Download Custom Assays to Study Nuclear Bi loogy

Custom Assays to Study Nuclear
Bi
l
Biology
By Mohamed Kodiha
B
M h
d K dih
July, 2011
From Apoptosis to Zebrafish Automated, Live Cell & High Content Imaging
A New Strategy to Analyze the
S b ll l Di
Subcellular
Distribution
t ib ti
off M
Molecules
l
l
1- Dynamic organization of eukaryotic cells
2- Signal transduction and spatial
distribution of signaling molecules
3 Cell
3C ll fractionation
f
ti
ti
Part 1.
Quantitative immunofluorescence tools
to measure fluorescence signals in the
1- Nucleus
2- Cytoplasm
3- Nuclear envelope (NE)
Automated Image Acquisition and Computer Based
Image Analysis
DAPI
TRITC
DAPI
TRITC
(HTS Images)
(Individual image to be analyzed)
Segments overlay
Segments overlay
(Segmentation)
(Kodiha et al., Science Signaling, 2008)
Multiwavelength Cell-Scoring Module to Quantify
Nuclear and Cytoplasmic Distribution
(Kodiha et al., Science Signaling, 2008)
Multiwavelength Cell-Scoring is Not
Al
Always
the
th Protocol
P t
l off Ch
Choice
i
(Kodiha et al., Science Signaling, 2008)
Translocation-Enhanced Module to Quantify Nuclear and
Cytoplasmic Distribution
1-DAPI staining identifies
the compartment of interest (nucleus).
2-Two
2
T
regions
i
((segments)
t ) th
thatt are ring
i
shaped are created either within or
outside of the compartment.
3-This segmentation is applied to the
image of the protein of interest.
(Kodiha et al., Science Signaling, 2008)
Disadvantages of the Translocation-Enhanced
Module
Size bar is 25 µm
(Kodiha et al., Science Signaling, 2008)
Multiwavelength
Translocation Module to
Quantify NE Fluorescence
1- A simplified
p
version of the
translocation-enhanced
module.
2- An outer region that co-localizes
with the NE is created.
3- Fluorescence intensity at the
NE is measured in one step.
(Kodiha et al., Sci. Signal., 2008)
Image Acquisition with ImageXpress Micro of Cells Grown on
Cover Slips
(Kodiha et al., Science Signaling, 2008)
Applications of Quantitative
Immunofluorescence
• M
Monitoring
it i
the
th subcellular
b ll l distribution
di t ib ti
off
cellular proteins under normal and stress
conditions.
Examples:
1-Transport factors
2-Individual components of signaling
cascades
Oxidative stress
inhibits Crm1mediated
di d nuclear
l
export
Size bar is 20 µm
(Crampton et al., Mol Biol Cell, 2009 )
Oxidative stress changes the levels of Crm1 and
multiple nucleoporins at the nuclear envelope
Oxidative stress changes the levels of Crm1 and multiple
nucleoporins at the nuclear envelope
Energy depletion and oxidative stress alter the distribution of AMPK
5'-AMP activated protein kinase (AMPK) localization under
normal and stress conditions
Control EtOH
Oxidative stress DEM
Kodiha et al., Am. J. Physiol: Cell Biology, 2007
Quantification of nuclear/cytoplasmic fluorescence of AMPK
Part 2.
Quantitative immunofluorescence tools
to measure fluorescence signals in the
Nucleolus
www.larousse.fr/.../medical/nucléole/14845
bsmc.insa‐lyon.fr/MOCEME/Modele_biologique.php
Nucleolar functions and dynamics
• Nucleolar functions
N l l f ti
• Dynamic nature of the nucleolus
• Proteomics vs. quantitative immunofluorescence
Computer‐based image analysis
?
Nucleolar markers relocate upon stress
Size bar is 20 µm
(Kodiha et al., BMC Cell Biol. 2011)
Detect dark holes filter
(Kodiha et al., BMC Cell Biol. 2011)
Median filter
(Kodiha et al., BMC Cell Biol. 2011)
Quantification of nucleolar fluorescence requires :
requires :
(Kodiha et al., BMC Cell Biol. 2011)
Identification of nucleoli based on DAPI
staining
(Kodiha et al., BMC Cell Biol. 2011)
Quantification of fluorescent signals
(Kodiha et al., BMC Cell Biol. 2011)
Identification of nucleoli based on Pol-II
staining
g
Identification of nucleoli using DAPI and Pol‐II staining
1‐ Improves the accuracy of nucleolar detection.
2‐ Recommended when visual inspection is not allowed (Ex. HTS setting)
(Kodiha et al., BMC Cell Biol. 2011)
Staining the nucleoli with 5‐
ethynyluridine (EU)
y y
( )
(Kodiha et al., BMC Cell Biol. 2011)
Quantification of nucleolar fluorescence in different
fluorescence in
cell lines
(Kodiha et al., BMC Cell Biol. 2011)
Comparison between the different methods
(Kodiha et al., BMC Cell Biol. 2011)
Results
Results
Quantitative immunofluorescence in high g p
g(
)
g
throughput screening (HTS) setting
Quantitative immunofluorescence in high throughput screening (HTS) setting
throughput screening (HTS) setting
(Kodiha et al., BMC Cell Biol. 2011)
Calculation of the Z‐factor
Conclusions
• We developed quantitative immunofluorescence methods that rely on computer based image analysis to measure fluorescence in the nucleus cytoplasm NE and nucleolus
fluorescence in the nucleus, cytoplasm, NE and nucleolus. • These strategies were applied successfully to analyze the subcellular distribution of nuclear transport factors and signaling molecules under normal and stress conditions.
• Our methods were further applied successfully to monitor the dynamic association of proteins and RNA with nucleoli under different physiological conditions.
under different physiological conditions.
• These techniques can be easily combined with automated high throughput screening (HTS) platforms
high throughput screening (HTS) platforms.
Acknowledgement
Dr. Ursula Stochaj
r. Claire rown
Dr. Claire Brown
Aleksandrs J. Spurmanis
Piotr Bański
Hicham Mahboubi