POGONATUM : morphology, anatomy, reproduction etc.
Presentation
1. A high-throughput fluorescence
resonance energy transfer
(FRET)-based endothelial cell
apoptosis assay and its
application for screening
vascular disrupting agents
X. Zhu, A. Fu, K. Q. Luo, 2012
ILANA J PORTER
An early high-
throughput application
of GFP-FRET
2. The importance of high-throughput
• Automation
• Can conduct
thousands of assays
• Drug discovery
http://www.penchovsky.site90.com/re
search.php
3. FRET - Fluorescence Resonance Energy Transfer
Donor fluorophore is
excited by light pulse
Energy is transferred to
acceptor fluorophore
Acceptor fluorophore
emits light
Citation for image
4. Importance of FRET measurements
• High spatial resolution (10 nm)
• Non-disruptive to cells
• Real-time measurements
of living cells
Previous applications
•Protein conformations
•Identifying signaling
pathways
•Protein-protein interactions
5. and Caspace-3
• Increased apoptosis associated with atherosclerosis
• Inhibition of apoptosis stimulates capillary growth
• Solid tumor growth and metastasis
• Possible target for tumor therapy
• Primary execution protease
• Cleavage sequence Asp-Glu-Val-Asp (DEVD)
Endothelial Apoptosis
http://commons.wiki
media.org/wiki/File:
Caspase_3.png
6. C3 FRET Sensor
• Analog for Caspace-3 substrate
• Fusion of FRET donor and acceptor
• Cleavage disrupts FRET
CFP
Donor
D
E V D
YFP
Acceptor
8. Moving to High-Throughput
• 96-well plate
• Minimum FRET levels after 12 hours UV
• FRET does not reduce from necrotic inducers
But only detects
cleavage of
substrate
analog, not
actual substrate
Are these
measurements
accurate?
Could there
be
competition?
14. High-throughput Reliability
• Z’ factor evaluates quality
of high-throughput assay
• Z’>0.5 is standard
• Z’ = 0.66 for the FRET assay
Citation here
16. Issue with Comparison
• CC50 is standard measurement for
cytotoxicity
• FRET50 is more sensitive
How can results from this
assay be compared to
previous research?
17. FRET is perfect for High-throughput
• Distinguish apoptosis
• Non-disruptive to live cells
• Z’ indicates reliable high-throughput method
• High sensitivity
18. Future Applications of this Screen
• Co-culture
• Endothelial protective drugs
• Prevent vascular Injury
• Screening of different cell lines
-Widely used tool of biochemists is high-throughput assay-utilizes robotics and automated plate-readers to perform thousands of experiments, automated.-Generally used for discovery of drugs
-FRET stands for-consists of two fluorophores, light emitting and absorping protein moieties-GFP derivatives-Distance dependent, see image-FRET efficiency is acceptor emission over donor emission, y/c
-FRET is an extremely powerful spectroscopic tool-Energy transfer only occurs when fluorophores are less than 10 nm apart-Extremely high spatial resolution, good temporal resolution-GFP and derivatives non-disruptive to protein conformations when inserted correctly, non toxic-Can be applied to in vivo measurements
-Endothelial cells line interior surface of blood vessels-Too much apoptosis occurs, atherosclerosis and thrombosis-Apoptosis is inhibited, stimulate growth of capillaries, which allows tumors to grow & metastasize-Endothelial cells are a target for tumor therapy-caspase 3 is an important protease involved in apoptosis
-C3 is a recombinant substrate for caspace 3-two fluorophores, cyan donor and yellow acceptor-connected by cleavage sequence-cleave c3, no FRET, signal changes from green to blue-Transformed into human umbilical vein endothelial cells
-irradiated cells for 3 min under UV light-incubated for different periods, then took FRET measurements-cells turn from green to blue, c3 is cleaved-shape becomes round and shrink, apoptosis-compare to paclitaxel, anticancer drug
-performed with 96-well plate, at same time and automated-shine UV for 3 minutes, decrease FRET after 3 hours-minimum FRET after 12 hours-necrotic inducers H2O2, cell death but not apoptosis-necrotic does not reduce FRET, no C3 cleavage -but detect analog --- oops
-MTT assay biochemistry method-determines cell viability, percent-longer UV incubation, lower FRET and lower Viability-for necrotic factors, FRET does not change but viability decreases-this FRET assay can specifically detect apoptosis not necrosis
-performed an activity assay of caspace 3-to prove that it was activated during UV apoptosis-not activated H2O2 necrosis
-western blot visualize proteins in cell-specific proteins stick to antibodies-PARP endogenous substrate for caspace 3-c3 is cleaved at the same time
-Pax is a known cancer drug-fig A dose dependent MTT cell viability assay-cc50 is cell cytotoxicity, 50% of cells die-fig B is dose dependent FRET emission ratio-FRET50 , 50% reduction, is lower. FRET is more sensisitve
-time dependent FRET emission for 3nM dose and 30nM dose-30nM has faster reduction of FRET-able to compare potency
-Z factor measures quality of high-throughput assay-this assay is reliable -Z = 1- [(3sigs + 3sigc)/|mus-muc|] stimulated and control
-applied the assay to various compounds they had-found dioscin, herbal compound-fig A dioscin reduced FRET in a dose and time dependent manner-fig B caspace 3 activity assay, activity enhanced by dioscin-fig C western blot, PARP cleavage is induced
-cc50 standard measurement-FRET50, new statistic introduced here-hard to compare to previous research-Can’t estimate dosage
-This assay can distinguish apoptosis from necrosis-can be performed in live cell-z’ indicated reliability of the method-fret measurement FRET50 more sensitive than previous methods
-co-culture means multiple types of cells-can distinguish these signals and measure them separately-measure opposite, anti-apoptotic drugs-prevent vascular injury, two possible causes-use a different cell line other than human umbilical vein endothelial cells
-co-culture means multiple types of cells-can distinguish these signals and measure them separately-measure opposite, anti-apoptotic drugs-
