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 immune-localization - fluorescently •ii • ImC-Ff avid ICC 1 1 Deparariiriu.ilion and dehydration j- Fix slid** Xylene Xjtene fcl *»tr i00%«9wwl. 4% PFAfor 10 1 UC* BU-jr-31 tow to emarol Of Methanol lice wW) rw lOmln ^^s^ Of Acetone (ice cold) foi 1Dnw 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 13 ' hr 1 •ti in PBfi 0.2% Tween J rjnwj