Nodes of regulation in cellular systems cell membrane signal transduction ligands receptors oligomerization transport signal transduction modified protein Golgi transcription factor transport ER transport activation protein posttranslational modification poly-ubiquitination degradation nucleus DNA transcription splicing pre-mrna processing pre-micro-rna mrna micro-rna transport mrna translation micro-rna 5 degradation
Specific cell ablation or cell labeling in transgenic mice loxp loxp Stop DTR (Diphteria toxin receptor) good promoter (e.g. pcaggs) loxp Stop cross-breeding with a cell-type specific Cre strain DTR is expressed only in specific cell types injection of diphteria toxin leads to specific killing of these cells loxp EYFP (enhanced yellow fluorescent protein) good promoter (e.g. pcaggs) > fluorescent labeling of a specific cell type of interest 13
Transfections Usually designates the incorporation of DNA into mammalian cells. DNA present in form of plasmids. Transient Transfection: plasmid remains outside of the genome and is slowly lost (degradation, dilution by cell division), exception: episomal replication e.g. SV40-Plasmids in COS-cells). The transfection efficiency varies but can reach close to 100% Stable Transfection: integration of foreign DNA into the genome (Efficiency: usually below 0.1%). Isolation of stably transfected clones requires selection genes (for antibiotic resistance, e.g. puromycin, G418 ). Plasmids are usually linearized before transfection to increase the possibility of correct integration. 41
Professional Design of sirna or shrna Design via company website http://www.thermoscientificbio.com/design-center/?redirect=true This delivers a list of several possible sequences (gene specific, checked by BLAST) with a score based on empirically determined criteria: Nature Biotechnology 22, 326-330, 2004 Check literature for functional sirna sequences For transduction of primary cells: lentiviral shrna constructs (also work in non dividing cells) there are also inducible lentiviral constructs available (http://tronolab.epfl.ch/) Many vectors can also be obtained from plasmid repositories: Addgene: http://www.addgene.org Belgian repository: http://bccm.belspo.be/db/lmbp_search_form.php 71
Molecular weight assessment after SDS-PAGE http://www.meduniwien.ac.at/user/johannes.schmid/sds-page.xls log[mw] migration distance starting from stacking gel/separation gel interface 80
EMSA s (Electrophoretic mobility shift assays) used to monitor active transcription factors (by binding to short, labeled oligonucleotides comprising the bound DNA sequence) Example: comp.: competitor: non-labeled ds-oligo of the same sequence (usually added in > 10-fold molar excess) competes with the labeled oligo for binding to the TF > reduces the specific signal mut.comp.: mutated competitor: should not compete for specific binding
EMSA Alternative: ABCD Assay (Avidin-Biotin Complex with DNA) TF dsoligo Biotin Streptavidin 87
Microscopy: Human vision and the concept of magnification image formation in the human eye 2-step magnification principle of a microscope with 2 lenses: objective and eye piece (occular) 185
Basics of optical resolution I Fine structures induce a diffraction of light (light of zero-order, 1st order...). Light diffraction on a small iris is more or less equal to diffraction on small cellular structures: sinθ(1) = 1.22(λ/d) θ... angle to the first light minimum λ... wavelength d... diameter of the iris for very small angles θ: θ(1) 1.22(λ/d) objects that are closer than θ(1) cannot be resolved as separate objects 186
Basics of optical resolution II The more orders of light are resolved the better is the resolution. The optical resolution that can be achieved is defined by the so called numerical Aperture (N.A.) of the objective. N.A. = i sin q i... Refraction index of the medium (e.g. 1.0 for air, up to 1.56 for oil) q... half of the objective opening angle (Aperture) 187
Phase contrast Unstained objects such as cells slow down the light (the phase of the passing light) by ¼ λ. Phase contrast rings in the objective can accelerate the light, which does not pass through cells by ¼ λ, the resulting difference of ½ λ causes an interference, which leads to contrast enhancement. ¼ λ ½ λ http://www.microscopyu.com/tutorials/java/phasecontrast/opticaltrain/index.html 194
Fluorescence Filter Cubes The filter cube consists of: 1. Excitation filter: just the correct excitation light (wavelength) passes the filter 2. Dichroic mirror: is reflective for the excitation light but transmittent for the emission light (the emitted fluorescence) separates excitation from fluorescence light sample 3. Emission filter: filters the emitted light so that just the correct wavelength (e.g. in double fluorescence) reaches the detector 207
Protocol of an immunofluorescence staining Fixation: 15 min 4% Paraformaldehyd 3x 5 min mit TBST wash (50mM Tris- HCl ph7.4, 150 mm NaCl, 0.1%Triton) Block: 1 h at RT with 3% BSA in TBS Incubation with 1. Ab: anti-iκb (rabbit polyclonal, sc-371 Santa Cruz) 1:300 in TBS/3% BSA, over night at 4 C (or 1 h at 37 C). 2x 5 min wash with TBST, 1x 5 min with TBS Incubation with Alexa488 goat antirabbit 1:2000 in TBS/BSA: 1 h at 37 C 3x 5 min wash with TBST, 1x 5 min with TBS Mounting 215
Confocal microscopy removes the blur from thicker objects http://zeiss-campus.magnet.fsu.edu/tutorials/opticalsectioning/confocalwidefield/index.html
Optical sectioning and 3Dprojections z-stack Acquisition of a z-stack (image slices along the z- axis) allows reconstruction of a 3D-projection, which can be shown as animation projection 3D rendering 220
Spectral imaging Resolving spectral information on a pixel-by-pixel basis Emission finger printing : emission scan of a microscopy sample ( lambda stack of images) at a given excitation wavelength (e.g. with Zeiss LSM META systems or with Leica confocal microscopes ) Alternative: Excitation scan (at a constant emission wavelength; e.g. using a monochromator light source) Combinations of excitation and emission finger printing (e.g using filter wheels) Increases the number of markers to be measured in parallel Can be used to discriminate fluorophores with overlapping spectra Can be used to discriminate specific fluorescence from autofluorescence Leica concept Zeiss META concept 221
Spectral imaging example I: CFP, GFP and YFP http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html 224
Spectral imaging example II: strongly overlapping dyes SYTOX Green (nucleus), Alexa Fluor 488 conjugated to phalloidin (filamentous actin network), and Oregon Green 514 conjugated to goat anti-mouse primary antibodies (targeting mitochondria). Invitrogen Spectra Viewer http://www.invitrogen.com/site/us/en/ home/products-and- Services/Applications/Cell- Analysis/Labeling- Chemistry/Fluorescence- SpectraViewer.html 225
Separation of specific fluorescence from autofluorescence by spectral imaging 226
FRAP: Fluorescence Recovery After Photobleaching FRAP at the membrane Non linear regression analysis y = span (1-e -kx ) + bottom An image is taken then a region of the cell is bleached by high laser intensity, followed by a time series of images after bleaching. Briefly after bleaching the region is significantly darker and then the fluorescence intensity increases again (fluorescence reoovery) due to diffusion of molecules into the bleached area. The kinetics of recovery depends on the diffusion coefficience; the extent of recovery (the plateau to which the fluorescence recovers) is a measure of the overall mobility (the fraction of mobile molecules versus molecules immobilized, e.g. to the cytoskeleton) FRAP in the cytosol: 236
FLIP: Fluorescence Loss in Photobleaching to determine the dynamic shuttling of molecules between different compartments of the cell A certain compartment A (e.g. the cytosol) 120 is repetitively bleached by the laser and 100 the fluorescence decrease in a different 80 compartment B is monitored by time lapse microscopy. Molecules that shuttle from B 60 to A are bleached in A > thus the 40 compartment B gets dimmer when there is 20 a dynamic distribution of molecules between A and B. 0 cytosol nucleus 0 2 4 6
FLIP to determine a nuclear export signal and a nucleolar localization signal NFκB inducing kinase truncated NIK without the export sequence: nuclear FLIP (bleach in nucleus outside nucleoli) 125 100 nuclear nucleolar 75 50 25 0 0 100 200 300 400 500 600 700 sec
FCS: Fluorescence Correlation Spectroscopy to determine diffusion coefficients and interactions between molecules. The sample is illuminated by the laser at a very small spot, the movements of molecules in this spot (in and out) cause fluorescence fluctuations, which are analyzed by correlation functions
Spectral crosstalk of donor and acceptor ECFP and EYFP-Scans 1.2 relative fluorescence 1.0 0.8 0.6 0.4 raw FRET-channel: Donor Excitation + Acceptor Emission 0.2 0.0 340 360 380 400 420 440 460 480 500 520 540 560 580 600 nm Excitation window of donor Emission window of acceptor Problems: 1. Co-excitation of the acceptor at the Donor-excitation wavelength > Non-FRET-Fluorescence in the raw-fret channel 2. Signal-overlap of donor into the acceptor channel > Non-FRET fluorescence in the raw-fret channel 249
3-Filter FRET Microscopy 3 Images are taken (under constant camera settings): 1. Donor (e.g. CFP-excitation and emission), 2. Acceptor (e.g.yfp-excitation and emission this signal is not affected by FRET 3. FRET-Filter (raw FRET: CFP-excitation and YFP-emission). A normalized FRET signal (image) can be calculated by using correction factors obtained with single stained samples: FRETc = I FRET -corr CFP x I CFP corr YFP x I YFP corr CFP : ca. 0.59 corr YFP : ca. 0.18 CFP / YFP neg. control CFP-YFP pos. control 251
FRET microscopy example corrected FRET = I FRET -corr CFP x I CFP corr YFP x I YFP sample Donor channel Acceptor channel FRET channel corr. factor corrected FRET CFP alone 100 0 60 0.6 0 YFP alone 0 100 20 0.2 0 non-bound 100 100 80 0 CFP + YFP bound CFP-YFP 100 100 160 80 neg. control Donor Acceptor corrected FRET normalized FRET Normalized FRET (normalized to diff. expression levels): = sample 252
FRET Microscopy by acceptor bleaching and monitoring donor recovery (do not use for CFP / YFP) Donor recovery after acceptor bleaching: An image of the donor in the presence of the acceptor is taken, then the acceptor is bleached (partially), followed by acquisition of a second donor image Donor FRET Acceptor Donor Acceptor 255
FRET analysis with self-written ImageJ macro neg. control 14 12 10 8 6 4 2 0 Negative Control sample IKK1+Myc 1 sample IKK2+ Myc 2 sample