L3: Cells as model systems Flashcards
what is needed to culture cells?
Culture Medium: Salt solutions; pH sensor;
Glutamine
Growth Factors: usually coming from the
addition of animal serum (Fetal Calf Serum;
Horse Serum; Chick Embryo Extract)
Supplements: Antibiotics; Insulin
Plastic vessels: Petri dishes, Culture flasks,
Multiwell plates; Microscopy slides
Substrates: Extracellular Matrix; Hydrogels;
Fibrin Matrix; Biocompatible Scaffolds
why culture cells?
ADVANTAGES
Defined system (know exactly what is there)
Easier to manipulate (adding drugs;
microscopy)
Potentially inexhaustible supply of cells – better
reproducibility of experiments
Fluorochromes
Why culture cells?
DISADVANTAGES
Potential of artefacts (especially in 2D culture)
Lack of physiological input
which cell types?
Primary cultures:
Isolated from animal tissues; usually limited life
span
Cell lines:
Immortalised; may have lost differentiation
markers
growth phase
lag phase, log face (feed in the middle) subculture at top and then plateau phase
undesireables
mould, yeast, bacteria, mycoplasma
how do flurochromes work?
excited by a shorter wave length and emit a longer wave length.
* Fluorophore electrons absorb a photon
* Briefly enter excited state
* Emit a photon with a lower energy, i.e. longer wavelength
* Stokes shift: difference in wavelengths
* Lifetime: time taken to emit a photon
* The quantum yield is an indicator of the efficiency of the
dye: ratio of emitted photons per absorbed photon
principles of fluorescent dyes
Fluorescein is a small green fluorescent dye
Isothiocyanate group (N=C=S) in derivatives enabled
binding to primary amines e.g. in antibodies or
proteins
Fluorescent dyes usually have multiple rings
e.g: phalloidin and F-actin
how to stain a nucleus?
DAPI/Hoechst
+ reliable
+ relatively cheap
+ can come with mounting medium
+ reasonably stable
- need UV/blue diode laser
- need UV compatible lenses
- living cells not necessarily keen on UV illumination
how to stain an organelle?
Mitochondria
MitoTracker
Cells have to be alive!
GFP
From the jellyfish Aequorea victoria
Emits green light after excitation with blue light
FRET
Cyan fluorescent protein (CFP) absorbs a photon and enters excited
state - emits a photon at ~470 nm (blue)
Yellow fluorescent protein (YFP) absorbs a photon and enters excited
state – emits a photon at ~545 nm (yellow)
Used to follow protein: protein interactions
FRET 3Can perform FRET using:
- Fluorescent proteins
- Fluorophore-labelled
antibodies
activation of pka by camp
Fusion Partners and Their Locations:
CFP (Cyan Fluorescent Protein):
Often used as the donor fluorophore, CFP is fused to one of the subunits of PKA. In many designs, CFP is attached to the regulatory (R) subunit of PKA.
YFP (Yellow Fluorescent Protein):
Serving as the acceptor fluorophore, YFP is typically fused to the catalytic (C) subunit of PKA.
Inactive State Configuration:
When PKA is inactive (without cAMP stimulation), the regulatory and catalytic subunits are bound together. This tight interaction places CFP (on the R subunit) and YFP (on the C subunit) very close to each other.
When CFP is excited, its energy is efficiently transferred to YFP, generating a significant FRET signal.
Activation by cAMP:
cAMP Binding:
When a cellular signal increases the concentration of cAMP, it binds to the regulatory subunits.
Conformational Change:
Binding of cAMP induces a conformational change in the R subunits, reducing their affinity for the C subunits.
Subunit Dissociation:
The catalytic subunits are released from the complex. Consequently, the physical distance between CFP and YFP increases.
Change in FRET:
With the increased separation, energy transfer from CFP to YFP becomes inefficient, leading to a decrease in the FRET signal. This decrease is monitored and interpreted as PKA activation.
📊 Visualizing the Mechanism:
Before cAMP Stimulus (Inactive PKA):
CFP is attached to the R subunit and in close proximity to YFP on the C subunit.
Upon excitation of CFP, energy is transferred to YFP, leading to high FRET efficiency.
After cAMP Stimulus (Active PKA):
cAMP binds to the R subunit, triggering a conformational change.
The C subunit (with YFP attached) dissociates from the complex.
The increased distance between CFP and YFP results in a decrease in energy transfer—observed as a lower FRET signal.
🔍 Summary:
CFP is typically fused to the regulatory subunit of PKA (acting as the FRET donor).
YFP is typically fused to the catalytic subunit of PKA (acting as the FRET acceptor).
The efficiency of FRET depends on the proximity of these subunits. When PKA is inactive, the fluorophores are close together, generating a high FRET signal. When PKA is activated by cAMP, the subunits separate, and the FRET signal decreases, which can be monitored in real time.
FRAP
Fluorescence recovery after photobleaching
used to investigate protein dynamics within a cell
like dynamics of Z-disc proteins
