Table of Contents
Paris Veltsos ................................................................................................................................................. 3
1 GFP origin and 5 different examples of its use ..................................................................................... 3
2 FRET & FRAP & expansion microscopy ................................................................................................. 3
3 TEM & SEM ........................................................................................................................................... 4
1. TEM.................................................................................................................................................. 4
2. SEM .................................................................................................................................................. 5
Advantages .......................................................................................................................................... 5
4 Confocal microscopy & CryoEM & vs x-ray crystallography.................................................................. 5
Confocal microscope............................................................................................................................ 5
CryoEM & X-ray .................................................................................................................................... 5
5 Signal recognition particle & ribosome cycle ....................................................................................... 6
Signal Recognition Particle (SRP) .......................................................... Error! Bookmark not defined.
6 Microsome use to confirm the signal hypothesis, ER functions and how to retain protein in ER ....... 7
Microsomes ......................................................................................................................................... 7
Endoplasmic reticulum (ER) ................................................................................................................. 7
How to retain protein in ER ................................................................................................................. 8
7 Co-translational vs post-translational protein translocation ................................................................ 8
Protein translocator ............................................................................................................................. 8
Membrane integration ........................................................................................................................ 8
8 Unfolded protein response ................................................................................................................... 9
9 Nuclear pore complex - structure and function ................................................................................. 11
Nucleus .............................................................................................................................................. 11
10 Specialised plant organelles ............................................................................................................. 12
Chloroplasts ....................................................................................................................................... 12
Specialised peroxisomes .................................................................................................................... 12
Vacuoles ............................................................................................................................................. 12
11 Clathrin coat & AP2 adaptor & LDL intake ........................................................................................ 13
Clathrin .............................................................................................................................................. 13
AP2 adaptor & LDL intake .................................................................................................................. 14
12 Membrane identity, Rab cascade & endosome maturation ............................................................. 14
13 Secretory vesicles ............................................................................................................................. 15
14 Phagocytosis & Macropinocytocis .................................................................................................... 16
Wen Juan Ma ............................................................................................................................................. 18
1 Introduction ........................................................................................................................................ 18
2 Synaptic vs endocrine signalling ......................................................................................................... 18
3 Cell-surface receptors ......................................................................................................................... 19
1
, 4 G-protein-coupled receptors .............................................................................................................. 20
G-protein-coupled receptors (GPCRs) ............................................................................................... 20
cAMP.................................................................................................................................................. 20
PKA..................................................................................................................................................... 20
Certain G-protein activate phospholipids .......................................................................................... 21
Ca2+ .................................................................................................................................................... 21
Nitric oxide (NO) ................................................................................................................................ 21
5 G-proteins ........................................................................................................................................... 21
6 Cell junctions ...................................................................................................................................... 21
Adherens junctions ............................................................................................................................ 22
Desmosomes ..................................................................................................................................... 22
Tight junctions ................................................................................................................................... 22
Gap junctions ..................................................................................................................................... 23
7 Transmembrane adhesion proteins in cell-cell ................................................................................... 23
8 Cadherins ............................................................................................................................................ 24
Cadherins ........................................................................................................................................... 24
Adherens junctions ............................................................................................................................ 25
9 Desmosomes ...................................................................................................................................... 25
10 3 superfamilies of adhesion proteins ............................................................................................... 26
11 ECM................................................................................................................................................... 26
Basal lamina ....................................................................................................................................... 27
12 Collagens........................................................................................................................................... 28
2. Collagens........................................................................................................................................ 28
13 Talin + cell walls ................................................................................................................................ 29
Talin.................................................................................................................................................... 29
Cell wall.............................................................................................................................................. 29
14 Integrins ............................................................................................................................................ 30
Structure: ........................................................................................................................................... 30
Functions ........................................................................................................................................... 31
The titles indicated in purple are extra information. You can also learn it if you want, but I didn’t learn it
and I don't think it's necessary.
2
, Paris Veltsos
1 GFP origin and 5 different examples of its use
Fluorescence
Chromophore =
- molecule that absorbs light (excitation) → emit photon (fluoresces) at a longer wavelength (less
energy) than the absorbed photon.
- = typically, aromatic rings.
GFP (Green Fluorescent protein)
- Chromophore isolated from a jellyfish
- Discovered in 1962 → mutations, enhancements in lab → cloned in 1992 → brought to
expression in 1994
- 238 amino acids → fluorophore = 3 amino acids in the middle
Probe = molecule targeting something specific
5 different examples of GFP use:
GFP Fused with peptide GFP Fused with Immunofluorescence Fused with nucleic acid Fused with protein
location signal promotor
Targets eg Allows to monitor gene Primary/ secondary With fluorescent Fuse GFP directly with
mitochondria or ER expression → to see when antibody fused with chemical analogues of protein of interest → to
& where a gene is active GFP RNA/DNA track protein
FRET & FRAP & expansion microscopy
FRET
= Fluorescence Resonance Energy Transfer
Purpose: to determine if molecules are close or not
Working:
2 molecules of interest are labelled with different fluorochromes with overlapping
emission and absorption spectra. If…
- Molecules don’t interact → colour emitted from first molecule (here blue)
- Molecules interact → resonance transfer energy to other molecule → other colour
emitted (here green)
Technique often used to study:
- Signalling molecule ↔ receptor interaction
- Macromolecular complex interaction
3
, FRAP
= Fluorescence Recovery After Photobleaching
Purpose: deliver data about:
- Diffusion coefficient
- Active transport rate
- Binding/ dissociation rate
Working:
1. proteins are labelled with GFP
2. Bleaching of a small area with a laser beam
3. Measure time before area is recovered
ExM
= Expansion microscopy
Working:
1. Fix cell + add fluorescent labels and gel monomers (for step 2)
2. Gel formation (with acrylate/acrylamide)
3. Proteinase digestion → so that the sample can expand
uniformly
4. Gel expansion in water (4-10 x)
5. Cell has become larger and can be observed with standard microscope
Advantages:
- Super-Resolution (±25 nm)
- Requires no costly hardware
- With deconvolution → you can create 3D
- Sample can be washed and re-probed with different antibodies
3 TEM & SEM
What? 2 types of e- microscopy → use e- instead of light → shorter wavelength → higher resolution
Conditions: vacuum, dead specimen
1. TEM
= Transmission Electron Microscopy
What? e- go through sample → analyses intern structure (2D)
Design similar to large inverted optical microscope:
- Illumination source = filament or cathode that emits e- (at the top of the column)
- Vacuum → (no scattering by collisions with air molecules)
- Magnetic coils (focus e- beam)
- Specimen stained with e—dense material → image from non-scattered e-
Sample preparation:
- Fixed or rapidly frozen
- Dehydrated
- Stained with heavy metals (eg uranium, lead, osmium) → to increase e- contrast
- Embedded in resin
- Cut into ultrathin sections (25–100 nm) → with fine glass/ diamond knife microtome
Make contrast:
4
Paris Veltsos ................................................................................................................................................. 3
1 GFP origin and 5 different examples of its use ..................................................................................... 3
2 FRET & FRAP & expansion microscopy ................................................................................................. 3
3 TEM & SEM ........................................................................................................................................... 4
1. TEM.................................................................................................................................................. 4
2. SEM .................................................................................................................................................. 5
Advantages .......................................................................................................................................... 5
4 Confocal microscopy & CryoEM & vs x-ray crystallography.................................................................. 5
Confocal microscope............................................................................................................................ 5
CryoEM & X-ray .................................................................................................................................... 5
5 Signal recognition particle & ribosome cycle ....................................................................................... 6
Signal Recognition Particle (SRP) .......................................................... Error! Bookmark not defined.
6 Microsome use to confirm the signal hypothesis, ER functions and how to retain protein in ER ....... 7
Microsomes ......................................................................................................................................... 7
Endoplasmic reticulum (ER) ................................................................................................................. 7
How to retain protein in ER ................................................................................................................. 8
7 Co-translational vs post-translational protein translocation ................................................................ 8
Protein translocator ............................................................................................................................. 8
Membrane integration ........................................................................................................................ 8
8 Unfolded protein response ................................................................................................................... 9
9 Nuclear pore complex - structure and function ................................................................................. 11
Nucleus .............................................................................................................................................. 11
10 Specialised plant organelles ............................................................................................................. 12
Chloroplasts ....................................................................................................................................... 12
Specialised peroxisomes .................................................................................................................... 12
Vacuoles ............................................................................................................................................. 12
11 Clathrin coat & AP2 adaptor & LDL intake ........................................................................................ 13
Clathrin .............................................................................................................................................. 13
AP2 adaptor & LDL intake .................................................................................................................. 14
12 Membrane identity, Rab cascade & endosome maturation ............................................................. 14
13 Secretory vesicles ............................................................................................................................. 15
14 Phagocytosis & Macropinocytocis .................................................................................................... 16
Wen Juan Ma ............................................................................................................................................. 18
1 Introduction ........................................................................................................................................ 18
2 Synaptic vs endocrine signalling ......................................................................................................... 18
3 Cell-surface receptors ......................................................................................................................... 19
1
, 4 G-protein-coupled receptors .............................................................................................................. 20
G-protein-coupled receptors (GPCRs) ............................................................................................... 20
cAMP.................................................................................................................................................. 20
PKA..................................................................................................................................................... 20
Certain G-protein activate phospholipids .......................................................................................... 21
Ca2+ .................................................................................................................................................... 21
Nitric oxide (NO) ................................................................................................................................ 21
5 G-proteins ........................................................................................................................................... 21
6 Cell junctions ...................................................................................................................................... 21
Adherens junctions ............................................................................................................................ 22
Desmosomes ..................................................................................................................................... 22
Tight junctions ................................................................................................................................... 22
Gap junctions ..................................................................................................................................... 23
7 Transmembrane adhesion proteins in cell-cell ................................................................................... 23
8 Cadherins ............................................................................................................................................ 24
Cadherins ........................................................................................................................................... 24
Adherens junctions ............................................................................................................................ 25
9 Desmosomes ...................................................................................................................................... 25
10 3 superfamilies of adhesion proteins ............................................................................................... 26
11 ECM................................................................................................................................................... 26
Basal lamina ....................................................................................................................................... 27
12 Collagens........................................................................................................................................... 28
2. Collagens........................................................................................................................................ 28
13 Talin + cell walls ................................................................................................................................ 29
Talin.................................................................................................................................................... 29
Cell wall.............................................................................................................................................. 29
14 Integrins ............................................................................................................................................ 30
Structure: ........................................................................................................................................... 30
Functions ........................................................................................................................................... 31
The titles indicated in purple are extra information. You can also learn it if you want, but I didn’t learn it
and I don't think it's necessary.
2
, Paris Veltsos
1 GFP origin and 5 different examples of its use
Fluorescence
Chromophore =
- molecule that absorbs light (excitation) → emit photon (fluoresces) at a longer wavelength (less
energy) than the absorbed photon.
- = typically, aromatic rings.
GFP (Green Fluorescent protein)
- Chromophore isolated from a jellyfish
- Discovered in 1962 → mutations, enhancements in lab → cloned in 1992 → brought to
expression in 1994
- 238 amino acids → fluorophore = 3 amino acids in the middle
Probe = molecule targeting something specific
5 different examples of GFP use:
GFP Fused with peptide GFP Fused with Immunofluorescence Fused with nucleic acid Fused with protein
location signal promotor
Targets eg Allows to monitor gene Primary/ secondary With fluorescent Fuse GFP directly with
mitochondria or ER expression → to see when antibody fused with chemical analogues of protein of interest → to
& where a gene is active GFP RNA/DNA track protein
FRET & FRAP & expansion microscopy
FRET
= Fluorescence Resonance Energy Transfer
Purpose: to determine if molecules are close or not
Working:
2 molecules of interest are labelled with different fluorochromes with overlapping
emission and absorption spectra. If…
- Molecules don’t interact → colour emitted from first molecule (here blue)
- Molecules interact → resonance transfer energy to other molecule → other colour
emitted (here green)
Technique often used to study:
- Signalling molecule ↔ receptor interaction
- Macromolecular complex interaction
3
, FRAP
= Fluorescence Recovery After Photobleaching
Purpose: deliver data about:
- Diffusion coefficient
- Active transport rate
- Binding/ dissociation rate
Working:
1. proteins are labelled with GFP
2. Bleaching of a small area with a laser beam
3. Measure time before area is recovered
ExM
= Expansion microscopy
Working:
1. Fix cell + add fluorescent labels and gel monomers (for step 2)
2. Gel formation (with acrylate/acrylamide)
3. Proteinase digestion → so that the sample can expand
uniformly
4. Gel expansion in water (4-10 x)
5. Cell has become larger and can be observed with standard microscope
Advantages:
- Super-Resolution (±25 nm)
- Requires no costly hardware
- With deconvolution → you can create 3D
- Sample can be washed and re-probed with different antibodies
3 TEM & SEM
What? 2 types of e- microscopy → use e- instead of light → shorter wavelength → higher resolution
Conditions: vacuum, dead specimen
1. TEM
= Transmission Electron Microscopy
What? e- go through sample → analyses intern structure (2D)
Design similar to large inverted optical microscope:
- Illumination source = filament or cathode that emits e- (at the top of the column)
- Vacuum → (no scattering by collisions with air molecules)
- Magnetic coils (focus e- beam)
- Specimen stained with e—dense material → image from non-scattered e-
Sample preparation:
- Fixed or rapidly frozen
- Dehydrated
- Stained with heavy metals (eg uranium, lead, osmium) → to increase e- contrast
- Embedded in resin
- Cut into ultrathin sections (25–100 nm) → with fine glass/ diamond knife microtome
Make contrast:
4
