1Aims/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?• 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 street2BiochemistryMolecular 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 tweezersCourse Organization• Lectures• Readings/Discussions• Minicourse structure• Relevant backgroundBiochemistryGeneticsMolecular Biology3MoleculesMolecular assembliesOrganellesCellsTissuesOrganismsQuestions of scale and methodsof detectionX-Ray DiffractionElectron MicroscopyLight MicroscopyEyeOrganelles4Histochemical Histochemical stain of small intestinal cross section - light microscopystain of small intestinal cross section - light microscopyLumenEpitheliumHigher power light microscopic view of intestinal epitheliumHigher power light microscopic view of intestinal epithelium*5Low power EM of Intestinal EpitheliumLow power EM of Intestinal Epithelium*Scanning EM Transmission EMHigher 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 occludens6Fast 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 HeuserHigher 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 occludens7Intercellular adhesionStaining for cadherinsthat mediate cell-celladhesion*8TEM - osmium tetroxide stainFreeze FractureTEM - metal shadowing~ 2 nm~ 2 nmIMPs - intramembranousparticles - integral membraneproteinsDifferent Sorts of Light Microscopy• Phase contrast• Nomarski• Birefringence• Immunofluorescence• fluorescently tagged antibodies• other tags allow EM visualization9Metaphase ChromosomesNomarskiPhase contrastBirefringence 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).10Resolving 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) Fly Embryo: 3 nuclear antigens stained red, green, or blue Fly Embryo: 3 nuclear antigens stained red, green, or
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