Cellular Communication Genomics Lectures 1 How does fluorescence work? 2 How does a fluorescence microscope work? How does a confocal microscope work? 4 Protein localization live imaging 5 GFP and membrane proteins It is good to have GFP tag localized inside the cell Fluorescent proteins on the market iiiiiffiiifffri mmm0333S3333333 Q. 0* o o * -St M -Si 3 2j "Q 3 ^ - » N 3 8 Excitation and emission 9 Transport among compartments Alberts et al. 2008 CYTOSOL I 1 NUCLEUS 1 PEROXISOMES 1 MITOCHONDRIA | PLASTIDS ENDOPLASMIC RETICULUM GOLGI SECRETORY VESICLES LATE ENDOSOME If LYSOSOME 1 EARLY ENDOSOME CELL EXTERIOR : = gated transport = transmembrane transport = vesicular transport Also RNA can be differentially localized RNA ZIP codes for localization 13 ZIP codes often motor protein bound Table l | Cross-species comparison of proteins involved in cytoplasmic mRNA localization Function/feature Zip code binding hnPNP protein (located in nucleus and cytoplasm) Cytoplasmic zip-code-bmding RNP Motor protein for PNP RMP motor adaptor mRNA/RNP anchor Yeast »• 1 '■■■■I .:■.-( lili - i She2 [ASH J, IS Iž?)06164 Mycí \ASH1, ISTZ7f* She3 (ASH1, /S727)6'63 BniV, Bude* [ASH if2 Drosophaa melanogastor Squid [gik^ Vertebrates hnRNP A2 [MBPf1 VghitíPoO- [Vg1)éz Vera/VgRBP- [Vgíf ZBP-1* (B-actin) Staufen (csft pros)65 Swalow' {bcd)tz Ypsilon-SchachteT (os*r)8: Kinesin I (osA)88 Uynein" {bcd),z Dynein lighl chain {bcdf1 Not yet identified Statifen (to}30 Xlsin mRMAs* (Vglf Oikar (as*)" mstaulen- i?)087"0 VI LIP' (irtf TB-RBP (Ca/UWflf/)* Kinesin' \MBP CaMKII)1'- 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 Localization of mRNA RNA hybridization in situ 16 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 17 18 Also mRNA can be differentially localized Ashl mRNA localized to the tip of the daughter cell Also mRNA can be differentially localized 20 Protein localization immunolocalization - fluorescently •ii • ImC-Ff avid ICC 1 1 Deparariiriu.ilion and dahydralion j- Fix slid** Xylene Xjtene fcl *»tr i00%«9wwl. 4% PFAfor 10 1 UC* BU-jr-31 tow to emarol Of Methanol lice wW) Iw lOmln ^^s^ Of Acetone (ice cold) foi IDrrrkv A ii : .;l 11 • 1 . ..: j>r Heal In rjirain bufler oH 5 5 -20 htbi ■«. >i A i Block v-rum c# 9 v i""1 mm t:> ' hr 1 •ti in PBfi 0.2% Tween 4 tm« (o< 5 rrwiuLei I alls A A O.Z% Triton (or ID minutes -4^H. -V-J^B;-. '|rn1 rwcossvy it f«pc rr • acesoiwormslharwl) 1 primary 1 1 antibodies QjgJlj I ■h in P BS 0.2% Twtren 4 tm« for £ mmutw W Jft fluorescent A A dye attached secondary f * f* antibodies j^^^^k^k i 22 Protein localization immunolocalization - immunogold 23 Transport among compartments Alberts et al. 2008 CYTOSOL I 1 NUCLEUS 1 PEROXISOMES 1 MITOCHONDRIA | PLASTIDS ENDOPLASMIC RETICULUM GOLGI SECRETORY VESICLES LATE ENDOSOME If LYSOSOME 1 EARLY ENDOSOME CELL EXTERIOR : = gated transport = transmembrane transport = vesicular transport Protein sorting - target peptides 25 Nuclear transport nucleoporins Nuclear import 27 Nuclear import protein with nuclear cargo delivered to cytosol localization signal (cargo) Ran-GDP DISSOCIATES _ FROM RECEPTORS cargo delivered protein with nuclear to nucleus export signal (cargo) NUCLEAR IMPORT NUCLEAR EXPORT Mitochondrial transport TIM and TOM complexes decide at which side of mitochondria the protein will be transported. TOM COMPLEX receptors SAM COMPLEX translocation channel outer mitochondrial membrane CYTOSOL inner mitochondrial membrane Hsp70 import -ATPase TIM 23 COMPLEX INTERMEMBRANE SPACE A MATRIX SPACE TIM 22 COMPLEX Advanced confocal techniques • FRAP • photoactivatable FP • FCS 30 FRAP Fluorescence Recovery After Photobleaching region of interest IM um 0 Pre-bleach Bleaching ROI Post-bleach_Fluorescence recovery 32 FRAP - bleaching curve 33 34 iFRAP - dissociation of premRNA from species 1 min__2 min__3 min FRAP - advantages not only proteins (also other dyes) FRAP - disadvantages • 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 derivatives FLIP Fluorescence Loss After Photobleaching continuous bleaching here bleaching process is repeated during the experiment for studying general protein turnovers in compartments less often used I J 38 FRAP derivatives FLAP Fluorescence Localization after Photobleaching Intermezzo: story from a conference 40 41 Photoactivable proteins Protein Repeated snort-term tracking Long-term tracking Ces or organelle 7 Dronpa PAmRFPI mEosFP PA-GFP PS-CFP2 Kaede KFP1 Monomer ■ Monomer Monomer Monomer Tel ramer Tetramer Reversible Irreversible Irreversibte Irreversible Irreversible Irreversible Irreversible Fluorescence changes during photoactivation [J ■ [] ■ II I High brightness ■HB BBB ■SB High contrast ✓ ✓ Dual labelling with red and green fluorescent proteins ✓ ✓ Low phototoxicity of the activation light Copyright ©{2005) Nature Publishing Group Nature Reviews | Molecular Cell Biology 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) 44 FLIM Fluorescence Life Time Imaging Microscopy Fluorochromes • excitation spectra • emission spectra • unique lifetime Lifetime sensitive to almost everything: • pH • ionic strength • polarity • other fluorochrome 45 FLIM - applications Protein-protein interactions (FRET-FLIM) (other lecture) s ation Spectroscopy It is counted, how many times the fluorescent molecule comes through the focal plane. Autocorrelation analysis: the way how to discriminate the diffusions speeds of particles. 48 FCS 49 FCS 51