,9 Thus, using LC3 (or GFP-LC3) we can now specifically monitor autophagic activity both biochemically and microscopically.10,11 One approach is to detect LC3 lipidation, since its unconjugated (LC3-I) and conjugated (LC3-II) forms can be easily separated by SDS-PAGE. Another widely used method to measure autophagic activity is to follow changes in LC3 or GFP-LC3 localization, because autophagosomes are easily detected as punctuate dots. However, a recent report12 showed that this protein, particularly if overexpressed by transient transfection, tends to incorporate into punctuate dot-like aggregates in an autophagy-independent manner. In addition, LC3-II labeled autophagosomes are degraded during autophagy, and therefore it is important to quantify the level of LC3-II or autophagosomes delivered to lysosomes by comparing their amounts in the absence or presence of lysosomal protease inhibitors. In summary, while LC3 serves as the only bona fide marker to measure autophagy in mammalian cells, quantification of autophagic activity using microscopy or Western blot analysis is rather inaccurate and work-extensive.11,13 Thus, development of a new reliable assay to measure autophagic activity in living cells is greatly advantageous. Here we have established a new quantitative assay to measure autophagic activity in living mammalian cells. We used GFP-LC3, a well-established auophagosomal marker, and followed its turnover by flow cytometry (FACS). We show that its fluorescent signal is reduced during amino acid starvation in a time-dependent manner, while autophagic or lysosomal inhibitors blocked this reduction. Other known inducers of autophagy, such as rapamycin or tunicamycin, also decreased the level of GFP-LC3. Moreover, this decrease occurred specifically with wild type LC3, but not with mutant LC3G120A. By utilizing this assay we determined the minimal nutrient requirement for the induction of autophagy. Thus, quantification of selective degradation of GFP-LC3 using flow cytometry

Introduction

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Autophagy is a major intracellular catabolic pathway that takes part in diverse biological events including response to amino acid starvation, protein and organelle turnover, development, aging, pathogen infection and cell death. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, was shown to selectively incorporate into autophagosome, thus serving as a unique bona fide marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming, labor-intensive and require much experience for accurate interpretation. Here we took advantage of the Fluorescence Activated Cell Sorter (FACS) to quantify the turnover of GFP-LC3 as an assay to measure autophagic activity in living mammalian cells. We showed that during induction of autophagy by rapamycin, tunicamycin or starvation to amino acids, fluorescence intensity of GFP-LC3 is reduced in a time-dependent manner. This decrease occurred specifically in wild type LC3, but not in mutant LC3G120A, and was inhibited by autophagic or lysosomal inhibitors, indicating that this signal is specific to selective autophagy-mediated delivery of LC3 into lysosomes. By utilizing this assay, we tested the minimal nutrient requirement for the autophagic process and determined its induction by deprivation of specific single amino acids. We conclude that this approach can be successfully applied to different cell-lines as a reliable and simple method to quantify autophagic activity in living mammalian cells.

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Key words: autophagy, LC3, FACS, amino acids, starvation

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Autophagy is a major intracellular catabolic pathway, primarily regulated by amino acids,1,2 where long-lived proteins and organelles are delivered by a double-membrane vesicle, called autophagosome, to lysosomal/vacuolar system for degradation and consequent recycling. Recent reports show that autophagy plays a role in diverse biological events including development, aging, immunity, pathogen infection and cell death, and its dysfunction has been associated *Correspondence to: Zvulun Elazar; Department of Biological Chemistry; The Weizmann Institute of Science; Rehovot 76100 Israel; Tel.: 972.8.9343682; Fax: 972.8.9344112; Email: [email protected] Submitted: 11/20/07; Revised: 03/19/08; Accepted: 03/20/08 Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/article/5939 www.landesbioscience.com

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FACS analysis of autophagic activity

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Utilizing FACS to quantify autophagic activity in living mammalian cells. LC3, a mammalian homologue of yeast Atg8, is a key factor of the autophagic process and a unique marker of mammalian bona fide autophagosomes, since its lipidated (phosphatidylethanolamineconjugated) form is predominantly associated with autophagic organelles.8 LC3 fused to GFP has been widely used to detect these organelles microscopically, both in cultured cells and in the whole organism.14,15 Quantifying the autophagic activity, however, is best accomplished by following the delivery of autophagosomes into lysosomes. As shown in Figure 1A, inhibition of lysosomal activity in starved cells by Bafilomycin A (Baf A)16 resulted in accumulation of vesicles labeled by either GFP-LC3 or endogenous LC3 (autophagic bodies) within lysosomes,15 indicating that a significant amount of this protein is delivered to lysosomes for degradation. To test this further, CHO cells stably expressing GFP-LC3 were starved for different time periods in the absence or presence of Baf A, and the amount of either GFP-LC3 or endogenous LC3 was analyzed using Western Blot. The level of both endogenous LC3 and GFP-LC3 was significantly reduced in a time-dependent manner, while addition of either Baf A (Fig. 1B and C) or wortmannin (data not shown) inhibited this process. Next, we tested whether the reduction in the level of GFP-LC3 can be detected in living cells microscopically. To this end, GFP-LC3 stably expressing cells were either incubated in amino-acid rich medium (αMEM) or starved (EBSS medium) for 6 hours in the absence or presence of Baf A or 3-methyladenine (3-MA) and then visualized every 3 hours by DeltaVision microscope. Amino acid starvation induced appearance of GFP-LC3 labeled autophagosomes, which was inhibited by 3-MA17 (Fig. 1D) or wortmannin (data not shown), while Baf A treatment resulted in accumulation of these organelles. As shown in Figure 1D and E, the level of fluorescence intensity decreased in a time-dependent manner in starved, but not in control cells, and treatment with Baf A or 3-MA significantly inhibited this process. Based on these results, we argue that disappearance of GFP-LC3 may serve as a functional indicator for autophagic activity in-vivo. Next, we took advantage of the Fluorescence Activated Cell Sorter (FACS) to monitor and quantify the turnover of GFP-LC3 in living starved cells. For that purpose, cells were incubated in EBSS medium for different time periods, and their average fluorescence intensity was measured and calculated compared to cells incubated in serum free-αMEM medium. As depicted in Figure 2A, a significant decrease in the level of GFP-LC3 was observed already after 3 hours, and 6 hours of starvation resulted in about 50 percent decay in fluorescence intensity. Treatment of starved cells with wortmannin, 3-MA or Baf A inhibited the reduction in the fluorescence signal, indicating that it is dependent on autophagy-mediated lysosomal degradation. To further confirm that this reduction is due to autophagic activity, we followed the fluorescence decay of GFP-LC3G120A, the mutated form of the protein which is unable to undergo the C-terminus cleavage and consequentially conjugation to the autophagosomal membrane.8 As shown in Figure 2B, no significant change in the level of GFP-LC3G120A intensity was detected. Similarly, fluorescence of GFP alone, serving as cytosolic marker, was

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not reduced following starvation period (Fig. 2C), indicating that only wild type GFP-LC3 is selectively delivered into lysosomes upon amino acid deprivation. Notably, similar time-dependent reduction in the level of GFP-LC3 was observed also in HeLa cells (Fig. 2D), demonstrating that starvation-specific disappearance of this protein is not limited to CHO cells. Next, we examined the ability of cells to recover from starvation. To this end, cells starved for 6 hours were transferred to full medium for different time periods and analyzed by FACS. As depicted in Figure 3A, the signal of GFP-LC3 was elevated during recovery, reaching its basal level within 4 hours. The rate of recovery in this system reflects both the synthesis of GFP-LC3 and the termination of the induced autophagic activity. Finally, we tested whether induction of the autophagic process by other known stimuli, such as rapamycin or tunicamycin treatment,18,19 is detectable by flow cytometry. As shown in Figure 3B, these treatments resulted in approximately 20% and 50% decrease in GFP-LC3 level for rapamycin and tunicamycin, respectively. In summary, selective degradation of GFP-LC3 may serve as a functional indicator of autophagy and we propose that determining the rate of its turnover using FACS technique is reliable and accurate method to monitor autophagic activity. Utilizing FACS to detect the effect of different amino acids on starvation-induced autophagy in CHO cells. Autophagy-mediated proteolysis is the only mechanism known among a number of cellular proteolytic systems to be subjected to nutritional regulation. The effect of specific amino acids has been extensively studied in the past and a group of specific single amino acids was shown to inhibit autophagy-mediated proteolysis.20-24 To test whether induction of autophagy by specific amino acids is detectable by FACS analysis, cells stably expressing GFP-LC3 were incubated for 5 hours in medium, systematically omitted of a specific single amino acid and the level of autophagic activity was examined using flow cytometry. As shown in Figure 4A, only specific amino acids inhibit autophagy. Thus, removal of either leucine, arginine, lysine or methionine from the medium resulted in a significant induction of autophagy. The removal of either alanine, aspartate, asparagine, cysteine, glutamate, glycine, isoleucine, proline or serine had no effect on induction of autophagy, while a minor effect was observed for the other amino acids (Fig. 4A). To complement these results we quantified autophagy by counting the number of GFP-LC3 labeled vesicles appearing either in cells incubated for 2 hours in medium lacking one specific amino acid (Fig. 4B, starvation), or in cells incubated in EBSS medium for 2 hours, and only then transferred to medium lacking a specific amino acid (Fig. 4B, recovery). The amount of GFP-LC3 labeled autophagosomes under starvation conditions was then set to 100% and the relative level of autophagy was calculated compared to this treatment (Fig. 4C). Based on this analysis we found that, similarly to quantification by FACS, only removal of either leucine, arginine, lysine or methionine from the medium resulted in a significant induction of autophagy. Consistently, when these amino acids were omitted from the recovery medium, recovery failed to occur (Fig. 4B and C). Thus, based on both methods we were able to divide the amino acids into three groups: amino acids that fail to inhibit autophagy; amino acids that inhibit autophagy; and a group of amino acids that inhibit autophagy to low level (Table 1). Notably, removal of all 20 amino acids resulted in over 4-fold increase in the number of autophagosomes per cell, and in over

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is shown here as a reliable and simple method to accurately measure autophagic activity in living mammalian cells.

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Figure 1. GFP-LC3 and endogenous LC3 are selectively delivered into lysosomes under amino acid starvation. (A) CHO cells were starved in the presence of Bafilomycin A (100 nM) and localization of GFP-LC3 (upper) or endogenous LC3 (lower) with LAMPI-labeled lysosomes was determined using confocal microscopy. White boxes represent enlarged areas. Scale bars, 5 μm. (B) Cells stably expressing GFP-LC3 were starved for 3- or 6- hour periods in the absence or presence of Baf A, and cell extracts were subjected to Western blot analysis using antibodies against GFP, LC3 and actin. The asterisk (*) indicates unrelated band. (C) Quantitative analysis of amount of GFP-LC3 or LC3 following different time periods of starvation. The amount of these proteins was normalized using loading control (actin) and mean ± s.d. of three independent experiments is presented below. (D) CHO cells stably expressing GFP-LC3 were incubated for 6 hours in αMEM medium, or in EBSS medium in the absence or presence of Bafilomycin A (100 nM) or 3-methyladenine (10 mM), and randomly selected fields of cells were taken at different time periods using DeltaVision microscope as described in Materials and Methods. Representative images of one experiment are shown below. White boxes represent enlarged areas. (E) Quantitative analysis of GFP fluorescence intensity was performed as described in Materials and Methods and mean ± s.d. of three independent experiments is presented below.

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50 percent decrease in the level of GFP-LC3 (Fig. 4). Removal of a specific single amino acid induced the formation of 65–75% of the total number of autophagosomes formed when all amino acids were absent from the medium (Fig. 4C). Similar results were obtained when the level of autophagic activity was quantified by FACS (Fig. 4A), showing that although induction of autophagy is selective to specific amino acids, only a combination of different amino acids leads to full autophagic response. Based on these results we further confirmed that employing FACS analysis to determine the rate of GFP-LC3 turnover is a sensitive and quantitative method to measure autophagic activity.

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Discussion LC3, a ubiquitin like protein, is conjugated to autophagosomal membrane, thus serving as a bona fide marker of autophagosomes in mammals. However, current use LC3 for quantification of autophagic activity is time-consuming, labor-intensive and require much experience for accurate interpretation.11,13 We took advantage of the fact that GFP-LC3, similarly to endogenous LC3, is specifically delivered into lysosomes in response to induction of autophagy. This selective disappearance of GFP-LC3 is well detectable in living cells using techniques that measure the fluorescence signal, such as FACS (Fluorescence-Activated Cell Sorter). We showed that

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Figure 2. The delivery of GFP-LC3 into lysosomes following amino acid starvation is detected and quantified by flow cytometry. (A) CHO cells stably expressing GFP-LC3wt were incubated in αMEM (control) or EBSS (starvation) mediums for 3 and 6 hours in the absence or presence of 3-methyladenine (10 mM), wortmannin (100 nM) or Baf A (100 nM). Relative level of GFP-LC3 was measured using flow cytometry and normalized to cells incubated in serum-free, amino acid-rich medium for similar time periods. Right represents the mean ± s.d. of at least three separate experiments. Left shows a histogram presentation of GFP intensity vs. cell count following 6 hours incubation under control (black line), starvation (red line), or starvation in the presence of inhibitors (blue, purple and green lines) conditions in a representative experiment. (B) CHO cells stably expressing GFP-LC3G120A or GFP alone (C) were incubated in αMEM (control) or EBSS (starvation) mediums for 3 and 6 hours and relative level of GFP-LC3G120A or GFP was measured using flow cytometry. Right represents the mean ± s.d. of at least three experiments. Left shows a histogram presentation of the analysis of GFP-LC3G120A or GFP relative level following 6 hours incubation under control (black line) or starvation (red line) conditions in a representative experiment. (D) HeLa cells stably expressing GFP-LC3wt were incubated in αMEM (control) or EBSS (starvation) mediums for 3 and 6 hours and relative level of GFP-LC3 was measured using flow cytometry. Left shows a histogram presentation of GFP intensity vs. cell count following 6 hours incubation under control (black line) or starvation (red line) conditions.

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the relative level of GFP-LC3 detected by FACS is reduced under different autophagy-induced conditions in a time dependent manner and is blocked by autophagy-specific inhibitors. Moreover, this reduction was specific to wild-type but not to a mutant form of LC3 which is defected in its ability to conjugate to the autophagosomal membrane. Clearly, the observed reduction in GFP-LC3 fluorescence signal reflected autophagic activity. Next, using FACS analysis, we examined the nutritional requirement for induction of autophagy by quantifying the level of autophagic activity in the absence of a single amino acid. Results showed that only specific single amino acids induce this process, although not to its maximal level. 624

Counting the number of GFP-LC3 labeled autophagosomes yielded similar results, indicating that FACS could be successfully applied in different cell-lines as a reliable, sensitive and simple method to quantify autophagic activity. The classic direct approaches of measuring autophagy in the past were quantitative electron microscopy and quantification of the degradation rate of long-lived proteins. Although electron microscopy is a sensitive and accurate way to detect the induction of autophagy, it is work-intensive and requires well-trained personnel and expensive equipment. Moreover, electron microscopy is limited to morphological observations of fixed cells. The measurement of

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of LC3 punctated structures that do not represent autophagosomes. Therefore, in the present study we used cells stably transfected with GFP-LC3, when the level of overexpression was 3-fold relative to the endogenous protein.15 By utilizing FACS to follow the turnover of GFP-LC3 as a means to monitor autophagy, we are able to assess not only conjugation of LC3 to autophagosomal membrane (LC3-II) but also formation of autophagosomes and their delivery and fusion with lysosomes. Notably, the reduction in the GFP-LC3 flourescence reflects its delivery into the lysosomes, rather than degradation, as treatment by lysosomal protease inhibitors (leupeptin, pepstatin, E-64D) did not inhibit fluorescence decay nor accumulation of GFP-LC3 vesicles (data not shown). Thus, GFP fluorescence is blocked by low pH of the lysosomal lumen following fusion with the lysosomes and, therefore, only factors affecting autophagosome formation, delivery, fusion with lysosome or lysosomal acidification, but not its proteolytic activity, will inhibit the decay in GFP-LC3 fluorescence intensity. This rational was recently used by Kimura et al., that utilized a mRFP-GFP tandem fluorescent tagged LC3.26 Since the decay in fluorescence intensity of GFP-LC3 reflects its delivery to lysosomes, rather than its degradation within these organelles, this method is more sensitive than other methods which are based on bulk protein or selective LC3 degradation. However, one should be aware of conditions that may affect the fluorescence intensity, which may lead to misinterpretation of autophagic activity. For example, conditions/drugs that affect protein expression (serum starvation, protein synthesis inhibitors), membrane permeability or overall fluorescence signal, may lead to autophagy-unrelated reduction in GFP-LC3 level. Therefore, to avoid this potential shortcoming, it is essential to inspect cells under the microscope prior to flow cytometry analysis. Since this analysis is based on disappearance of GFP signal, and not on appearance of autophagosomes labeled by LC3, it cannot be performed on LC3-immunostained cells. Moreover, as cell fixation releases part of the cytosolic proteins, this assay can be carried out only on living cells. Finally, this method specifically quantifies the activity level of autophagic machinery, such as the rate of autophagosome production and delivery into lysosomes, but not bulk protein degradation, and, therefore, this system cannot detect defects in substrate incorporation into autophagosomes. Quantifying the autophagic activity in response to omission of single amino acid from the medium allowed us to determine the minimal nutritional requirement for induction of this process. As shown, only removal of specific amino acids induced this process in CHO (Fig. 4) and HeLa cells (data not shown), although not to the full extent of the autophagic response. In the past, the effect of amino acids on autophagy has been intensively studied by measuring bulk protein degradation22,24 or by detecting the induction of mTOR signaling pathway.27 These studies identified a group of amino acids (leucine, asparagine, glutamine, histidine, tyrosine, pthenylalanine, proline, methionine and tryptophan) that directly regulate intracellular protein degradation while alanine was shown to co-regulate this activity. Here, using a specific autophagic marker, we also demonstrate that removal of single amino acids leads to induction of autophagy in mammalian cell-lines. The fact that we have also identified leucine and methionine as prime regulators of autophagy serves a “proof of concept” for this assay, and show that these amino acids are common to different cell-types regulators of this process.

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Figure 3. Use of flow cytometry to monitor autophagy induced by different stimuli. (A) Cells were starved for 6 hours and transferred to full medium (αMEM + 10% FCS) for 2, 3 or 4 hours. The cells were collected and the relative level of GFP-LC3 was measured using flow cytometry. Values represent the mean ± s.d. of three experiments. (B) CHO cells stably expressing GFP-LC3wt were incubated either in EBSS (starvation) medium, or αMEM (control) media in the presence of rapamycin (200 nM) or tunicamycin (5 μg/ml) for 3, 6 or 12 hours and relative level of GFP-LC3 was measured using flow cytometry. Values represent the mean ± s.d. of three experiments. Asterisk (*) indicates significance at p < 0.001.

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degradation of long-lived proteins, although used as a functional assay to evaluate autophagy, is affected by autophagy-unspecific total protein degradation. The discovery of MAP1-LC3 (LC3) as the first marker of autophagosomes in-vivo provides a new tool to study the molecular basis of autophagy in mammals. Although LC3, or GFP-LC3, are widely used to monitor autophagy, there are several difficulties in quantifying and interpreting data from their analysis. A common way to quantify autophagic activity is either by measuring the lipidated form of LC3/GFP-LC3 (LC3-II) by Western blot or by counting LC3/GFP-LC3 labeled vesicles. However, formation of lipidated LC3 does not always correlate with formation of autophagosomes. In addition, this analysis represents only a steady-state between formation and degradation of LC3-II labeled autophagosomes. To quantify the level of LC3-II or autophagosomes delivered to lysosomes, one should compare their amount in the absence or presence of lysosomal protease inhibitors.25 Finally, recent report12 showed that GFP-LC3 has a tendency to aggregate, especially as a result of transient transfection, leading to appearance www.landesbioscience.com

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Figure 4. Induction of autophagy following removal of a specific single amino acid from the medium. CHO cells stably expressing GFP-LC3 were incubated in amino acid-rich medium (20AA), in the absence of all amino acids (EBSS), or in the absence of one amino acid (-Xxx) and analyzed by flow cytometry (A) or by fluorescence microscopy (B and C). Recovery (B, lower) represents cells pre-incubated for 2 hours under starvation conditions, then transferred for 2 hours to a medium lacking the indicated amino acid and analyzed by fluorescent microscope. (C) Quantitative analysis of number of autophagosomes counted in cells treated in (A) as explained in Materials and Methods. The amino acids that inhibit autophagy are marked in bold. Asterisk (*) indicates significance at p < 0.001.

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Future studies on the effect of different amino acid compositions on autophagic activity may provide new insights into identification of the sensory pathway that triggers autophagy. Although several precautions have to be taken to successfully interpret FACS analysis, its application to detect and quantify selective autophagy-mediated disappearance of GFP-LC3 offers numerous advantages. By allowing semi-automation, FACS analysis may be used to evaluate more samples with much larger cell sample sizes (population of 1 x 105 or more) than practically possible through conventional immunofluorescence microscopy. FACS analysis is also an objective methodology and, hence, reproducible and does not require experience in interpreting the visual appearance of the cells. In flow cytometry, different parameters (including size, granularity and fluorescence intensity) are made on individual cells. This allows to quantify sample heterogeneity, identify cell subpopulations, detect contaminants or distinguish between viable and non-viable cells. We showed that this method is quantitative, sensitive to various stimuli, 626

drugs that induce or block autophagy, and can be used to quantify the level of autophagic activity in response to omitting a single amino acid from the medium. Finally, the fact that FACS analysis is performed in living cells can be exploited to sort and collect specific cell populations that are of interest. Thus, this technique enables to perform large-scale screens for identification of yet unknown factors/ signals involved in autophagy.

Materials and Methods Antibodies and reagents. Minimal essential medium (αMEM), Earle’s balanced salt solution (EBSS), valine-free αMEM medium and fetal calf serum (FCS) were obtained from Biological Industries (Beit Haemek Laboratories, Israel). Medium containing all amino acids (20AA) was prepared by addition of all amino acids into EBSS medium at concentrations as in αMEM medium. When needed, a single amino acid was omitted from the medium (-Xxx). Bafilomycin A1 (Baf A) was provided by LC Laboratories. Wortmannin and

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Table 1 Classification of amino acids according to their effect on autophagic activity Minor effect

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Lys

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Met

Cys

Thr



Glu

Trp



Gly

Tyr



Iso

Val



Pro



Ser

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3-methyladenine were provided by Sigma Aldrich. The following antibodies were used: mouse monoclonal anti-actin (Sigma); mouse monoclonal anti-LAMPI (Developmental Studies Hybridoma Bank, University of Iowa); mouse monoclonal anti-green fluorescence protein (GFP) (BabCo); rhodamine-conjugated goat anti-mouse IgG; FITC-conjugated donkey anti-rabbit IgG (Jackson Immuno Research Laboratories); and horseradish peroxidase (HRP)-coupled goat antibody against mouse or rabbit IgG (Bio-Rad). Anti-LC3 antibody was produced as previously described.15 DNA construction. pEGFP-LC3 vector encoding human LC3 fused with GFP was prepared as previously described.15 The point mutation from glycine to alanine at position 120 of LC3 (LC3G120A) was created by PCR-based site-directed mutagenesis using LC3 sense primer (5'-CAGGAGACGTTCGCGATGAAATTGTCA-3') and LC3 antisense primer (5'-TGACAATTTCATCGCGGACGTCTCCTG-3'). Cell culture and transfection. CHO or HeLa cells were grown on αMEM medium supplemented with 10 % FCS and 1% penicillin-streptomycin (Sigma) at 37°C in 5% CO2. Subconfluent cells were transfected with 5 μg DNA/10 cm plate using Lipofectamine. Stable clones of GFP-LC3 transfected cells were selected in 1 mg/ml geneticin (G418). To obtain starvation conditions, cells were washed three times by PBS and incubated in EBSS medium for the indicated time periods. Total cell extracts were made using RIPA extraction buffer (10 mM Tris [pH 7.5], 10 mM NaCl, 1.5 mM MgCl2, 1% DOC, 1% Triton X-100) with protease inhibitors mixture (Sigma-Aldrich). Microscopy. Immunofluorescence analysis was performed by confocal microscopy as was previously described.15 For living cell imaging experiments, GFP-LC3 stably transfected CHO cells were grown at 37°C on single thickness four-well Lab-Tek chamber glass slides (Nunc). In each chamber, cells were incubated under different conditions (αMEM medium, EBSS medium in the absence or presence of Bafilomycin A or 3-MA) for 6 hours at 37°C in 5% CO2. In each treatment, 15 fields of about 15 cells each were randomly chosen based on transmitted-light DIC images, followed by fluorescence images taken every 3 hours with GFP filter using DeltaVision instrument (Applied Precision Instruments) with a 40 x 1.4 N.A. planApo objective (Olympus) and SoftWoRx program.

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Classification was performed based on statistical analysis of two different methods (FACS analysis and counting of GFP-LC3 labeled vesicles), performed in at least four separate experiments with p-value less than 0.01% (p < 0.001).

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Twelve z-series optical sections were captured at 0.4-μm step sizes and a single section at best focus of nuclei was chosen for statistical analysis. The analysis of the level of GFP fluorescence intensity was performed using ImageJ program (NIH Image) and calculated as mean fluorescence intensity per cell area in each image. The value obtained at first time point (0.5 h) of each treatment was set to 100%, and the mean fluorescence intensity at the next time points (3 and 6 hours) was calculated accordingly and presented as mean ± s.d. of at least 200 cells. FACS analysis. Subconfluent cells were incubated as indicated in figure legends. The cells were harvested with trypsin/EDTA, washed twice with PBS, and resuspended in 0.5 ml of PBS. Analysis of 1 x 105 cells per sample was performed on a FACScan flow cytometer (Becton Dickinson), and the data of viable cell counts was plotted as GFP fluorescence intensity. Quantification analysis. The level of LC3/GFP-LC3 proteins detected by Western blot in separate experiments (three at least) was analyzed using ImageQuant image program and normalized to actin as loading control. The largest number obtained was set to 100% and the relative protein level in cells undergoing other treatments was calculated accordingly and presented as mean ± s.d. of three independent experiments. For FACS analysis, the level of GFP fluorescence intensity in each treatment was normalized to the level of control sample (cells incubated in serum-free, amino acid- rich medium for similar time periods). The level of control sample was set to 100% and the relative level of GFP-LC3 in other treatments incubated for similar time periods was calculated accordingly for each treatment in each experiment. The graphs represent mean ± s.d. of at least three separate experiments. The number of GFP-LC3 vesicles was counted in at least 20 cells, the number of vesicles appeared in starved cells (incubation in EBSS medium) was set to 100% and the relative level of vesicles in other treatments was calculated accordingly for each treatment. The graphs represent mean ± s.d. of at least four separate experiments. The statistical analysis of the results shows p < 0.001.

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Acknowledgments

Z.E. is an incumbent of the Sholimo and Michla Tomarin Career Development Chair of Membrane Physiology. This work was supported in part by the Israel Science Foundation by the Weizmann Institute Minerva center, and by the Josef Cohn Minerva Center. References 1. Mortimore GE, Poso AR and Lardeux BR. Mechanism and regulation of protein degradation in liver. Diabetes Metab Rev 1989; 5:49-70. 2. Blommaart EF, Luiken JJ and Meijer AJ. Regulation of hepatic protein degradation. Contrib Nephrol 1997; 121:101-8. 3. Huang J and Klionsky DJ. Autophagy and human disease. Cell Cycle 2007; 6:1837-49. 4. Boland B and Nixon RA. Neuronal macroautophagy: from development to degeneration. Mol Aspects Med 2006; 27:503-19. 5. Shintani T and Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science 2004; 306:990-5. 6. Huang WP and Klionsky DJ. Autophagy in yeast: a review of the molecular machinery. Cell Struct Funct 2002; 27:409-20. 7. Ohsumi Y. Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol 2001; 2:211-6. 8. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y and Yoshimori T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J 2000; 19:5720-8. 9. Kabeya Y, Mizushima N, Yamamoto A, Oshitani Okamoto S, Ohsumi Y and Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on formII formation. J Cell Sci 2004; 117:2805-12.

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2008; Vol. 4 Issue 5