AfCS Microscopy Laboratory Department of Molecular Pharmacology Stanford University Tobias Meyer
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Transcript of AfCS Microscopy Laboratory Department of Molecular Pharmacology Stanford University Tobias Meyer
AfCS Microscopy LaboratoryDepartment of Molecular PharmacologyStanford University
Tobias Meyer
Lead Scientists: Nancy O’Rourke Grischa Chandy
Mary Verghese Liz Gehrig WeiSun Park Sarah Lim Takako Mukai
Won Do Heo Jen Liou
The two Goals of the AfCS Microscopy Core
1. To identify the location of signal transduction processes by investigating subcellular localization and by monitoring receptor triggered translocation processes
2. To dissect dynamic properties of signaling networks by using perturbation strategies combined with fluorescence microscopy based screening tools (antibodies, fluorescent probes, FRET and protein translocation assays)
Fluorescence microscopy strategies
Why fluorescence microscopy?
1. To measure subcellular localization of signaling proteins
2. To monitor receptor triggered translocation of signaling proteins (GFP conjugation)
3. To measure dynamic signaling parameters such as delays, cooperativity, coincidence
4. Microscopy-based readouts of signaling processes enable one to perform large scale perturbations screens using RNAi, dominant negative and constitutively active signaling constructs (for example translocation or FRET as readout)
Requirements for effective microscopy studies in the AfCS
cell model
A. Cells require distinguishable nuclei, cytosol, organelles, membrane structures (flat cells and morphologically differentiated cells are best suited for microscopy)
B. Cells have to be adherent
C. Cells have to be readily transfectable with expression constructs and RNAi molecules (or constructs)
D. Single cell assays have to be available to measure important receptor-triggered cell functions in these cells
Raw cells seem to be OK
The strategy for measuring protein localization is based on dual
fluorescence imaging using proteins of interest versus marker proteins
A marker set of expressable CFP-YFPconstructs has been developed (and keeps being refined)
Subcellular markers are based on expression constructs of CFP and YFP conjugated marker and signaling proteins as well as on dual immunostaining using antibodies
Quantitative co-localization tools still need to be further developed
Recycling endosome marker in live cells (VAMP3/cellubrevin)
Correlation – 0.92
CFPPLA2
YFP ER
Correlation – 0.93YFPPM
YFPDAGK
Correlation analysis for co-localization
Marker
Marker
Microscopy Localization Screens Using Grouped Protein Families &
Protein Domains
Y kinases 106
Y phosphatases 56
Ras 141
S/T kinases 395
SH3-domain 143
PDZ-domain 96
EF-hand 83
C2-domain proteins 73
C1-domain, DAG binding 45
PH-domain proteins 193
SH2-domain 87
PTB-domain 24Signaling lipid metabolizing enzymes ?
Full length signaling proteins:Signaling Domains:
Advantages of protein family approach:
1. Related kinases function as internal controls
2. Gives mechanistic and structural insights into localization
3. Facilitates molecular biology; enables systematic creation of perturbation constructs
G. Manning et. al., (Science 298:1912).
Examples of protein kinase localizations in resting WEHI cells
… intermediate filaments, centrosomal localizations
Why GFP conjugated protein domains?
1. To understand mechanisms of localization
2. To identify minimal fluorescent translocation biosensors
3. Also useful as perturbation constructs
C1-domains monitor localized diacylglycerol signals
C2-domains monitor local Ca2+/PS-signals
PH-domains monitor local changes in phosphoinositide concentration
SH2-domains monitor local tyrosine phosphorylation
Creation of more than 100 CFP and YFP conjugated PH-domains
PH-domain from the protein kinase ITK
1. Measurements of dynamic systems parameters
2. Measurement protocols of sufficiently high throughput to dissect the modular structure of signaling network
Single-cell fluorescence measurements are ideally suited for:
1. Measuring rates, bistability, cooperativity, delays, coincidence and other dynamic signaling parameters
2. High Throughput Perturbation Screens to define pathways,cross-talk and modular structures in signaling networks
Challenges for the AfCS
Now that the parts list of cells and connection maps start to exist, two things are needed:
Measured cell functions
Microscopy-based perturbation screens to dissect the signaling
networkReceptor inputs
1. RNAi
2. Expression of suppressing and activating signaling constructs
3. Monitoring of internal signaling steps
4. Monitoring of cell functions
Fluorescent single cell perturbation screens
What are perturbation strategies that the AfCS should employ?1. RNAi2. Expression of perturbation constructs 3. Small molecule inhibitors and activators
What are the advantages of single cell assays?1. Identification of signaling states, oscillations, delay times,
coincidence measurements2. Partial cell transfection does not reduce the quality of the
data; improved signal-to-noise
Why is a perturbation strategy required to understand signaling networks?
Perturbation screens are the only way to assess the relevance of different players in a signaling system
Time courses of protein kinase C and protein domain translocation
C1A-domainPKC
Rel
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C2-domain
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PAF PAF PAF
50 s
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averages
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Time (s)
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Delay times between Ca2+ increases and start of secretion
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Time (s)
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CFP-C2
GLUT4-YFP
GLUT4-YFP
CFP-C2
CFP-C2
GLUT4-YFP
1/9 th of field of view
antigen
antigen
Readouts for a microscopy-based perturbation screen
Fixed-cell assays (higher throughput) Using activation-dependent and other antibodiesFor example: phosphospecific antibodies, nuclear localization, increased protein expression, polymerized actin, cell size
Live-cell assays (lower throughput) 1. Protein translocation (for example to nucleus or to plasma
membrane)2. FRET or small molecule biosensors3. Functional assays such as cell migration, exocytosis-endocytosis, phagacytosis
Fixed and live cell fluorescence imaging assays: Existing assays as well as future assays developed by Cell lab, Antibody lab & Microscopy lab
Perturbation expression constructs: These can be created by systematically mutating signaling proteins in a protein family (for example S/T protein kinase) as well as by creating sets of constructs that express signaling protein domains (for example PH-domain)
RNAi screens: For microscopy based assays, this can be done using a new in vitro dicing technique developed by James Ferrell’s group that significantly cuts the cost of RNAi and enables high throughput microscopy-based perturbation screens
Overall strategy for a microscopy based perturbation screen
Dissecting the signaling network: Perturbation
constructsWhy using perturbation constructs based on proteins that are
low abundant or absent in a cell type?
Every perturbation construct that affects a pathway is (like a specific drug) a tool to dissect the structure of a signaling network
Example: K to R mutation in the ATP binding pocket often yields a dominant negative enzyme; a large set of these protein kinase mutants has been created (320 currently planned)
Constitutively active kinases can be created for many protein kinases and hundreds of other perturbation constructs can be made using available signaling literature
Specific steps to implement a microscopy-based perturbation screen for signaling
networksProposal:
1. Automated image acquisition and analysis. This microscope for fixed cell assays could be acquired immediately using existing funds in this years budget of the microscopy core ($170’000).
2. Creation of a large set of perturbation constructs (~ 2000-3000). These constructs can be made using existing literature. The time for completion is ~ 2 years using collaborative efforts. The cost is ~ $250 per construct.
3. Creation of a mouse library of in vitro diced RNAi for all known mouse signaling proteins (3000-4000 constructs). This can be done by 3 people in 1 year at a cost of less than $300’000