Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Save to My Library
Look up keyword
Like this
1Activity
0 of .
Results for:
No results containing your search query
P. 1
Quantitative Measurement of Autophagy Using a Novel Flow Cytometry Assay

Quantitative Measurement of Autophagy Using a Novel Flow Cytometry Assay

Ratings: (0)|Views: 5 |Likes:
Autophagy is an intracellular catabolic pathway which causes cellular protein and organelle turnover, and is associated with diverse diseases such as Alzheimer’s disease, cancer, and Crohn’s disease, in addition to aging. It is a tightly regulated process that plays a normal part in cell growth, development, and cellular homeostasis. Autophagy functions as a housekeeping mechanism through disposal of aging and dysfunctional proteins and organelles by sequestering and priming them for lysosomal degradation (Figure 1). Increasing evidence suggests that not only apoptosis, but also autophagy, can contribute to cell death and greatly influence general cell health. Malfunctions of autophagy can adversely impact longevity and the capability of cells to function at full capacity. In cancer cells, autophagy can compensate for hypoxic conditions and nutrient starvation; on the other hand, activation of cell death via autophagy can kill tumor cells. As a result, there is great interest in assays that can efficiently screen for activators and inhibitors of autophagy.
Autophagy is an intracellular catabolic pathway which causes cellular protein and organelle turnover, and is associated with diverse diseases such as Alzheimer’s disease, cancer, and Crohn’s disease, in addition to aging. It is a tightly regulated process that plays a normal part in cell growth, development, and cellular homeostasis. Autophagy functions as a housekeeping mechanism through disposal of aging and dysfunctional proteins and organelles by sequestering and priming them for lysosomal degradation (Figure 1). Increasing evidence suggests that not only apoptosis, but also autophagy, can contribute to cell death and greatly influence general cell health. Malfunctions of autophagy can adversely impact longevity and the capability of cells to function at full capacity. In cancer cells, autophagy can compensate for hypoxic conditions and nutrient starvation; on the other hand, activation of cell death via autophagy can kill tumor cells. As a result, there is great interest in assays that can efficiently screen for activators and inhibitors of autophagy.

More info:

Categories:Types, Research
Published by: EMD Millipore Bioscience on Jul 10, 2013
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less

02/20/2014

pdf

text

original

 
Quantitative Measurement o Autophagy Using a Novel FlowCytometry Assay
Application NoteAbstract
Autophagy is an intracellular catabolic pathway whichcauses cellular protein and organelle turnover, and isassociated with diverse diseases such as Alzheimer’sdisease, cancer, and Crohn’s disease, in addition to aging.It is a tightly regulated process that plays a normal partin cell growth, development, and cellular homeostasis.Autophagy unctions as a housekeeping mechanismthrough disposal o aging and dysunctional proteinsand organelles by sequestering and priming them orlysosomal degradation (Figure 1). Increasing evidencesuggests that not only apoptosis, but also autophagy, cancontribute to cell death and greatly inuence general cellhealth. Malunctions o autophagy can adversely impactlongevity and the capability o cells to unction at ullcapacity. In cancer cells, autophagy can compensate orhypoxic conditions and nutrient starvation; on the otherhand, activation o cell death via autophagy can killtumor cells. As a result, there is great interest in assaysthat can efciently screen or activators and inhibitors o autophagy.
EMD Millipore is a division o Merck KGaA, Darmstadt, Germany
Mark Santos, Kevin Su, Luke Armstrong, Angelica Olcott, Jason Whalley, and Matthew HsuEMD Millipore
Figure 1.Autophagy maintainscellular homeostasis.
Autophagy is a constitutiveand dynamic cellularcatabolic process requiredin living cells. Autophagy isresponsible or degradingcellular proteins and iscurrently the only knownprocess or degrading cellularorganelles, recycling them toensure cell survival.
Aging OrganellesCytosolic Proteinsfor breakdownNutrient DepletionCytosolic Proteins (eg. LC3)LC3AutophagosomeLysosome
mTor
1. Induction andLC3 translocation2. Autophagosomeformation3. Docking & fusionwith the lysosyme4. Autophagosomebreakdown
PLASMA MEMBRANE
 
2
Types o cellular stress, such as nutrient limitation,hypoxia, oxidative stress, and DNA damage (genotoxicstress), can induce autophagy, oten via inhibition o mTOR. Autophagy induction signaling prepares cellsto construct a double membrane vesicle known asthe autophagosome by catalyzing the scaolding o Atg proteins (such as LC3) to the pre-autophagosomemembrane, which enguls aging organelles andrecyclable proteins. In the fnal step o autophagy, theouter membrane o autophagosome uses with thelysosome that provides the hydrolytic enzyme machineryand the contents are degraded and recycled.Here, we describe a novel ow cytometry assay orstudying autophagy by using a GFP-LC3 reporter cellline and a selective permeabilization method. Selectivepermeabilization enables discrimination betweencytosolic GFP-LC3 (LC3-I) and GFP-LC3 which has beenlipidated and sequestered into the autophagosomes(LC3-II) as it trafcs to the lysosome or degradation.This discrimination is achieved by selectively “ushingout” any cytosolic LC3, while all lipidated LC3 remains“protected” by enclosure within the autophagosome.Because only the protected LC3-II molecules yielda uorescent signal, the assay enables specifcmeasurement o autophagosomes (Figure 2). Theability to track uorescently labeled autophagosomesthus provides a tool or measuring autophagy inindividual cells.
Introduction
EMD Millipore’s FlowCellect™ GFP-LC3 ReporterAutophagy Assay Kits provide a quantitative solutionor studying autophagy and measuring the potency o autophagy inducers using ow cytometry. These kits haveour unique eatures to aid in the detailed evaluation o autophagy by ow cytometry:
•
Selective permeabilization solution discriminatesbetween cytosolic LC3 rom autophagic LC3 byextracting the soluble cytosolic proteins, whileprotecting LC3 which has been sequestered into theautophagosome
•
Monomeric GFP is used as a reporter to acilitate thetranslocation o the usion protein. Other orms o GFP orm dimers and aggregate when overexpressedin the cells, making it difcult to extract rom thecytoplasm and impossible to measure translocationby ow cytometry
•
Included autophagy detection reagent preventslysosomal degradation o LC3, allowing itsquantifcation by ow cytometry and prolonging thesignaling event or robust measurement.
•
The monomeric GFP used in our LC3 usion protein isattached on the 5’ end (N-terminal usion), protectingthe GFP rom ATG4 cleavage, allowing its visualizationwithin the autophagosomes (Figure 3).
Figure 2.Selective permeabilization aids in discriminating cytosolic rom autophagic LC3.
Discrimination between cytosolic GFP-LC3-Irom autophagosome-associated GFP-LC3-II is achieved by disrupting the plasma membrane with a selective permeabilizationsolution. Selective permeabilization releases cytosolic LC3, which is then fushed away during washing steps. GFP-LC3-II trappedin the autophagosome remains intact and its fuorescence can be measured.
Accumulationof AutophagosomesSelectivePermeabilizationSelectivePermeabilization
 
3
Our system utilizes a cell line stably expressing GFP-LC3, transected either into a Chinese hamster ovary(CHO) or human osteosarcoma (U20S) cell background.A stable GFP-LC3 reporter system is ideal or screeningo autophagy-modulating compounds. LC3 tagged at itsN-terminus with GFP allows tracking the translocation o LC3 rom the cytosol and into the autophagosome moreclearly and defnitively than monitoring endogenousprotein by antibody-based methods. Especiallywhen endogenous LC3 levels are below the limits o immunodetection, using exogenous LC3 constructsshows a clear beneft. Another advantage o using astable GFP-LC3 reporter system is the ability to analyzea larger number o cells, because nearly 100% o thepopulation expresses tagged LC3. Analyzing more cellscan yield greater statistical signifcance to experimentalobservations, or more productive research and betterdecision-making.
Figure 3.N-terminally used (but not C-terminally used) GFP-LC3 isa valid marker or autophagy.
According to Klionsky (2011),the location o GFP usion to LC3 is critical or measuremento LC3 translocation to serve as a marker or autophagosomes.I GFP is used to the C-Terminal (or 3’ end), ollowing Atg4cleavage GFP is removed and subsequently GFP is now lost.But GFP usion to the N-terminus (or 5’ end) will retain GFP,making this construct a suitable marker to track autophagicactivity.
Having the ability to measure compound activity iscrucial to screening and rank-ordering compounds indrug discovery campaigns. Here, we have demonstratedthe utility o our above-described ow cytometry assayor measuring the autophagy-modulating activity o compounds. First, we show identifcation o an autophagyinducer (rapamycin) or an autophagy inhibitor (dynasore).Second, we describe the detailed compound analysisand rank-ordering o autophagy inducers (STF-62247and PI-103) based on the mean uorescence intensitiesgenerated by titration o these known autophagy-inducing compounds. We show quantitative activitymeasurement or STF-62247 and PI-103 via doseresponse curves to derive EC
50
values.Using the FlowCellect™ GFP-LC3 Reporter AutophagyAssay on a ow cytometry platorm may enableidentifcation o new autophagy targets andpathways, providing insight into aging, cancers, andneurodegenerative disease.
Methods
Monitoring autophagosomes by ow cytometry
To measure autophagy by LC3-II recruitment into theautophagosomes, we used cell lines stably expressingGFP-tagged LC3, transected either into a CHO or a U20Scell background. Cells were harvested and placed into a96-well assay plate, either in nutrient deprived/starvedconditions or let in normal ed conditions as a control.Cells were then treated with a lysosomal degradationinhibitor or 2 hours. Cells were then washed with aselective permeabilization buer at room temperatureto extract all cytosolic LC3-I, ollowed by one wash withassay buer to remove any residual permeabilizationbuer rom the cells. Data were acquired using a guavaeasyCyte™ ow cytometer to measure the uorescencesignal rom autophagosome-bound GFP-LC3-II.
Identifcation o an autophagy inducer andinhibitor using the FlowCellect™ GFP-LC3 ReporterAutophagy Assay
In order to demonstrate that a GFP reporter-based systemis a viable tool or compound hit identifcation by owcytometry, GFP-LC3-expressing CHO cells were pretreatedwith either rapamycin (to induce autophagy) or dynasore(to inhibit autophagy) or 5 h. A lysosomal degradationinhibitor was also added to the cells simultaneously (i treatment with dynasore) or 45 minutes ater rapamycinaddition (or a total o 5 hours incubation time) toprevent the autophagosome degradation by the lysosome.
C-terminal fusion
Free GFP
: not amarker of thephagophore orautophagosome
N-terminal fusion
ATG4LC3ATG7LC3ATG3LC3G-PELC3GG*GR
GFP
R
GFP
ATG4LC3ATG7LC3ATG3LC3G-PELC3GG*GR
GFPGFPGFPGFP
GFP-LC3-PE
:a marker of thephagophore orautophagosome

You're Reading a Free Preview

Download
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->