Davide Mazza - [email protected] 1 San Raffaele Scientific Institute Centro Di imaging Sperimentale School of photonics Cortona, March 30 – April 3, 2014 Protein dynamics in living cells by fluorescence microscopy From ensemble average experiments to single molecule imaging xVivo – The inner life of cell http://www.xvivo.net/animation/the-inner-life-of-the-cell/ 3 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale 28. P. J. Mulholland et al., Nature 452, 202 (2008). 16. A. Front. D. Rosemond, C. 9,M.229 Pringle, A. Ramírez, webshence (from leaf litterbasis toon detritivores to fish)activities. cou1. comA. M. Helton et al., Ecol. Environ (2011). expression and is the of cellular understanding of transcription assembly, 29.transcriptional M. J. Feio, T. Alves,regulation M. Boavida, A. Medeiros, metrics based fish,allinvertebrate, or algalLittle 23 January 2012; accepted 26 April 2 M. J. Paul, J. L. Meyer, Limnol. Oceanogr. 47, 278 1 2. C. J. Vörösmarty et al., Nature 467, 555 (2010). hightheprocess rates might be irreconM. A. S. Graça, Freshw. Biol. 55, 1050 (2010). informationpled existswith about kinetics of this process in live cells and cross-talk with transcription-coupled processes. , as munities) are typically least sensitive. Consequently, 10.1126/science.1219534 3. E. S. Bernhardt (2002). et al., Science 308, 636 (2005). 30. W. F. Cross, J. B. Wallace, A. D. Rosemond, S. L. Eggert, cilable goals in stream management. Second, Here we report accurate in vivo measurements of the mammalian understanding of gene expression regulation comes from studies using 4. P. H. Gleick, Science 302, 1524 (2003). litter breakdown—and potentially other functionEcology 87, 1556 (2006). streamFor managers currently rely primarily struc5. N. Adv. Ecol.inReseach 44, 2of (2011). Pol etIIal.,engaged the steps of active transcription. We purified proteins. instance, the subunits of theonelongating Pol II Friberg al measures such as whole-ecosystem metabotural measures to assess stream ecosystem health. 6. D. Hering et al., Sci. Total Environ. 408, 4007 thank the European Commission and previously developed a method for the Acknowledgments: in vivo labelingWe of mRNA are well known2 and thenutrient crystal structure of thisprimary enzymeproduction explains lism, (2010). In particular, changes inspiraling, biologicalorcommunity the Swiss State Secretariat for Research and Education for 3,4 transcripts containing a series of repeated stem-loops (from phage much of itsstructure behavior in vitro . mRNA transcription can be decon7. M.reO. Gessner, E. Chauvet, Ecol. Appl. 12, 498 (26–28)—can be used to complement, not funding the RivFunction research project (European Union (invertebrates, fish, and algae) have long (2002).MS2), which are specifically bound by ancontract MS2 EVK1-CT-2001-00088), coat protein fusedwhich to was supported under structed intounderpinned a succession of steps: promoter assembly, and place,stream established procedures to clearance assess stream bioassessment schemes be8. J. Hilton, M. O’Hare, M. J. Bowes, J. I. Jones, 16. The assay the Fifth Framework Programme. The constructive comments green fluorescent protein (GFP) by elongation and termination. The process of consists of a human cell escape5, followed ecosystem highlightsrethe needSci.forTotal Environ. cause they provide ahealth. reliableThis time-integrated 365,E.66Purvis, (2006). Kyle W. Karhohs, Jeremy Caroline Mock, Ericwhich Batchelor,* by three anonymous reviewers, substantially improved transcriptional initiation involves several structural changes line etharboring gene323 array into which the these stem-loops have been 9. the C. Perrings al., Sciencea 330, (2010). sponse to stressors such as organic pollution or differential diagnoses in environmentalinassesspaper, are greatly appreciated. All basic data are Alexander Loewer,† Galit Lahav‡ 6. Early in initiation, 17. Trends 10. the M. O. Gessner et al., Ecol.now Evol.used 25, 372 polymerase as the nascent transcript elongates integrated We have this system to follow the synthesismaterials. of acidification (5), but biogeographical constraints available in the supplementary This paper is ment, as is standard practice in medicine. ImporREPORTS IS STUDYING THE DYNAMICS OF YOUR PROTEIN IMPORTANT? (2010). 7,8 dedicated to the memory of of our colleague Björn Malmqvist, make this approach difficult to standardize at . These abortive cycles polymerase can produce abortive transcripts RNA in real time. Our method allows direct measurement Pol II breakdown and some other Man, functionally I. B. Holland, C. Cole,etK. al., Kuchler, F. 3.tantly, Materials litter and methods are presented as supporting phenotype in tissues other than the stem and 11. J. S.C.P.Moore Ecol.C. Lett. 7, 584 (2004). who sadly passed away in 2010. Higgins, Eds. (Academic Press, London, 2003), pp. material on Science Online. leaf and accumulation of residual surface observed wax large scales (10). Litter breakdown can help here Cells through molecular that often show complex d have been with aTindall, single prokaryote polymerase initiation eventsJ. as well asJ. R. elongation in isolation fromsignals the other steps 12. J. B. Wallace, S. L. transmit Eggert, L. information Meyer, Webster, 335–355. 4.based H. Powell, R. P. Schultz, Arch.be Neurol. 32, 250 methods can implemented at (RNAP) relatively on the stem of cer5-2 knockout line suggest 15. Thanks to G. Haughn, M. Smith, T. Hooker, and (1975). 9,10 because biogeography is a minor issue (for examScience 277, 102 (1997). The dynamic of the tumor suppressor p53 varies depending releasing thein order promoter of patterns. transcription. By usingbehavior a deterministic computational model . insightful O. Rowland for their comments. The 5.transcripts A. M. cost Rashotte, M.resource A. Jenks, K.escaping A. Feldmann, little orwithout input (29) to13. assess that additional wax export mechanismsseveral must Supplementary Materials J.ofL. the Tank, E. J. Sciences Rosi-Marshall, N. A. Griffiths, financial support Natural and Phytochemistry 57,similar 115 (2001).species of the genus ple, black alder or exist in plants. Chemical The analysis of the muin response to double-strand DNA breaks, it shows a series constrained by extensive dataSoc. sets tested with transcriptionof repeated pulses. elongation step can beofregulated byand pausing various times, Engineering impacts Research Council of Canada, 6.effects ABC transporter motifs were predicted byother PROSITE for pollution ecosystem S. A. Entrekin, M. L.Canadian Stephen, J. N. Am. Benthol. 29, andwww.sciencemag.org/cgi/content/full/336/6087/1438/DC1 tant wax demonstrated that CER5, like many for11,12 Innovation, BC Knowledge Develas referenced in (13). are common throughout most of Europe and Foundation the computational we identified a sequence of heretofore precisely timed drug addition inhibitors, weare were model, able to extract features of transcription .and(2010). as substrate demonstrated using polymerases in Materials and Methods 118 I –transporters, CARRIES INFORMATION ABOUT UNDERLYING MOLECULAR MECHANISMS. opmentvitro Foundation, the UBC Blusson fund 7.that M. Jasinski, Ducos,concern E. Martinoia, M. Boutry, Plant areE.prokaryotic of to environmental managers ABC has broad speciHolarctic), and marked gratefully acknowledged. We thankE.the Salk InPhysiol. 131, 1169 (2003). changes in breakdown ficity and is capable of transporting a variety Figs. S1 to S6 14. J. R. Webster, F. Benfield, Annu. Rev. Ecol. Syst. 17, pulses to instead produce a sustained p53 response. This leads to the expressio unexplored and provide a guide for application of the method to For eukaryotic cells, madeJ. O.to D.calculate endo-Analysis ńchez-Ferna ´have stitutethe for Genomic Laboratory for pro8.and R.attempts Sa ndez, T.been G. E. Davies, stakeholders. of wax substrates. We conclude that in plants, ratetransport occurred the portion of13(2001). the pollution Table S1 567 (1986). Interactions, processes, oligomerization states… viding sequence-indexed Arabidopsis T-DNA insertion Coleman,in P. A. Rea,rising J. Biol. Chem. 276, 30231 setThegenes. ofCER5downstream genes and also Reference alters cell fate: Cells that experience p53 pu genous elongation run-on assays as in other eukaryotes, proteins of the WBC/ mutants (project funded byother NSF). gene Freshw. Biol. 9.speed C. T. Increasing Otsuusing et al., J. Exp. Bot. 55, 1643 (2004). , reverse-transcription human pressure is accelerating en(31) 15. V. Gulis, K. Suberkropp, 48, 123 gradient, inanalyses which structural has been submitted to Genbank, and the accession 10. The of R.established Sańchez-Fernańdez et al. (8) agreemeasures ABCG subfamily are key components of 14 15 DNA damage, whereas cells exposed to sustained Databases S1 and S2 p53 signaling frequently und (2003). (RT)-PCR (such or fluorescence in situ hybridization (FISH) specific no. ison AY734542. with these relationships; however, they erroneously change throughout theand world, threatasvironmental water chemistry, hydromorphology, lipid transport systems. duplicated WBC15/WBC22 in their 2001 work. This Supporting Online Material 16. A. D. Rosemond, C. M. Pringle, A. Ramírez, Our results show that protein dynamics can be an important part of a signal, d mRNAs, andmetrics these have yielded apparent elongation estimates was corrected in (14). RESULTS ening water security fororhumans andranging aquatic www.sciencemag.org/cgi/content/full/306/5696/702/ II – CELLULAR OUTCOMES DEPEND ON PROTEIN DYNAMICS based on fish, invertebrate, algal comJanuary 2012; accepted 26 April 2012 M. J. Paul, J. L. Meyer, Limnol. Oceanogr. 47, 278 11. G. L. Scheffer et al., Cancer Res. 60, 2589 (2000). DC1 –1 influencing fate decisions. 23 J. are W. Jonker et al., Proc. Natl. Acad. U.S.A. 99,no Materials References and Notes from 1.1 tomunities) 2.5 12. kilobases (kb) min Kinetics of Pol cellular II transcription . ToSci.stretches date, assay been biodiversity (2). Large of theandhas landscape typically least sensitive. Consequently, Methods 10.1126/science.1219534 (2002). 15649 (2002). 1. L. Kunst, A. L. Eg. Samuels, Prog. Lipid Res. 42, 51 (2003). Figs. S1 toinflammation, S4 Circadian rhythms, Response to toline DNA 13.in L. Falquet al., Nucleic Acids Res. 30, 235 (2002). 2. M. Koornneef, C. J. Hanhart, F. Thiel, J. Hered. 80,measure breakdown—and other Europe andpotentially other parts ofII functionthe world areincharwith adamage… stable integration of approximately 200 developed tolitter theet various steps of Pol transcription a We used a cell 5 July 2004; accepted 3 September 2004 14. P. A. Rea et al., in ABC Transporters from Bacteria to 118 (1989). 17, eachto al measures such today as whole-ecosystem metaboacterized by highly industrialized, inten- repeats of ells some instances, dynamical pro use cassette molecular networks a gene at asignaling single locus containing 256 living cell. For instance, although abortive initiation is widely believed 18 lism, nutrient spiraling, or primary production sively managed agriculture the large-scale sense,repeats interpret,andanda respond stimuli. cillation to occur, the dynamics of this event are unknown, and including whether upstream lacO minimal to cytomegalovirus (CMV)frequency or signal d (26–28)—can used to complement, not reapplication of fertilizers. This, in combination with shown toaalter gene expression (1 Recent advances in time-lapse microscopy initiating polymerases are be committed to entering processive elongapromoter coupled to a tetracycline-operator cassette controlling sized free I0B" binds to nuclear NF-0B, place, established procedures to assess stream leading to export of the complex to the other sources suchDNA, as atmospheric de- gene or intoitscontrol cellular different have many mRNA signaling Oscillations in NF-.B Signaling tion or whether they maynutrient dissociate from the and the(10). probthatrevealed encodes athat functional with molecules 24 MS2 repeats 3¢ cytoplasm This complex, but not free ecosystem health.has This highlights the need fornutrient 16,19 Jeremy Purvis, Kyle W. Karhohs, Mock, detect Eric Batchelor,* position, resulted in widespread point to a ric show complex dynamical (1–13). ability of each event. Furthermore, no assay exists that can measure untranslated region (Fig.Caroline 1).behaviors We could theInlocusThese using examples the I0B", is the targetpolfor E. I0B" phosphorylation Control the Dynamics differential diagnoses of in environmental assessby IKK (11, Alexander 12). Loewer,† Galit Lahav‡ elongation speed template within a live cell. Accurate lactose repressor fused to red (Fig. 1b,e,h,k) or cyan (Fig. 1n,r,v) Oscillations in the temporal response of ment, on as ais chromatin standard practice in medicine. Impor- p53 Dynamics Control Cell Fate WHY C A R T Ip53 C L EDynamics S Control Cell Fate Gene tantly, Expression NF-0B activity litter breakdown and some other functionally have been observed by 15shift JUNE 2012 onlyVOL 336 SCIENCE www.sciencemag.org electromobility assay (EMSA) in Cells through molecular signals that often show complex dynamical D. E. Nelson, A. E. C. Ihekwaba, M. Elliott, R. Johnson, based methods canJ. be implemented at relatively studies of I0B$ andtransmit ( knockoutinformation mouse em1 1Department of1 Anatomy and 1 Structural 1 1 Einstein College of Medicine, Bronx, New York 10461, USA. 2Laboratoire de Ge ńe´tique ćulaire, Biology, Albert Centre on the stimulus; C. A. Gibney, B. E. Foreman,little G. Nelson, V. See, input C. A. Horton, bryonic fibroblast cell populations and have patterns. The dynamic behavior of the tumor suppressor p53Mole varies depending cost or resource (29)5inEcole order to assess 3The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan 1 1 4 UMR-8541, ´ National de la Recherche Scientifique, Normale Supe rieure, 75005 Paris, France. been simulated in a computational model D. G. Spiller, S. W. Edwards, H. P. McDowell, J. F. Unitt, in response to double-strand DNA breaks, it shows a series of repeated pulses. Using a effects pollution and other3 ecosystem impacts 4Integrative 6 7 Ganof 7 Israel. (13).Inc., In theLos absence time-lapse 94024, single-cellUSA. Correspondence should be addressed to R.H.S. University, Ramat 52900, Bioinformatics, Altos,ofCalifornia E. Sullivan, R. Grimley, N. Benson, D. Broomhead, computational model, we identified a sequence of precisely timed drug additions that alter p53 2 1 analysis, it has remained unclear whether that are of concern to environmental managers ([email protected]). D. B. Kell, M. R. H. White * asynchronouspulses single-cell oscillations occur a sustained p53 response. This leads to the expression of a different to instead produce and stakeholders. Impossibile visualizzare l'immagine. La memoria del computer potrebbe essere insufficiente per aprire l'immagine oppure l'immagine in 2007; single cells following NF-0B stimulation Received 18 April; accepted June; published doi:10.1038/nsmb1280 potrebbeby essere danneggiata. Riavviare il computer e aprire di nuovo il file. Se viene 28 visualizzata di nuovo la x rossa,involves potrebbeonline essere Signaling the transcription factor nuclear factor kappa B (NF-.B) its 5 August 1,2, Yaron 1,3, Valeria 1, Yehuda 3, Shailesh 1, necessario eliminare l'immagine e inserirla di nuovo. of downstream genes de and also alters cell Cells Mthat experience p53 pulses recover from Xavier Shav-Tal(15), Turris Brodyfate: Shenoy is accelerating en(8, Darzacq 14). Likeset calcium signaling NF-0B release from inhibitor kappa B (I.B) inIncreasing the cytosol, human followed pressure by translocation 4 1 & Robert H Singer Robert D Phair could be a complex dynamic oscillator using DNA damage, whereas cells exposed to sustained p53 signaling frequently undergo senescence. into the nucleus. NF-.B regulation of I.B! transcription a delayed vironmental changerepresents throughout the world, threatperiod and/or amplitude to regulate transcripnegative feedback loop that drives oscillations in NF-.B translocation. SingleOur results show that protein dynamics can be an important part of a signal, directly ening water security for humans andWeaquatic tion of transcription target genes. in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic cell time-lapse imaging and computational modeling of NF-.B (RelA) imaged 796 VOLUME 14 influencing NUMBER 9cellular SEPTEMBER 2007 NATURE STRUCTURAL & MOLECULAR BIOLOGY fate decisions. We have used fluorescence imaging biodiversity (2). Large stretches of the landscape localization showed asynchronous oscillations following cell stimulation that measurements of promoter binding, initiation andofelongation. Photobleaching of fluorescent RNA polymerase II revealed several NF-0B (RelA) and I0B" fluorescent fusion decreased in frequency with increased I.B! transcription. Transcription of kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent in Europe and other parts of the world areproteins char(11, 16) to study oscillations in target genes depended on oscillation persistence, involving cycles of RelA MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model RelA N-C localizationells (N-Cuse oscillations) in signaling networks to acterized today byconsequences highly industrialized, intensome min instances, dynamical properties such as osmolecular –1, much faster phosphorylation and dephosphorylation. The functional of NFthat fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases than HeLa (human cervical carcinoma) cells and Davide Mazza Raffaele Institute .B signaling may thus depend on number, period, and amplitude of oscillations. sively managed agriculture and the large-scale cillation frequency or signal duration, have been respond to previously documented, andsense, entered ainterpret, paused stateand for unexpectedly longstimuli. times.San Transcription onsetScientific was inefficient, with onlyCentro 4 SK-N-AS cells Ehuman S-type neuroblasto1% of with polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both expression polymerase and Di imaging Sperimentale [email protected] application of fertilizers. This, in combination shown to alter gene (1, 3, 6, 8, 11, 13–16) Recent advances in time-lapse microscopy ma cells that have been associated with mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of cytoplasm of unstimulated cells by binding NF-0B is a family of dimeric transcription deregulated NF-0B signaling (17)^. In SKother nutrient sources such as atmospheric de- processes have inrevealed that many signaling molecules or to control cellular differentiation (7, 12, 17). vivo. N-AS cells expressing RelA fused at the C to I0B proteins. NF-0B–activating stimulitranscriptional factors (usually RelA/p65:p50) that regulates 1 14402 1 1 In vivo dynamics of RNA polymerase II transcription C position, hasthe resulted widespread nutrientterminus pol- toshow complex dynamical activate inhibitorin kappa B kinase (IKK) cell division, apoptosis, and inflammation the red fluorescent protein behaviors (1–13). In These examples point to a rich mode of regula- WHAT INSTRUMENT TO USE? OPTICAL MICROSCOPY looks like a good candidate. Cells are transparent to visible light. Relatively low phototoxicity (but beware of near UV radiation). With sufficient resolution (~200nm) to visualize intracellular compartments What kind of microscopy? WIDEFIELD ILLUMINATION Sensitive Fast Out of focus blur 5 TIRF ILLUMINATION Sensitive No out of focus blur Limited to surface Davide Mazza [email protected] LASER SCANNING (e.g. CONFOCAL) No out of focus blur Not that fast Not that sensitive San Raffaele Scientific Institute Centro Di imaging Sperimentale The missing link. Light microscopy Viability Electron microscopy © T. Hatano, NIG, Japan Immunofluorescence © CBIM, Imperial College, UK © M.Neguembor, HSR. Specificity 6 Resolution Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale GFP: bridging viability and specificity Viability Specificity Resolution Von Dassow, 2009 GFP-tagged Histone H2B in live zebrafish embryo 7 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Some dynamics can be measured directly Example: massive translocations between different compartments NF-kB translocation (Sung and Agresti, 2009) 8 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Mobility of dispersed particles Tracked particles one image every 5 sec! whole movie ~ 500s! Linear fit of mean square displacement D = 0.0096 µm2/s A0 =0.239 4.5 4 3.5 MSD [µm] 3 2.5 2 1.5 1 0.5 Aggregates of Ab heavy chain. (w/ M. Mossuto and R. Sitia, HSR) 9 0 0 Davide Mazza [email protected] 20 40 60 Time[s] 80 100 San Raffaele Scientific Institute Centro Di imaging Sperimentale Equilibrium: a molecular Where’s Waldo It can be difficult to understand what individual molecules do, even when selectively labelling your protein of interest. Tumor suppressor p53-GFP in live fibroblasts (30 min after dex stimuli) 10 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Binding to ~immobile scaffolds is a widespread event in the cells chromatin cytoskeleton In the nucleus, these interactions regulate transcription, translation and DNA repair. B 2 J/m2 UV p53-Venus (AU) 800 Interfering with protein 400 dynamics cellular fate. 0 mulus-dependent dynamics of p53 in single cells allows interfering with Batchelor et al 0 5 10 15 20 5 10 15 20 Time (h) p53-Venus (AU) 400 ng/ml NCS 800 400 C p53-Venus (AU) 0 5 10 15 200 50 10 5 15 10 20 15 200 5 10 15 20 TimeTime (h) (h) Time (h) Time (h) F First pulse 800 400 400 ng/ml NCS 100 ng/ml NCS 200 ng/ml NCS 400 ng/ml NCS 0 0 2009) 5 10 (Batchelor 0 5et al.,Nat. 10 15Cancer 20 Rev, Time (h) 15 20 Time (h) 0 5 10 15 20 Time (h) 0 5 10 Tim B 8 J/m2 UV p53-Venus (AU) D 100 ng/ml NCS p53-Venus (AU) A 800 400 0 0 5 010 515102015 20 Time (h) hours Time after irradiation (h) 800 400 0 0 5 10 15 200 50 10 5 15 10 (h) TimeTim (h hours Time after irradiation D 2 4 800 6 CELL DEATH 400 8 (Batchelor et al., Mol Sys Biol, 2011) 10 02012) (Purvis et al., Science, 0 5 10 15 0 5 10 15 20 1.4 Time (h Time (h) f p53-Venus (AU) Time (h) 0 5 10 15 20 Time (h) 2.2 DNA DAMAGE 1.8 REPAIR p53-Venus (AU) 100 ng/ml NCS The toolbox of cellular dynamicists* Photoperturbation microscopy Correlation microscopy Single Molecule Imaging * http://en.wiktionary.org/wiki/dynamicist 13 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Photo-perturbation techniques Modify the fluorescence properties of a subpopulation of molecules by using a pulse of intense light. Fluorescence Perturbation techniques (FPT) Photobleaching Techniques Photoactivation Techniques i-FRAP Inverse FRAP FRAP Fluorescence Recovery after Photobleaching FLIP Fluorescence Loss Into Photobleaching San Raffaele Scientific Institute Centro Di imaging Sperimentale Instrumentation for FRAP A Widefield microscope + CCD + High intensity source (laser) A Confocal Laser Scanning Microscope (CLSM) OR with Acusto-optic or Electro-optic modulator devices 15 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Instrumentation for FRAP A Widefield microscope + Fast image collection system + High intensity source (laser) A Confocal Laser Scanning Microscope (CLSM) OR with Acusto-optic or Electro-optic modulator devices 16 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Fluorescence recovery after photobleaching Fluorescence Recovery Normalized Intensity After Photobleaching (FRAP) p53 dynamics in living cells (whole movie 30 s) Time 17 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Qualitative analysis of FRAP data F(t) Immobile Fraction FASTER PROTEIN ER W O SL EIN T O PR t 50 > t 50 > t 50 18 Mobile Fraction t Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Can we be more quantitative? YES BUT WE NEED SOME MATH Free diffusion Axelrod, D et al.(1976), Biophys J 16, 1055--1069. Soumpasis, D. M. (1983), Biophys J 41, 95--97. Anomalous sub-diffusion Saxton, M. J. (2001), Biophys J 81, 2226--2240. Diffusion in heterogeneous systems Siggia, E. D. et al. (2000), Biophys J 79(4), 1761--1770. Diffusion and binding problems (hit and run) Sprague, B. L.; et al. (2004) Biophys J, 86, 3473--3495. Mueller et al. , B. L. et al. (2008), Biophys J. 19 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Selecting a model for the FRAP experiments Equations deduced from the choices made !! ! ∗ = !!! !−!!"!+!!""! !" ! !! = +!!∗!"!−!!""!!! ! ! ! ! ! !" With proper initial and boundary conditions lead to model for experimental data 20 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale What model to be chosen? Different models can fit the same FRAP curve equally well Normaized Intensity 1.0 0.9 Different in-vivo FRAP studies result in different binding estimates (and different biological interpretations) One binding state + diffusion 0.8 0.7 0.6 0.5 residence time = 3.7 s 0.4 0 Normaized Intensity 1.0 2 4 6 Time [s] 8 10 12 Two binding states (no diffusion) 0.9 TF # of binding states Residence time Bound fraction GR1 1 (non-specific) 13 ms 85 % Max2 2 (non-specific / specific) 6 s / 14 s 95 % p533 1 (non specific) 2.5 s 43 % AR4 1 (specific) 90 s 20 % 0.8 Many of these differences are not biological but due to different protocols/models used to analyze FRAP data 0.7 0.6 1st residence time = 0.26 s 2nd residence time = 4.6 s 0.5 1 Sprague et al., Biophys J, 2004 3 Hinow et al., Biophys J, 2006 0.4 0 2 4 6 Time [s] 8 10 12 2 Phair et al., Mol Cell Biol 2004 4 Farla et al., J Cell Sci, 2005 “With four parameters I can fit an elephant, and with five I can make him wiggle his trunk.” J. von Neumann O. Levenspiel, Chemical Innovation, 2000 22 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale And this is only one of the problems of photo-perturbation techniques 1. Bulk Experiments. Only information on the average behavior Rare events are lost Needs modelling 2. Requires “high” levels of tagged protein (µM concentration range) Potential problems with overexpression. 3. Photoperturbation. Potential photodamage of the cells. 23 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Also FRAP is a local measurement Can answer “how fast do protein move” at position x,y,z. Won’t tell you how fast protein moves from A to B. 24 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Expanding the scope of FRAP Fluorescence loss into photobleaching (FLIP) Fluorescence Perturbation techniques (FPT) Photobleaching Techniques Photoactivation Techniques i-FRAP Inverse FRAP FRAP Fluorescence Recovery after Photobleaching 25 FLIP Fluorescence Loss Into Photobleaching Normalized intensity time Davide Mazza [email protected] Time San Raffaele Scientific Institute Centro Di imaging Sperimentale FLIP Example: Search of p53 for its targets FLIP detects a slow-down in the p53 search upon DNA damage 1 10 µ m Normalized Intensity 0.9 0.8 0.7 0.6 0.5 0.4 Ctrl 10 Gy IR 0.3 0.2 (P. Rainone) 0 20 40 Time [s] 60 80 And this is only one of the problems of photo-perturbation techniques 1. Bulk Experiments. Only information on the average behavior Rare events are lost Needs modelling 2. Requires “high” levels of tagged protein (µM concentration range) Potential problems with overexpression. 3. High doses of irradiation during photobleaching. Potential photodamage of the cells. 4. Local measurement. 27 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Photoactivation/Photoconversion/ Photoswitching Different mechanisms for “turning on” FPs fluorophores 28 The first engineered photoactivatable protein (PA-GFP, Patterson, 2002) shows change in abs spectrum upon activation Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Little summary Dendra2 photoactivation in HEPg2 cells Fluorescence Perturbation techniques (FPT) Photobleaching Techniques Photoactivation Techniques i-FRAP Inverse FRAP with Dr. M. Crippa 29 FRAP Fluorescence Recovery after Photobleaching Davide Mazza [email protected] FLIP Fluorescence Loss Into Photobleaching San Raffaele Scientific Institute Centro Di imaging Sperimentale And this is only one of the problems of photo-perturbation techniques 1. Bulk Experiments. Only information on the average behavior Rare events are lost Needs modelling 2. Requires “high” levels of tagged protein (µM concentration range) Potential problems with overexpression. 30 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale SOMETIMES LESS IT’S BETTER DECREASING CONCENTRATION OF FLUORESCENT PROBE FRAP FLIP Fluorescence fluctuation techniques Probe concentration µM µM nM pM to nM Photobleaching Yes Yes No No 1 µm >1 µm 0.2 µm 0.02 µm Local measurement Yes No Yes/no No Bulk technique Yes Yes Yes No Resolution 31 Davide Mazza [email protected] Single molecule San Raffaele Scientific Institute Centro Di imaging Sperimentale Fluorescence fluctuation spectroscopy (FCS) Slower protein Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Getting non-local information from fluctuation measurements Correlating fluorescence fluctuations between different pixels in time. STICS: Spatiotemporal image correlation spectroscopy (Hebert et al., Biophys J, 2005) RICS: Raster Image Correlation Spectroscopy (Digman et al., Biophys J, 2005) Pair correlation spectroscopy (Digman and Gratton, Biophys J, 2009) (credit: M. Digman) 33 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Modelling of FCS faces the same problems of FRAP Temporal Image correlation spectroscopy of p53 in living cells 34 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale Modelling of FCS faces the same problems of FRAP 35 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale SOMETIMES LESS IT’S BETTER DECREASING CONCENTRATION OF FLUORESCENT PROBE FRAP FLIP Fluorescence fluctuation techniques Probe concentration µM µM nM pM to nM Photobleaching Yes Yes No No 1 µm >1 µm 0.2 µm 0.02 µm Local measurement Yes No Yes No Bulk technique Yes Yes Yes No Resolution 36 Davide Mazza [email protected] Single molecule San Raffaele Scientific Institute Centro Di imaging Sperimentale A nice add-on Advantage: if sample sparse – we can localize single molecules with precision higher than the resolution limit. Localization precision depends on the number of photons N detected over background σ ~ 1/√N Reduction of out of focus signal WIDEFIELD ILLUMINATION INCLINED (HILO) ILLUMINATION TIRF ILLUMINATION Images of 100nm fluorescent beads in aqueous solution - Frame rate 100fps Tokunaga et al., Nat. Methods 2007 Single plane illumination for SMT Supplementary 1 Gebhardt etFigure al. Nat. a Meth 2013 b C1 M3 2=;3%/>329; ;0%808 D3 @A310$;3<A1 3;;?&3%9134%$ 456021370 $&3>>4> 89&:;0$ 819<0 /0102134%$ 456021370 D1 !"#$%& !''$( #)!$%& #*"$%& L1 D2 D4 L2 T1 T2 T3 c M5 M1 +,--. M2 ! Setup of the reflected light sheet microscope. (a) Scheme of the setup. Lasers are collimated (telescope T1 – T3) and aligned (mirrors M1 and M2 and dichroic beamsplitters D1 and D2). A cylindrical lens telescope and cylindrical lens C1 create an expanded and collimated line that overfills the back aperture of the illumination objective. After the illumination objective, laser beams are reflected off an AFM cantilever and focused to a diffraction limited sheet. Alternatively, laser beams are focused by L1 and reflected by dichroic D4 for wide field illumination. Fluorescent light is collected by the imaging objective Davide Mazza San Raffaele Scientific Institute 40 and focused by L2 onto an electron-multiplying CCD (EMCCD) chip. (b) Photograph of the illumination Centro Di imaging Sperimentale [email protected] objective. A home built xyz-device is clamped to the objective and holds the AFM cantilever. (c) Close up of the illumination objective and the metal device that holds the AFM cantilever. (d) bright field image Intranuclear single molecule tracking. I - Bright and photostablefluorescent label. II - Possibility of tuning concentration of label without changing the actual concentration of the TF à FRAP, FCS and SMT at the same expression levels. Post-translatonal labeling system (HaloTag) SMT of tumor suppressor p53 p53 show a saltatory motion, alternating between a free and a bound state p53-wt in living H1299 cell nucleus (25fps – shown15fps– FOV 12x12 mm) Automatic tracking (Grier and Crocker algorithm) – hand checking of detected tracks Kymograph analysis of binding events Bright field + fluo time SMT Movie 25 fps – displayed RT Kymograph analysis of binding events Bright field + fluo Kymograph (bkg subtracted) time SMT Movie 25 fps – displayed RT Binding properties of p53 to chromatin Distribution of residence times for bound p53 •  Distributed exponentially (for > 95%). •  Bound fraction ~ 20% à Mostly free. •  Average is ~2s à Transient binding. Bound fraction 18% ± 3% Average residence time 1.7 ± 0.2 s if you really like modelling… Time [s] Cross validation with ensemble techniques Displacements [µm] Bound fraction 22% ± 5% Average residence time 1.8 ± 0.2 s (Gebhardt J.C.M. et al., Nat. Meth., 2013) (Mazza D. et al., Nat Meth., 2013) p53 binding dynamics are modulated over time upon IR Average residence time on chromatin [s] 10 Gy IR 0hrs 1.5hrs 2.5hrs 6! 5! 4! 3! 10 Gy IR! 2! 1! 0! 1! 2! 3! 4! Time after irradiation [hrs]! 5! 4.5hrs time Upon IR p53 binding shows “oscillatory” dynamics UV result in modulation of p53 binding with a distinct temporal profile Average residence time on chromatin [s] 8 J/m2 UV 0hrs 1.5hrs 2.5hrs 6! 5! 4! 3! 10 Gy IR! 2! 8J UV! 1! 0! 1! 2! 3! 4! Time after irradiation [hrs]! 5! 4.5hrs time Upon UV p53 binding shows a sustained increase formed in a custom-made cuvette [19] (Hellma) by a plan apochromat illumination objective (10x, NA 0.28, Plan Apo, Mitutoyo) as depicted in Fig. 1. Work in progress PolII-GFP p53-HaloTag With H. Mueller and J.G. McNally (submitted) acquisition rate 5 fps – displayed 15 fps Fig. 1. (a) Schematic representation of the instrument. Illumination light was focused into the sample chamber by a 10x apochromat objective to form a thin light sheet in the focal plane (z0). The sample could be positioned coarsely by a 3-axes translation stage (X, Y, Z) and more accurately by an additional z piezo stage (Zp). Fluorescence was collected by a high NA 40x water-immersion objective, cleared up by notch and bandpass filters (NF, BF) and detected with a camera (EMCCD). A cylindrical lens (C) was used to shape the PSF for 3D localization. Fast image analysis was used to determine this information in real-time and feed it back to the z piezo stage in an active feedback loop to keep a particle near the focal plane. (b) Detailed view of the specimen within the sample chamber. Fluorescent particles located below (1) or above (3) the focal plane resulted in an elliptical PSF as observed on the camera chip (z0’). Focusing the excitation light with an air objective through the 2 mm glass wall (BK7) of the sample cuvette into aqueous solutions introduced spherical aberration, which resulted in focal shifts when moving the sample chamber in x-direction. These shifts could automatically be compensated by moving the objective with a motorized translation stage coupled to the xaxis of the sample stage. Chromatic aberrations in the illumination path caused by the refractive index mismatch between air, glass wall and aqueous medium were well below the Rayleigh limit for all light sheet configurations. The sample cuvette was placed in a custommade holder making it accessible for e.g. a micro-injection device. It was magnetically attached to a motorized 3-axes stage (3x M-112.12S, Physik Instrumente) equipped with an additional closed loop piezo z-stage for fast and accurate positioning perpendicular to the image plane (P-611.ZS, Physik Instrumente). Fluorescence was collected through the coverslip bottom of the sample cuvette (0.17 mm) by a long working distance water immersion apochromat objective (CFI Apo LWD Lambda S 40x NA 1.15, wd 0.60 mm, Nikon). Scattered excitation light entering the detection pupil was rejected by placing appropriate notch filters (NF01-[488, 532 and 633]U-23.7-D, Semrock) and, if necessary, additional band pass emission filters into one of the filter turrets. For (Spille et al. Opt Expr. 2012) fies the constant focus step $z between planes. Scale bars, 1 Mm. BRIEF (~67%) of this type of grat- shift is introduced by aCOMMUNICATIONS carefully calculated geometri tically to ~93%) by using a tion of the MFG pattern (Fig. 1d) and is dependent on us e image in the N × N arr Objective Tube lens MFG CCG prism f an MFM amicroscope with f order so that each duplicate ifferent MFG to image the a focus shift $z × (mx + N × my) (Fig. 1c). The ma y Primary Fourier Sample image plane MFG distortion determines the step size $z. To obtaining 5 plane × 5 = 25 planes, the lay Final ens, Excitation lasers image . (488 and 560 nm) mm) Glane)is 14 14 sat it d 13 CCG Prism f b 12 ne 13 11 cona b c f109 12 r 8 deep f 238 nm 7 PSF MFG –4∆z –3∆z –2∆z 11 parate bjece focus –∆z z = 0 +∆z –2 –1 0 1 2 ch it x (m) Time (s) T +2∆z +3∆z +4∆z 7 plane I 2.5 rise 6 MFG. 5 660 nm 2.0 Primary 4 ion, 1.5 image 3 Fourier orrect 1.0 2 plane –2 –1 0 1 2 void Final image 1 z (m) 0.5 ays of g h Meth, 2013) 0 d (Abrahamsson et al., Nat sine he MFG. z 2 e hase T 1Time (s) 1 2 0 0 40 0 40 0 20 0 1, 0 20 0 00 0 1, 80 60 0 Distance (µm) 20 0 y (µm) 0 1, 00 1, 0 20 0 0 Amplitude (a.u.) 00 0 20 0 max ion (m) 4 z (µm) min 80 0 40 20 0 min .u.) 1 60 x (µm) max 0 Amplitude (a.u.) Camera 2 Viability Specificity Resolution Experimental Imaging Center San Raffaele Research Institute NOWMilan HIRING. - Italy Marco E. Bianchi Alessandra Agresti Samuel Zambrano Giovanni Pietrogrande Advance Microscopy and Nanoscopy Unit Carlo Tacchetti Teresa Leva Paolo Rainone How does a single molecule look like How do we recognize a single molecule? Single molecules bleach suddenly. Individual GFP molecules spattered on a coverslip time The payoff is high! (2) tumor suppressor p53 Histone H2B Single cell analysis of: - Dynamic behavior of molecules - Interactions. - Oligomerization. - Number of molecules. Mazza et al, Nucl Ac Res, 2012 w/ Bianchi ME, Chromatin Dynamics unit Acquisition speed up to 100 images/s (3x faster than video rate). Epi, TIRF and inclined illumination. Localize individual molecules with high precision: 20 nm (10x higher than resolution limit) SMT of a stably bound protein Histone H2B is tightly bound to chromatin H2B displacements as function of time Movie collected 25 fps (displayed 15 fps) Field of view 13 x 13 mm2 Bound Fraction: 98% SMT of tumor suppressor p53 p53 show a saltatory motion, alternating between a free and a bound state p53-wt in living H1299 cell nucleus (25fps – shown15fps– FOV 12x12 mm) Automatic tracking (Grier and Crocker algorithm) – hand checking of detected tracks Binding properties of p53 to chromatin Distribution of residence times for bound p53 •  Distributed exponentially (for > 95%). •  Bound fraction ~ 20% à Mostly free. •  Average is ~2s à Transient binding. Bound fraction 18% ± 3% Average residence time 1.7 ± 0.2 s Example: transcription factor dynamics during cellular reprogramming Scientific Report 2011 Overexpression of three different transcription factors (MASH1, LMX1a, NURR1) induces direct reprogramming of Fibroblasts into Dopaminergic neurons. 62 ffaele Scientific Institute What does it happen to the transcription factors during the reprogramming? Davide Mazza [email protected] Scientific Report 2011 (Vania Broccoli) San Raffaele Scientific Institute Centro Di imaging Sperimentale Example: transcription factor dynamics during cellular reprogramming When all three factors are expressed, the dynamics of each of them are slower than when only one of them is expressed Normalized Intensity 1.0 0.8 0.6 0.4 1_day_solo 1_day_all 0.2 50 Mash - Only 40 Mash - All 30 20 10 0 Lmx1a dynamics Time to reach 80% recovery [s] Time to reach 80% recovery [s] Mash1 dynamics 14 12 10 8 6 4 2 0 Lmx1a - Only Lmx 1a - All 0.0 0 10 20 30 40 50 Time [s] (T. Leva and M. Perino) 63 Davide Mazza [email protected] San Raffaele Scientific Institute Centro Di imaging Sperimentale GFP: bridging viability and specificity Isolated from Aqueorea Victoria (Shimamura, 1960) 64 Applied as genetically encoded marker (Prasher and Chalfie, early 90’s) Davide Mazza [email protected] Improved and modified (Tsien et al., ongoing) San Raffaele Scientific Institute Centro Di imaging Sperimentale