How to visualize genes and their products Kamil Růžička FGP CEITEC MU CG920 Genomics Lecture 9 Outline • reporter genes • promoter fusions • visualizing proteins • visualizing RNA • dynamics of protein imaging: FRAP, photoactivable proteins, FLIM, FCS 2 3 Promoter activity monitoring promoter here can be reporter terminator 1-10 kb prior to ATG LacZ, GUS Luciferase GFP 4 Reporter genes • LacZ, GUS • Luciferase • GFP some need external substrate, some do not 5 LacZ, GUS – rhapsody in blue (in case of GUS – X-Gluc) promoter LacZ terminator soluble insoluble 6 LacZ, GUS LacZ/ GUS: worm, mouse – LacZ, plants - GUS 7 Luciferase (similar to chemiluminiscence) promoter luciferase terminator What’s the difference between fluorescence and luminiscence? 8 Luciferase 9 How does fluorescence work?energy 10 How does a fluorescence microscope work? 11 Stokes shift George G. Stokes 12 How does a confocal microscope work? What are advantages of confocal microscopy? 13 Live imaging Martin Chalfie GFP discovery - Nobel Prize 2008 Roger TsienOsamu Shimomura 14 Fluorescent proteins on the market (Tsien’s fruits) 15 Excitation and emission 16 Multicolored fluorescent protein image (neurones) 17 Promoter-GFP promoter GFP terminator 18 Promoter activity monitoring choice of suitable reporter • LacZ, GUS • Luciferase • GFP accessibility, sensitivity, accuracy… 19 Promoter activity monitoring • LacZ, GUS – easy assay, also on sections, easy imaging – substrate must diffuse, kills the organism • luciferase – good quantification, very sensitive, no autofluorescence – substrate must diffuse, special machine, dark • GFP – good sensitivity, colocalization with other dyes/promoters possible, no substrate needed – only in vivo, autofluorescence, thin transparent sample; it should be ER localized in plants 20 Luminiscent mouse better than phluorescent mouse 21 Promoter activity monitoring Pros: • easy to clone, easy to visualize • usually some signal seen – cheers you up! • 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.) • length of promoter given arbitrarily 22 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 23 GFP and membrane proteins here can be GFP It is good to have GFP tag localized inside the cell (plants) 24 Expression of isoforms cDNA1 cDNA2 YFP YFP Kriechenbaumer et al 2011Not the best option available – can you guess? endoplasmic reticulum cytosol 25 Isn’t this better? YFP GFP 26 Expression of isoforms 27 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) 28 Why to visualize all this stuff pSHR :: GFP pSHR::SHR::GFP Nakajima et al, Nature 2001 promoter translational 29 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 30 Why to visualize all this stuff pSHR :: GFP pSHR::SHR::GFP Nakajima et al, Nature 2001 BANG! SHR moves from stele to endodermis 31 Protein immunolocalization (of given source organism) Most favorite animals: - rabbit (too many rabbits) - mouse (low volume) - goat - chicken - rat - sheep - donkey - guinea pig secondary: anti-rabbit from no-rabbit, anti-mouse from no-mouse, etc. 32 Protein immunolocalization immunolocalization - fluorescently fluorescent dye attached primary antibodies secondary antibodies 33 Protein immunolocalization immunolocalization • FITC (obsolete) • CY3, CY5 • Alexa (488, 568, 633) Fluorescent dyes conjugated to secondary (examples): 34 www.zeiss.com/microscopy 35 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 36 GFP tag partially retains PIN1 in endoplasmic reticulum (-> artifact) PIN1-GFP anti-PIN1 37 Protein localization - immunogold immunolocalization - immunogold electron microscope 38 Immunogold collocalization Nopp140 (5 nm gold particles) nascent DNA (10 nm) Philimonenko et al 2000, and an unfortunate Cell paper 39 Pros/cons Pros: • direct • nothing can beat the resolution Cons: • very tricky (needs rather expert) • huge experience for interpretation needed 40 Can we visualize posttranslational modifications? 41 Also RNA can be visualized 42 Localization of mRNA RNA hybridization in situ 43 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 44 45 Also mRNA can be visualized in vivo Ash1 mRNA localized to the tip of the daughter cell 46 λN22 system – RNA imaging in vivo nuclear localization signal promoter viral RNA binding protein 47 Drawbacks of λN22 system - we have SPINACH GACGCAACUGAAUGAAA UGGUGAAGGACGGGUCC AGGUGUGGCUGCUUCGG CAGUGCAGCUUGUUGAG UAGAGUGUGAGCUCCGU AACUAGUCGCGUC + Paige et al. 2012 RNA fusion aptamer blue-DNA green-RNA 48 Other vegetables than SPINACH Paige et al. 2012; Song et al. 2014 49 Advanced confocal techniques 50 (slightly) Advanced confocal techniques • FRAP • photoactivatable FP • FCS 51 FRAP region of interest (ROI) Fluorescence Recovery After Photobleaching 52 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 53 FRAP – bleaching curve What does the curve tell? 54 FRAP – bleaching curve 55 iFRAP inverse FRAP 56 iFRAP – dissociation of premRNA from specles 57 FRAP derivatives FLIP Fluorescence Loss After Photobleaching • bleaching process is repeated during the experiment • for studying general protein turnovers in compartments • scientific question here: is there a fraction of protein which does not leave the bright green patches continuous bleaching here 58 FRAP derivatives FLAP Fluorescence Localization after Photobleaching • two fluorochromes on one protein– one bleached, non bleached as control 59 Perhaps better scheme than previous YFP bleachedCFP not bleached prebleach after bleach RED=CFP-YFP Dunn et al. 2002 60 FRAP - advantages • not only proteins (also other dyes) • tells you more than simple life imaging movie 61 • 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 62 aquaporin PIP2 undergoes lateral diffusion photoactivation (UV) Photoactivable fluorescent proteins 63 Photoactivable proteins Dronpa, Kaede, Eos – probably the most popular 64 Photoactivable proteins Advantages: - elegant, can be convincing Disadvantages: - very weak signal - each material needs optimization 65 Remarks • your material is 3D • protein de novo synthesis in some experiments (e.g. cycloheximide stops translation) 66 FLIM Fluorescence Life Time Imaging Microscopy Fluorochromes • excitation spectra • emission spectra • unique lifetime 67 FLIM - applications 68 FLIM - applications Protein-protein interactions (FRET-FLIM) Lifetime sensitive to almost everything: • pH • ionic strength • solution polarity • other fluorochrome 69 FLIM Trautmann et al. PicoQuant Application note 2013 indeed, salt changes fluorophore life time (American cockroach glands) 70 FLIM - discrimination of autofluorescence Q: What might be the easier experiment to confirm autofluorescence? Dovzhenko, TrautmannPicoQuant Application note 2013 (be careful with the interpretation) 71 FLIM • experience needed • special module on your confocal needed 72 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. 73 FCS Schwille und Haustein t [ms] G(t) antibunching rotational movement photophysical processes (triplet state, …) diffusion 74 FCS (FCCS) fluorescence cross-correlation spectroscopy Digman and Gratton 2011 75 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 76 Photon bunching, if someone asks 77