‘principles of cellular organization: genes, proteins, and ......molecular biology background: •...
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ControlControl TreTreatedatedChallenge: Challenge: Cancer metastatic cascade
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Tony Ng
Randall Division of Cell & Molecular Biophysics
Cancer Cell Biology and Imaging, Division of Cancer Studies
‘‘Principles of cellular organization: Principles of cellular organization: genes, proteins, and global functiongenes, proteins, and global function’’
Chemistry/Chemical Biology
Mathematical/networkModelling
Bio / Nano-Photonics
Instrumentation & Data Processing
HTP PLATFORM
Bioinformatics
Interaction prediction
Molecular & Cell Biology
Understanding cell biology quantitatively requires multidiscipliUnderstanding cell biology quantitatively requires multidisciplinary collaboration nary collaboration
AmeerAmeer--Beg, Barber, Coolen, Festy, Beg, Barber, Coolen, Festy, Fraternali, Fraternali,
HailesHailes, , HeintzmannHeintzmann, Irving, Ng, , Irving, Ng, Richards, Richards, SuhlingSuhling, Tabor, Thomas, , Tabor, Thomas,
Vojnovic, et al.Vojnovic, et al.
http:www.opticalproteomics.orghttp:www.opticalproteomics.orgBiostatistics/stratification
Biomedical application
CancerAlterations in gene expression induces formation of a primarytumour.
1. Outside factors- Smoking- UV light
2. Genetic disease
3. Viral infection
Cancer• Understand the cellular processes causing
cancer– Identify changes in genes and protein
expression– Identify mutations in candidate genes and
proteins altering their function
Methods to study cancer
• Molecular Biology
• Protein Chemistry
• Cell Biology
• Molecular Biology
– Monitoring transcription and translation of DNA to RNA to protein
– Regulation of Gene expression
Methods to study cancer
• Protein Chemistry
– Physical behaviour of proteins
– Determination of protein expression
Methods to study cancer
• Cell Biology
– Study of cell behaviour
– Characterise protein function in cells
Methods to study cancer
Molecular Biology
Background:• Several whole genomes have been fully
sequenced, allow comparison of entire genomes, Genomics, and thus predict all proteins, as well as evolution of species Bioinformatics.
• Human Genome contains 23.000 genes compared to 28.500 in mouse.
Translate DNA to protein
Monitor gene expression
• Real-time Polymerase Chain Reaction
• cDNA array
PCRPolymerase Chain Reaction:
PCRPolymerase Chain Reaction:
PCR
- Monitor the incorporation of fluorescent dye during the reaction as an indication of production/amplification of gene of interest at each PCR cycle
cDNA array• Attach probes corresponding to larger amount of mRNA of interest• Commercially available
cDNA array
cDNA array
Gene array
Gene array1. Isolate four different cell lines with various
degree of metastatic probabilities for lung cancer
Breast CarcinomaCell line
Gene array2. Compare genotype of the four cell lines
Upregulated
Downregulated
Four cell lines
Gene array3. List of 54 genes with altered expression specific for
primary breast cancer to metastasise to lung.
• Compared expression of these 54 genes in primary tumours obtained from human breast cancer patients and developed a prognostic “gene signature” for breast-to-lung metastasis
Gene expression in cancer
• Very powerful technique, highly sensitive• Screen numerous genes in high-
throughput fashion. Large amount of data.
• Downside:– Gene expression levels is not always comparable to
protein expression levels. mRNA vs protein stability. – Protein function is regulated “post-translationally”
Protein Chemistry
Background:• Large macromolecules • The main machinery for all functions within the cell and
perform highly specialised duties determined by their amino acid composition
• Regulated function by modifications, phosphorylation, GDP/GTP loading, lipid anchoring, cellular localisation (membrane, cytoplasm, nucleus).
Protein Chemistry
Background:• Chain of amino acids (polypeptide)
– Number of amino acids varies from 10s to 20,000aa, average ~ 500 aa
• 20 different amino acids• Each protein has it’s own specifications
– Molecular weight (Daltons)– Isoelectric point (pH at which the molecule is neutral)
• Dimensions: ~ 10 nm3
Protein Chemistry
Protein ChemistrySDS Poly Acryl Gel Electrophoresis (SDS-Page):
- Analyse Protein Expression
Protein Chemistry
Mw
SDS Poly Acryl Gel Electrophoresis
Visualised by Dye (Coomasie)
Western Blot
Visualised by Chemiluminescence
Transfer to membrane
Incubate with antibodies of interest
- Analyse Protein Expression
Antibodies• Source for immune response in
order to neutralise foreign objects • Each antibody recognises
one specific antigen• Important tool in biochemistry
due to specificity• Produced in mouse or rabbit
by repeatedly injecting antigen (i.e. protein) of interest for a immune response, and isolate the serum.
Protein Chemistry- Analyse Protein Expression
Isoelectric focusing
Mw2 Dimensional SDS Page:
Protein Chemistry- Analyse Protein Expression
Control
Treated
Protein identification by Mass spectrometry
• Monitor protein expression, which is the cell machinery and ultimately the end-product of gene expression.
• Downside:– Low sensitivity, i.e. needs any cells for
detection– Limited by antibody specificity
Protein Chemistry
>Cancer Cell Biology
• Characterise the cellular function of expressed proteins:
– Alter cell proliferation
– Induce metastasis,i.e. increased cell migration
Structures within the cell
Cells in tissue culture• Grows at 37 degrees at controlled pH, supplied with calf serum,which contains vital supplement of various proteins and growth factors
• Divides every 16-24 hours
Imaging proteins in cells
• Immunofluorescent staining– detect localisation of endogenous proteins
• Fusion proteins with fluorescent proteins– detect spatial and temporal localisation of
exogenous proteins (live imaging)
• Immunofluorescent staining– detect localisation of endogenous proteins
• Specific fluorescent dyes or fluorescently conjugated antibodies against protein of interests on fixed samples
– DAPI, FITC/Cy2, TRITC/Cy3, Cy5
Imaging proteinsin cells
Imaging proteinsin cells
• Fusion proteins with fluorescent proteins– detect spatial and temporal localisation of
exogenous proteins (live imaging)
• Expression from plasmid after lipid transfection or microinjection
– Cyan, Yellow, Green, Red fluorescent proteins
Imaging proteinsin cells
18min18min
60min60min
88min88min
120min120min
131min131min
180min180min
260min260min
260+min260+min
EGFREGFR--GFPGFP Activated Activated ββ1 1 IntegrinIntegrin((mAbmAb 12G10)12G10)--Cy3Cy3
EGFEGF
Plasmid•Circular DNA approximately 5000 nucleotides•Easy to produce in large quantities in bacteria•Easy to “manipulate” (=cloning)
GFP-fusion protein
Excitation maximum at 488nm
Emission maximum at 507nm
GFP-fusion protein
CXCR4 is upregulated in many tumours. We wanted to characterise the recycling, and possibly manipulate and induce downregulationof the receptor
Receptor recycling using CXCR4-GFP
Cell migration• in vitro models for cancer cell metastasis
– characterise proteins promoting migration– identify signals expressed in tissues, which
attract cancer cells
Cell migration• Identify and characterise proteins involved
in cell migration– Detachment– Translocation– Protrusion
Cell migration“Wound assay”- easy to perform- measuring “wound closure”
Cell migrationRandom migration- observe behaviour- measuring cell speed
Cell migrationChemotaxsis- what attracts cells/ promotes migration - measuring cell direction
Gradient
Altering protein expression levels
• Knock out mice. – Delete single genes within the mouse
genome.
• RNA interference– Decrease the mRNA level in tissue culture
cells.
Altering protein expression levels
• Knock out mice. 1. Gene isolated and
manipulated in vitro2. Isolate stem cells from
embryos3. Introduce “new” gene into
stem cells4. Insert stem cells into
blastocytes, and implant back into “pseudo”-pregnantfemale mouse
5. Chimera offspring, heterozygote6. Cross (breed) with other chimera
homozygote
Altering protein expression levels
• …. knock-out mice– Expression of …. is necessary for cell migration
Wildtype mouse
embryonic fibroblasts
Knock-out mouse
embryonic fibroblasts
Altering protein expression levels
• RNA interference
Scramble RNAi Cdc42 RNAi
Western blotfor expression levels
– RNA interference of Cdc42
Altering protein expression levels
Scramble RNAi Cdc42 RNAi
– RNA interference of Cdc42
Altering protein expression levels
Compare cell tracks of individual cells- expression of Cdc42 is important for cell migration
• Analyse cell behaviour upon altering expression of individual proteins
– Secondly, to identify signalling pathways involved, i.e. other proteins and their function affected due to altered expression
Altering protein expression levels
Analyse protein-protein interactions, proteomics
• Proteins function in larger complexes
– Identify known members within these complexes can indicate previous unknown functions of the protein of interest
Analyse protein-protein interactions
Complexity of the Proteome Complexity of the Proteome ––
The need for an imaging approachThe need for an imaging approach
Protein ‘modifications’ +
interactions in time and space
= Function
Dynamics!Dynamics!
Analyse protein-protein interactions
• Immunoprecipitation (-protein chemistry)– Analyse protein complexes
• FRET/FLIM (-cell imaging)– Analyse protein-protein interaction in cells
Analyse protein-protein interactions
• Immunoprecipitation– Analyse protein complexes
X
Y
What interacts withprotein X?
Analyse protein-protein interactions
• Immunoprecipitation– Analyse protein complexes
Mass spectrometry and/or
Western Blot analysisto identify protein Y
X
Y
Incubate with immobilised antibodies
towards protein Xand isolatecomplexes
Analyse protein-protein interactions
• FRET/FLIM Fluorescent/Förster resonance Energy Transfer, FRET by Fluorescent Life Time Measurements, FLIM– Analyse where protein-protein interactions
occur within cells
Analyse protein-protein interactions
• FRET, Fluorescent Resonance Energy Transfer– causes excitation of acceptor if within the proximity of 9 nm
Donor Acceptor
Förster Resonance Energy Transfer (FRET)
donor2-photon excitation
acceptor
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FRE
T Ef
ficie
ncy
RR
R/R0
Fraction of excited donors transferring to acceptor
FRET
Energy absorbed by donor can Energy absorbed by donor can resonantly transfer to acceptor.resonantly transfer to acceptor.
Energy appears as donor fluorescence, Energy appears as donor fluorescence, acceptor fluorescence, or are lost acceptor fluorescence, or are lost through nonthrough non--visible processes.visible processes.
Multi-photon fluorescence lifetime techniques
Laser
ScannerFilters &
DescannedDetectors
Non-descannedDetectors
Sample
Dichroic mirrors
Dichroic
TCSPC Histogram
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time bin
coun
ts
TCSPC Histogram
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time (ns)
Inte
nsity
τ = 2 ns
Excitationpulse
Nextpulse
TRI2 software- analyse fluorescent lifetime
Photon count
Average fluorescentlifetime Time-Domain
Cell expressing ezrin-GFP
Developed by Dr Paul Barber at Gray Cancer Institute
Control
TRI2 software- analyse fluorescent lifetime
Photon count
Average fluorescentlifetime Time-Domain
Cell expressing ezrin-GFP
Treated
FRET
Active(low GFP lifetime,
low RFP anisotropy)
Inactive(high GFP lifetime,
high RFP anisotropy)
sensorligand
Conformation sensing - Two examples
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FRET population
time (s)
Rac/Cdc42
FRET
PAKInactive(high GFP lifetime
low FRET Pop)
Active(low GFP lifetime,high FRET Pop)
GDP-bound
Leo Carlin, Ton CoolenLeo Carlin, Ton Coolen
A delayed negative feedback loop in immunosurveillance