How to visualize genes and their products Kamil Růžička FGP CEITEC MU Genomics Lectures Outline • reporter genes • promoter fusions • visualizing proteins • visualizing RNA • dynamics of protein imaging: FRAP, photoactivable proteins, FLIM, FCS Promoter activity monitoring promoter here can be reporter terminator 1-10 kb prior to ATG LacZ, GUS Luciferase GFP Reporter genes • LacZ, GUS • Luciferase • GFP some need external substrate, some not LacZ, GUS – rhapsody in blue (in case of GUS – X-Gluc) promoter LacZ terminator soluble insoluble LacZ, GUS LacZ/ GUS: worm, mouse – LacZ, plants - GUS Luciferase (used principle of bioluminiscence) promoter luciferase terminator What’s difference between flurescence and luminiscence? Luciferase How does fluorescence work?energy How does a fluorescence microscope work? Stokes shift George G. Stokes How does a confocal microscope work? What are advantages of confocal microscopy? Live imaging Martin Chalfie GFP discovery - Nobel Prize 2008 Roger TsienOsamu Shimomura Fluorescent proteins on the market (Tsien’s fruits) Excitation and emission Multicolored fluorescent protein image (neurones) Promoter-GFP promoter GFP terminator Promoter activity monitoring choice of suitable reporter • LacZ, GUS • Luciferase • GFP accessibility, sensitivity, accuracy… Promoter activity monitoring • LacZ, GUS – easy assay, also on sections, easy imaging – substrate must diffuse, kills the organism • luciferase – good quantification, very sensitive, no autophluorescence – substrate must diffuse, special machine, dark • GFP – good sensitivity, colocalization with other dyes/promoters possible, no substrate needed – only in vivo, autophluorescence, thin transparent sample Luminiscent mouse better than phluorescent mouse Promoter activity monitoring Pros: • Cons: • Promoter activity monitoring Pros: • easy to clone, easy to visualize • “always works” • can be used in less accessible organs Cons: • limited information about gene product (mRNA, protein etc) • needs cloning and transformation • neglects regulatory elements (introns, UTRs etc.) Translational GFP fusions your genepromoter here can be GFP here can be GFP terminator your genepromoter terminator your genepromoter terminator here can be GFP N-terminal fusion C-terminal fusion fusion inside the coding sequence GFP and membrane proteins here can be GFP It is good to have GFP tag localized inside the cell (plants) Expression of isoforms cDNA1 cDNA2 YFP YFP Kriechenbaumer et al 2011Not the best option available – can you guess? endoplasmic reticulum cytosol Isn’t this better? YFP GFP Expression of isoforms Fluorescent protein fusion Pros: • Cons: Fluorescent protein fusion Pros: • in vivo imaging Cons: • not always functional • transformation needed • transparent material (you can sometimes fix GFP signal, however) • sometimes GFP artifacts (tag doesn’t allow proper targeting) Why to visualize all this stuff pSHR :: GFP pSHR::SHR::GFP Nakajima et al, Nature 2001 promoter translational Why to visualize all this stuff pSHR :: GFP pSHR::SHR::GFP 1 2 3 1 2 3 1 – epidermis 2 – cortex 3 – endodermis 4 – stele promoter translational 44 Why to visualize all this stuff pSHR :: GFP pSHR::SHR::GFP Nakajima et al, Nature 2001 BANG! SHR moves from stele to endodermis Protein immunolocalization (of given source organism) Most favorite animals: -rabbit (too many rabbits) -mouse (low volume) -goat -chicken -rat -sheep -donkey -guinea pig 2ndary: antirabbit from no-rabbit, antimouse from no-mouse, etc. Protein immunolocalization immunolocalization - fluorescently fluorescent dye attached primary antibodies secondary antibodies Protein immunolocalization immunolocalization • FITC (obsolete) • CY3, CY5 • Alexa (488, 568, 633) Fluorescent dyes conjugated to 2ndary (examples): www.zeiss.com/microscopy Protein immunolocalization Pros: • • Cons: • • Protein immunolocalization Pros: • no need to clone or transform or cross • direct (if no tag used) • allows sectioning (less accessible tissues) Cons: • fixed material only • excellent antibodies only, sometimes tricky GFP tag partially retains PIN1 in endoplasmic reticulum (-> artifact) PIN1-GFP anti-PIN1 Protein localization - immunogold immunolocalization - immunogold electron microscope Immunogold collocalization Pros/cons Pros: • direct • nothing can beat the resolution Cons: • very tricky (few labs, mainly specialized) • huge experience for interpretation needed • immunogold colocalization – only theoretical? Can we visualize postranslational modifications? Also RNA can be visualized Localization of mRNA RNA hybridization in situ Visualization of mRNA RNA hybridization in situ Pros • classical technique in developmental biology • no transgenes needed Cons • tedious, tricky, no success guaranteed • only on fixed samples For shorter RNAs (miRNA etc.): • LNA probes needed Also mRNA can be visualized in vivo Ash1 mRNA localized to the tip of the daughter cell λN22 system – RNA imaging in vivo nuclear localization signal promoter viral RNA binding protein Also mRNA can be visualized in vivo Drawbacks of λN22 system - we have SPINACH GACGCAACUGAAUGAAA UGGUGAAGGACGGGUCC AGGUGUGGCUGCUUCGG CAGUGCAGCUUGUUGAG UAGAGUGUGAGCUCCGU AACUAGUCGCGUC + Paige et al. 2012 RNA fusion aptamer blue-DNA green-RNA Other vegetables than SPINACH Paige et al. 2012; Song et al. 2014 Transport among compartments Alberts et al. 2008 Advanced confocal techniques (slightly) Advanced confocal techniques • FRAP • photoactivatable FP • FCS FRAP region of interest (ROI) Fluorescence Recovery After Photobleaching FRAP you can quantify fluorescence.. (ImageJ is our friend) mean min max A 90.404 49 113 C 8.556 3 8 D 39.934 19 63 FRAP – bleaching curve What does the curve tell? FRAP – bleaching curve iFRAP inverse FRAP iFRAP – dissociation of premRNA from specles FRAP derivatives FLIP Fluorescence Loss After Photobleaching • bleaching process is repeated during the experiment • for studying general protein turnovers in compartments • is there a fraction of protein which does not leave the bright green patches? continuous bleaching here FRAP derivatives FLAP Fluorescence Localization after Photobleaching • two fluorochromes on one protein– one bleached, non bleached as control Perhaps better scheme than previous YFP bleachedCFP not bleached prebleach after bleach RED=CFP-YFP Dunn et al. 2002 FRAP - advantages • not only proteins (also other dyes) • tells you more than simple life imaging movie • your cells are moving • high energy needed to bleach the ROI – long time needed to bleach – can damage your material • usually only one ROI can be observed – time consuming • for gourmets perhaps awkward (although more reliable and robust) FRAP – pitfalls aquaporin PIP2 undergoes lateral difussion photoactivation (UV) Photoactivable fluorescent proteins Photoactivable proteins Dronpa, Kaede, Eos – probably most popular Photoactivable proteins Advantages: -elegant, can be convincing Disadvantages: -very weak signal -each material needs optimization Remarks • your material is 3D • protein de novo synthesis in some experiments (e.g. cycloheximide stops translation) FLIM Fluorescence Life Time Imaging Microscopy Fluorochromes • excitation spectra • emission spectra • unique lifetime FLIM - applications FLIM - applications Protein-protein interactions (FRET-FLIM) (other lecture) Lifetime sensitive to almost everything: • pH • ionic strength • solution polarity • other fluorochrome FLIM Trautmann et al. PicoQuant Application note 2013 indeed, salt changes fluorophore life time (American cockroach glands) FLIM - discrimination of autofluorescence Dovzhenko, Trautmann, PicoQuant Application note 2013 (be careful with the interpretation) FLIM - discrimination of autofluorescence Q: What is easier experiment to confirm autofluorescence? Dovzhenko, TrautmannPicoQuant Application note 2013 FLIM • need to have experience • need to have special module on your confocal FCS Fluorescence Correlation Spectroscopy t + τ It is counted, how many times the fluorescent molecule comes through the focal plane. autocorrelation analysis Autocorrelation analysis: the way how to discriminate the diffusions speeds of particles. FCS Schwille und Haustein FCS (FCCS) fluorescence cross-correlation spectroscopy control membrane bound GFP and RFP (crosscorrelation curve) free GFP and RFP FCCS control membrane bound GFP and RFP (crosscorrelation curve) channel crosstalk threshold FCCS control membrane bound GFP and RFP (crosscorrelation curve) receptor with two labels channel crosstalk threshold FCCS receptor with two labels the crosscorelation curve is above threshold -> EGFR protein dimerizes Liu et al., 2007 FCS • special confocal module and objectives needed • interpretation tricky Literature • Paige et al., RNA Mimics of Green Fluorescent Protein, Science 333, 642-646, 2011 (SPINACH and other vegetables) • https://www.micro-shop.zeiss.com/index.php?s=452278238ae52c&l=en&p=de&f=f&a=d (comprehensive and broad list of phluorochromes) • http://www.illuminatedcell.com/ - nicely done pages about plant cell imaging • Ishikawa-Ankerhold et. al. Advanced Fluorescence Microscopy Techniques — FRAP, FLIP, FLAP, FRET and FLIM, Molecules 2012, 17, 4047-4132 • Sambrook & Russell Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001), 13.5 pounds weight • Ctirad Hofr – Pokročilé biofyzikální metody v experimentální biologii (přednáška)