17.61 Eukaryotic Cell7.61 Eukaryotic CellBiology: Principles andBiology: Principles andPracticePractice20062006R. Hynes and M. KriegerR. Hynes and M. KriegerAims/Focus of the Course• Principles and Approaches of Modern Cell Biology• Molecules Cells Tissues Organisms• How do we know what we think we know?• If we don’t know, how do we find out?• What are the appropriate techniques/approaches?• What are their strengths/limitations?Experimental focus2• genomics  sequence  proteome• what do the proteins do?• how do they know where to go?Cell Biology in ContextHow do cells work?How do they communicate?How does one study them?Cellular organization & polarity are keySo are cell adhesion/migration• development • immunology• neurobiology• pathophysiology (cancer, cardiovascular disease etc.)a two-way streetAll these have cells as their focusBiochemistryMolecular Biology Applied to cell structure & functionGeneticsClassical cell biology – microscopy / EM – both ↑↑.– cell fractionation21stC Confluence of Techniquesin Cell BiologyGenomics / Evolution – in order to interpret the proteome need insight into how proteins function in cellsStructural biology – gives deeper insight into protein anatomyProteomics – mass spectroscopy, 2D gels etc.Reverse genetics – overexpression /mutagenesis – antisense, RNAi – knockouts/knockins etc.– real time imaging– FRET, GFP, optical tweezers3Course Organization• Lectures• Readings/Discussions• Minicourse structure• Relevant backgroundBiochemistryGeneticsMolecular BiologyMoleculesMolecular assembliesOrganellesCellsTissuesOrganismsQuestions of scale and methodsof detection4X-Ray DiffractionElectron MicroscopyLight MicroscopyEyeOrganellesHistochemical Histochemical stain of small intestinal cross section - light microscopystain of small intestinal cross section - light microscopyLumenEpithelium5Higher power light microscopic view of intestinal epitheliumHigher power light microscopic view of intestinal epithelium*Low power EM of Intestinal EpitheliumLow power EM of Intestinal Epithelium*Scanning EM Transmission EM6Higher power EM of Higher power EM of Intestinal EpitheliumIntestinal EpitheliumMvMv==microvillimicrovilli, expand, expand surface area, surface area, transporters transportersTW=terminal webTW=terminal webD=D=desmosomedesmosomeZA=ZA=zona adherenszona adherensZO=ZO=zona occludenszona occludensFast freeze, deep etch, metal coated image of Fast freeze, deep etch, metal coated image of microvilli microvilli and TM,and TM,MvTW2.5 nm actin filamentsJ.J. Heuser Heuser7Higher power EM of Higher power EM of Intestinal EpitheliumIntestinal EpitheliumMvMv==microvillimicrovilli, expand, expand surface area, surface area, transporters transportersTW=terminal webTW=terminal webD=D=desmosomedesmosomeZA=ZA=zona adherenszona adherensZO=ZO=zona occludenszona occludensIntercellular adhesionStaining for cadherins thatmediate cell-cell adhesion8*TEM - osmium tetroxide stainFreeze FractureTEM - metal shadowing~ 2 nm~ 2 nmIMPs - intramembranousparticles - integral membraneproteins9Different Sorts of Light Microscopy• Phase contrast• Nomarski• Birefringence• Immunofluorescence• fluorescently tagged antibodies• other tags allow EM visualizationMetaphase ChromosomesNomarskiPhase contrastBirefringence10 This image of a newt lung cell shows the metaphase cell stained for This image of a newt lung cell shows the metaphase cell stained for centrosomes centrosomes(magenta), microtubules (green), chromosomes (blue) and intermediate filaments (red).(magenta), microtubules (green), chromosomes (blue) and intermediate filaments (red).Resolving power: smallest detail resolved inimaging an ideal specimen, ~0.1 nm for EM,~200 nm (0.2 µm) for LMMinimal resolvable separation of incoherentlyilluminated points: dmin = 0.61!/ nsin("): ! =wavelength, n = refractive index (reason youuse oil), " = aperture angle of lensActual Resolution: detail actually revealed inthe image of a given specimen, requirescontrast, not just resolution -> stains tohighlight or contrast portions of specimen ofinterestStains: metals, fluorophores, reactionproducts (peroxidase, alkaline phosphatase),particle labels (ferritin, gold), shadowing,negative stain, autoradiography, antibodies(immunofluorescence, immunoEM).11Intermediate filaments: Keratin (red) and nuclear Intermediate filaments: Keratin (red) and nuclear lamin lamin (blue) (blue)12Fly Embryo: 3 nuclear antigens stained red, green, or blue Fly Embryo: 3 nuclear antigens stained red, green, or blue How many proteins are there in a cell?13How many proteins are there in a cell?# of genes in genome?25-30,000Amount of protein/cell?~0.8 ngLet’s assume 10,000 expressed in a given cellThat converts to 1010 molecules of 50kDa per cellIf all were equally abundant - 106 (1010/104)of each per cellOf course, they vary widely in size and abundance2D Gel Analysis14Abundant proteins are easy to purify -Actin is 10% of many cells - 10x purification -> purityBUT - most proteins are not as abundantA major cell surface receptor - ~106 molecules per cellor 10-4 of total cellular proteinSo need 104 x purificationAssume 10% yieldSo 1g of cell protein (109 cells = 100 x 100mm dishes)will yield 1g x 10-4 x 0.1 = 10ug1pmol = 50ng of a 50kDa proteinBut what if it were 100-1000 x less abundant?15A minor cell surface receptor ~100-1000 copies per cell -need 107-108 fold purification.Animal tissue often a better source than cellsAlternative approaches:-affinity purificationmass spectrometryclone the gene firstexpression or functional cloninggenomicsthen express the proteinbacteria, yeastanimal cellsanimals (transgenesis)Purification of Membrane Proteins: Purification of Membrane Proteins: immunoaffinity immunoaffinity chromatographychromatographyPeptide-ElutedFractionsCellLysateFlow ThroughWash20011697664531kDamSR-BI-t11234567816Analyses of protein functionBiochemistry - purification and assayExpression - transformation, transgenesisInhibition - genetics (forward and reverse) - drugs, antibodies, - “dominant negatives” - RNAi, antisense - knockouts, mutantsMembranesPlay key roles in cell functioncompartmentalizationtransportintercellular signallingcell adhesionMembrane proteins have special properties