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Dynamic Proteomics

Paper review of same
by

Abraham Anderson

on 23 March 2010

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Transcript of Dynamic Proteomics

Person > 100 trillion Cells > unique RNA & Protein dynamics
differential response to stimuli
eg. cell death or drug resistance
variability masked by most observational approaches Apparatus:
H1299 (lymph drived) non-small-cell Lung carcinoma cell line
anti cancer drug campothecin
CD taging to generate a clone libary, each with unique fluorescently tagged protein
Monitoring proteomics with time-lapse fluoresence microscopy
Measure level and location of many proteins in individual cells CD tagging detail:
retroviral infection of cell line yielding ~1200 clones
each clone with different protein tagged with a flour
eYFP aka Venus - source mutant jellyfish (Aequorea victoria) GFP
mCherry - discosoma anameone Timelapse Fluorescence Microscopy
12 well optical plates in automated TFM (focus, temp, CO2, & humidity)
Custom Matlab image analysis software to discern cells from background; nucleus from cytoplasm; mitosis, cell death, etc.
for 1260 tagged proteins

Results
Most proteins decrease after CPT addition & functionally related proteins have similar trends
cytoskeletal degredation associated with loss of motility
not everything happens at the same time
Ribosomal changes happen early on
DNA repair mechanism activates later with apotosis and programmed cell death Summary
most HT experiements monitor pooled molecular dynamics from 1000's of cells
individual cell tracking allows observation of cell specific behavior
in oncology setting, drug resistance /adaptation can be observed
Spatiotemporal features of proteins in a subpopulation of drug resistant cells can be moniored to inform therapeautic development.
Recent publication on ERK2 response to EGF





Is there a similar approach for RNA? How does a drug affect the dynamics of the proteome and how does this differ for individual cells? Model system: Cancer cell line & CPT & Real Time Proteomics anti-cancer drug camptothecin (CPT)
quinoline alkaloid from the Chinese "Happy tree" Camptotheca acuminata
mechanism: TOP1 poison Experimental design:
1.) Grow cells
2.) Add 10uM CPT @ 24hrs
3.) Track cells for additional 48hrs
[1 image every 20 min]

Drug induces loss of motility & growth (in about 10 hours), leading to death. Fig S2 -immunoblot Fig S3 - cell death at 36hrs Drug target responds very early
Less than 1 hour
CPT dose-dependent Translocations were rare (<2%) & limited to CPT mode of action: ribosomal proteins, chromatin remodeling, etc.

Oxidative stress response proteins thioredoxin & T. reductase-1 showed nucleolar translocation at about 8 hours. Suggest oxidative stress & DNA damage.
Other proteins like Prefoldin-5 exhibited temporary changes. Differential Cell Behavior:
Normal SD was 10-60% of mean and partly due to cell cycle (20%)
CPT increases variability by ~30%
Heterogeneous trends after 20-30 hours post CPT
Related to cell death DDX5 siRNA knock-down (~80%) doubled death rate
DDX5 may be antiapoptotic here, but was not bimodal for other drugs http://www.reefpedia.com/index.php/Category:Discosoma Protein descriptions:
Top1 - DNA Topoisomerase 1
NCL - Nucleolin ... synth & maturation of ribozomes
DNMT1 - DNA (cytosine-5-)-methyltransferase 1
ARID1B - Chromatin remodeling
RPL39 - ribosomal protein L39
FAU - ubiquitin-like protein & ribosomal protein S30 fusion
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UPF3 - mRNA nuclear export & mRNA surveillance (truncated ORFs)
DDX5 - putative RNA helicase
C3ORF19 - ??
LPIN2 - triglyceride metabolism &/or trancription coactivator one of six subunits of prefoldin, a molecular chaperone complex that binds and stabilizes newly synthesized polypeptides "cells binned according to the time between
their last division and the time of drug addition—
an in silico synchronization approach"
Full transcript