BIOL 331 Midterm: BIOL 331 Midterm 1 Review

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5 Aug 2016
Biology 331: Advanced Cellular Biology
midterm 1 review
Unit 1: Visualizing Cells
Average eukaryotic cell dia. 10-20 µm
Optical microscopy cannot resolve details that are less than the wavelength of visible light (0.4-0.7 µm)
Bacteria and mitochondria are the smallest entities visible by light microscopy (0.5 µm wide)
The objective lens collects a light ray cone to create an image and the condenser lens focuses this cone of light
onto each point of the specimen
Limit of resolution: minimum separation at which two objects appear distinct; depends on wavelength of light and
numerical aperture (NA) (which is the light collecting ability) of the lens used (wavelength on top, NA on bottom)
oLarger NA = better resolution, brighter image, shorter working distance
oBest limit of resolution is about 0.2 µm (shortest visible wavelength, high NA)
Cells are colourless and translucent: enhance contrast by staining so light passing through will undergo phase shift
Phase contrast microscopy: increases the phase shift of light
Differential interference contrast microscopy: uses interference to increase contrast
Dark-field microscopy: light comes in from the side, only scattered light enters the objective. Cells are bright
Electronic imaging systems are used to overcome difficulties in detecting very dim light – very useful in
fluorescence microscopy
Sample preparation: samples must be thin enough for light to pass through
1. Fixed: killed, immobilized, preserved; use formaldehyde to form covalent bonds and cross link proteins
2. Embedded: tissues place in a supporting medium such as wax or plastic resin
3. Sectioned: using a microtome that makes even slices
oHowever, these 3 methods are distorting the cells, live cell imaging is the way to go
Stains: dyes absorb light of certain wavelengths, and also introduce contrast by reducing the amplitude of light
pass through which reduces brightness
Fluorescence microscopy allows for the visualization of specific molecules within cells that are below the
resolution of the microscope because fluorescence produces light
oFluorescent molecules absorb light at one wavelength and emit at a different, longer wavelength; emitted
wavelengths can be viewed through a filter in which the object appears to glow against a dark background
oFluorescent stains are specific for certain cell components; DAPI stains DNA nuclei, or fluorescent molecules
are bound to specific proteins within a cell; FITC-conjugated phalloidin binds to filamentous actin (green)
Immunofluorescence: antiserum containing antibodies are either polyclonal or monoclonal. Antibodies can be
conjugated to enzymes (ELISA), colloidal gold (electron microscopy), fluorescent molecules
oConjugated (labelled) secondary antibodies are used in indirect immunocytochemistry
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oto study protein-protein interactions, use antibodies to label proteins of interest and use a Western Blot to
determine which proteins are associated with it
Light microscopy: light from above and below the plane is observed as out-of-focus blur; fix by confocal (optical
solution) or image deconvolution (computation solution)
oZ stack: series of optical sections taken at different depths in specimen
oZ stack projection: reconstructed image using Z stack images using computational methods to deblur images
Confocal microscopy: uses a laser as a light source and goes through illuminating pinhole. Emitted light from the
specimen is perfectly lined with the pinhole where the laser is coming through, can take crisp images
oUsed in Z stack series and image is projected computationally
oConfocal can penetrate deeper into a specimen than deconvolution can (150 µm vs 40 µm)
Multi-photon imaging: a variation of fluorescence microscopy; uses two or more photons of low energy that are
less than a femtosecond away (red and infrared) and can penetrate deeper (250 µm) by using a longer wavelength
excitation (700 nm and above). Also, less damage to specimen
Green fluorescent protein (GFP): a protein that is natively fluorescent  use to probe protein-protein interactions
oCan be used to report gene expression or serve as a tag for specific cell, tissue, etc.
oSpecific amino acid sequences can be added to GFP that can be targeted to specific subcellular locations
oCan do live-cell image using GFP because any cell can be tagged without fixation or cross-linking methods
and the movement and morphology can be observed
Fluorescence Resonance Energy Transfer (FRET): GFP and GFP spectral variants are used to evaluate protein-
protein interactions; two proteins of interest are tagged with different fluorophores. The emission spectrum of one
must overlap with the excitation spectrum of the other in close proximity (2-5 µm) in order for there to be a
transfer of energy. i.e. excitement in the blue but emission of green indicates protein interaction
Photoactivation: involves inactive photosensitive precursor and using a microscope with a laser or UV light to
control when/where to activate molecule to become fluorescent
oPhoto activation can visualize protein trafficking – proteins diffuse across cell
Fluorescence Recovery After Photobleaching (FRAP): upon exposure to light/laser source, fluorescent markers can
be photobleached to determine migration of flu-labelled molecules
oExample: galactosidly transferase – enzyme that cycles between golgi and ER
The Electron Microscope: can resolve ultrastructure of the cell; resolution is 1 µm (200x better than light)
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Transmission Electron Microscope: uses a vacuum to contain electrons which pass through a hole to create an
electron beam and scatter/pass through when hits dense structures (cells) thus producing an image
oSamples must be thin so electrons can pass through and fixed with glutaraldehyde (cross links proteins) and
osmium tetroxide (binds and stabilizes lipids/proteins). Samples must also be embedded (dehydrated) and
permeated with resin, then sliced and stained with electron dense material to achieve contrast
oCyro-ultramicrotome: flash freeze tissues and section cells then view with TEM to avoid chemical fixation
oImmunogold EM: secondary antibodies attached to small particles of colloidal gold for crisp imaging
o 3D EM reconstruction (tomography): much like CT scan, beams are emitted at various angles
Scanning Electron Microscope: produces images of 3D surface of specimen, electrons do not go through the
specimen; they are always scattered. Specimen must be fixed and coated with heavy metal, resolution = 10 nm
Large macromolecules like DNA or large proteins can be visualized if mixed with an aqueous solution of heavy
metals. Molecules to be visualized must be biochemically purified. The electron dense metal is absorbed by the
background but the molecules of interest, such as protein filaments, are not
Unit 2: Membrane Structure and Membrane Proteins
Membrane Structure
Cell membranes: separate cell from its environment and creates intracellular compartments (euk). All have
common structure of lipids and proteins held together by non-covalent interactions
Membranes are fluid, dynamic structures that are impermeable to most water soluble molecules
oIon gradients across membranes are used to make ATP and drive transmembrane solute movement
oTransmembrane proteins: served as cell surface receptors that transport specific molecules across the
membrane and synthesis ATP. 30% of all proteins in eukaryotic genomes are membrane proteins
Lipid bilayer: lipid molecules spontaneously assemble to form bilayers, all are amphiphilic – have hydrophobic
tails and hydrophilic heads. Animal cell membranes are half lipid/half protein by mass
Phospholipids: are the most abundant membrane lipids with a polar head group and 2 hydrocarbon, nonpolar tails
oOne tail is usually saturated and the other is unsaturated (cis, double bonds, C=C)
oMain phospholipids are the phosphoglycerides (three carbon glycerol backbone)
Phosphatidyl-serine: has overall negative charge which gives membranes a characteristic property
Cholesterol: a lipid with a rigid steroid ring structure that has a polar head group (-OH) and nonpolar hydrocarbon
tail. Only present in eukaryotes and confers steric hindrance in a membrane
Spontaneous assembly of bilayers or micelles is driven by hydrophobicity and hydrophilicity – ability to H-bond is
important and the number of tails determines the shape of the lipid i.e. micelle (1 tail) vs bilayer (2 tails)
oLiposomes are synthetic lipid bilayers which can be used to measure motion of phospholipids via labelling
with a flu-probe or by an electron spin resonance – observe lots of lateral movement and rotation
Fluidity depends on composition and temperature
oPhase transition: when membranes change from liquid to gel state at “freezing point”
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