BIOL2200- Midterm Exam Guide - Comprehensive Notes for the exam ( 14 pages long!)
UQ
BIOL2200
MIDTERM EXAM
STUDY GUIDE
ROHAN TEASDALE: PROTEIN TRAFFICKING
Microscopy and Visualising Cell Components
- Greater amplification = more signal lost
- The Airy Disk: various points that pass-through objective which make up the image seen = caused by
diffraction/scattering of light when it passes through specimen; if too much overlap of airy disks = blur of
signals
- Resolution: limit up to which two small objects are still seen as separate entities; measure of resolving
power = do
- Prokaryotes = simple structure; eukaryotes = many membrane limited organelles (EM good for this)
Microscopy method
Functions
Light Microscope
Projection of light is captured through refraction
Double light
fluorescence
microscopy
Visualises relative distributions between two proteins; spatial relationship and overlap of
sigals a e see sigals at oloalise
Fluorescence Spectra
- Dyes used absorb light at one set wavelength and emit light at another set of
wavelengths
- max. absorbance = most energetic state = excitation = best absorbance wavelength
- max. emission can also be measured
- stokes shift = difference between peak emission and peak absorption wavelengths;
allows for analysis
- usually max. absorbance > max. emission
--> for this reason special filters are used to restrict bandwidths and differentiate
between the two; also, allow for multiple dyes to be used simultaneously
Live cell imaging
- Live cells stained with fluorescent probes which can bind chemicals/metabolites using
Ca and pH
- he ell ist fied, GFP is used, here fusio protei is ade ad traked/loalised in
a live state
FRET
- multiple GFPs are used to visualise protein-protein interactions which emit light at
specific wavelengths, where one can excite another via energy transfer when they are
sufficiently close
- procedure answers whether proteins are close together in a cell
Confocal Microscopy
-produces in-focus optical section through thick cells
-strong signal (HD image) requires a big input of light BUT this distorts resolution = trade-
off between light excitation and image blur (due to superimposition of images from
molecules above and below)
Types:
- point-scanning confocal microscope: provides high-res images in both 2D and 3D; slow
- spinning disk confocal microscope: real-time location of fluorescent reporter can be
captured (highly dynamic ones too); sensitivity not great because light source gets
exhausted (cannot look deep into tissue) but very fast
- two-photon microscopy: explores thicker samples i.e. live animals; lasers pulse at
wavelengths allowing for successful imaging
Photobleaching
(FRAP)
- burn fluroforms to analyse kinetics of a molecule
Laser Ablation
- itegrit of the ilusio is disrupted ith a laser that results i the host ells iate
immune system to respond and induce cell death
- zap-> rupture -> cell dies -> contents spill out and visualised
Super resolution
microscopy
- images formed by airy disk generation and light generated only from max. point where
the area that is excited is much smaller = more localised
Electron Microscopy
- higher resolution/magnification than light microscopy
- samples always processed meaning live imaging impossible and processing artefacts can
occur
find more resources at oneclass.com
find more resources at oneclass.com
Vesicular Transport within the secretory system of cells
Trafficking pathways: Analogy = pathway is bus, proteins are passengers, ticket is specificity.
- Secretory pathways/exocytosis: transport from inside ER to outside of cell
- Endocytosis/uptake: material from outside and internalising it
- Anterograde: essentially the same as secretory; a forward pathway that takes stuff out of cell
- Retrograde: from TGN via cisternae into the ER
- Vesicular transport allows for the transport of membrane proteins between intracellular compartments
- Each vesicular transport step involves unique combo of proteins: Small GTPases, SNAREs and Coat Proteins
- Critical that pathway maintains homeostasis and integrity, where a lot of recycling of lipids and membrane
takes place; disruption causes collapse of function
- Faces of different cellular membranes are conserved during membrane budding and fusion
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
The airy disk: various points that pass-through objective which make up the image seen = caused by diffraction/scattering of light when it passes through specimen; if too much overlap of airy disks = blur of signals. Resolution: limit up to which two small objects are still seen as separate entities; measure of resolving power = do. Prokaryotes = simple structure; eukaryotes = many membrane limited organelles (em good for this) Visualises relative distributions between two proteins; spatial relationship and overlap of sig(cid:374)als (cid:272)a(cid:374) (cid:271)e see(cid:374) (cid:894)sig(cid:374)als (cid:272)a(cid:374)(cid:859)t (cid:272)olo(cid:272)alise(cid:895) Dyes used absorb light at one set wavelength and emit light at another set of wavelengths. Max. absorbance = most energetic state = excitation = best absorbance wavelength. Stokes shift = difference between peak emission and peak absorption wavelengths; allows for analysis. -> for this reason special filters are used to restrict bandwidths and differentiate between the two; also, allow for multiple dyes to be used simultaneously.
