Cellular Communication Kamil Růžička FGP CEITEC MU Genomics Lectures How does fluorescence work? http://www.physics.emory.edu/%7Eweeks/confocal/fluorescence3.gif How does a fluorescence microscope work? How does a confocal microscope work? http://www.physics.emory.edu/%7Eweeks/confocal/confocalsketch2.gif Protein localization live imaging Martin Chalfie Martin Chalfie GFP discovery - Nobel Prize 2008 Roger Tsien Osamu Shimomura GFP fusions your gene promoter here can be GFP here can be GFP terminator your gene promoter terminator your gene promoter 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 Fluorescent proteins on the market http://home.medewerker.uva.nl/m.a.hink/bestanden/fps.gif Excitation and emission https://encrypted-tbn2.gstatic.com/images?q=tbn:ANd9GcQi7YvHd1p3-d9IHlYTh_Ocp01sgHGVqBXNaypdiM1XHPh atyOz http://www.biotek.com/assets/tech_resources/54/600fig22.jpg http://www.lamondlab.com/images/gfpspectra.jpg Multicolored fluorescent protein (neurones) brainbow1 Transport among compartments Alberts et al. 2008 Also RNA can be differentially localized An external file that holds a picture, illustration, etc. Object name is nihms174811f1.jpg Object name is nihms174811f1.jpg RNA ZIP codes for localization Mikko Frilander ZIP codes often motor protein bound Mikko Frilander Delivering at the correct address 1. Localisation complex assembly starts already in the nucleus 2. Cytoplasmic mRNP is "matured“, nuclear export proteins removed, additional proteins attached. 3. mRNP is associated with motor and transport system and delivered to the destination 4. Delivered mRNP is anchored to the destination (for storage) or translated directly. Mikko Frilander http://www.bioc.rice.edu/bios576/gene_exp/Lane1_files/image004.jpg Localization of mRNA RNA hybridization in situ http://www.bioc.rice.edu/bios576/gene_exp/Lane1_files/image002.jpg Localization of mRNA RNA hybridization in situ •classical technique, no alternative in developmental biology •results often clear •can be done without generating transgenic lines • •tedious •only on “dead” samples • λN22 system nuclear localization signal promoter viral RNA binding protein Also mRNA can be differentially localized Ash1 mRNA localized to the tip of the daughter cell Also mRNA can be differentially localized Also mRNA can be differentially localized Protein localization immunolocalization - fluorescently http://siren.punkt-international.eu/uploads/pics/triple_immunolocalization.jpg fluorescent dye attached primary antibodies secondary antibodies Protein localization immunolocalization - immunogold http://www.lifesci.sussex.ac.uk/home/Julian_Thorpe/immuno.jpg Immunogold quantification of amino acids and proteins in complex subcellular compartments electron microscope Transport among compartments Alberts et al. 2008 Protein sorting – target peptides Nuclear transport nucleoporins Nuclear import Nuclear import Mitochondrial transport TIM and TOM complexes decide at which side of mitochondria the protein will be transported. Advanced confocal techniques •FRAP •photoactivatable FP •FCS FRAP region of interest Fluorescence Recovery After Photobleaching FRAP FRAP – bleaching curve iFRAP inverse FRAP iFRAP – dissociation of premRNA from specles FRAP - advantages •not only proteins (also other dyes) • • •your cells are moving •high energy needed to bleach the ROI –can damage your material –long time needed to bleach •usually only one ROI can be observed – time consuming – FRAP – disadvantages FRAP derivatives FLIP Fluorescence Loss After Photobleaching •bleaching process is repeated during the experiment •for studying general protein turnovers in compartments •less often used continuous bleaching here FRAP derivatives FLAP Fluorescence Localization after Photobleaching •two fluorochromes on one protein– one bleached, non bleached as control • Intermezzo: story from a conference •even top scientists can be wrong Photoactivable proteins photoactivation (UV) non activated activated overlay Photoactivable proteins Photoactivatable fluorescent proteins Dronpa, Kaede, Eos – probably most popular Photoactivable proteins Advantages: -most elegant, most 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 Lifetime sensitive to almost everything: •pH •ionic strength •polarity •other fluorochrome FLIM - applications Protein-protein interactions (FRET-FLIM) (other lecture) FLIM - applications lifetime decreased by site specific IgG injection phosphorylation assay 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 control membrane bound GFP and RFP (crosscorrelation curve) free GFP and RFP FCS control membrane bound GFP and RFP (crosscorrelation curve) channel crosstalk threshold FCS control membrane bound GFP and RFP (crosscorrelation curve) receptor with two labels channel crosstalk threshold FCS receptor with two labels the crosscorelation curve is above threshold -> EGFR protein dimerizes Liu et al., 2007