How to visualize genes and their products Kamil Růžička FGP CEITEC MU Genomics Lecture Series 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 (principle of chemiluminiscence) 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 Many 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; free GFP sometimes moves Luminiscent mouse better than phluorescent mouse Promoter activity monitoring Pros: • Cons: • 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 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 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 Nopp140 (5 nm gold particles) nascent DNA (10 nm) Philimonenko et al 2000, and an unfortunate Cell paper Imunohistochemistry pros/cons Pros: • direct • nothing can beat the resolution Cons: • very tricky (needs rather expert) • huge experience for interpretation needed Can we visualize postranslational modifications? Can we visualize postranslational modifications? Stanislas et al. 2016 antibodies against phosphate pS4 -> phosphorylation is required for PIN1 to stay on the membrane Yes, we can. If missing other model organism than Arabidopsis 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 Similar system in single molecule resolution Grünwald and Singer 2010 export of βactin mRNA from nucleus (smFISH and stem loops) Big science of single molecule microscopy Vera et al. 2016 (Singer lab) Alternative to λN22 system etc. - 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 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 • scientific question here: 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 (photoswitchable) 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 Q: What is easier experiment to confirm autofluorescence? Dovzhenko, TrautmannPicoQuant Application note 2013 (be careful with the interpretation) FLIM • need to have experience • need to have special module on your confocal Light sheet microscopy Tomer et al. Nat Methods 2012 Light sheet microscopy Pros: – less bleaching: better tissue penetrance, better resolution and sensitivity – 3D structures fast Cons: – equipment price, availability – sample preparation could be slower – data handling 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 • Single molecule analysis of gene expression (Vera et al. 2016): https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5149423/ • 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)