L17: Investigating Intra-Cellular Signalling Flashcards
What parts does signal transduction typically consist of?
- a mesasge outside the cell
- some sort of receptor for the message
- some sort of response/ change inside the cell
What ways are there to examine a signal transduction system?
- the top down: start w the msg and dig down the signal cascade until you reach outcome
- the bottom up: start w the effect and dig up the cascade until you reach the msg
What are the main approaches for examining signal transuction system?
- work w purified proteins and try to reconstruct system in vitro
- use isolated cells from tissue/ cell culture
- use whole animals
- use humans
Name pros of using purified proteins and reconstrucuting sytem in vitro?
- ease of manipulation
- easy to control conditions
- reproducible
- molecular level analysis
- multiple data points
Name cons of using purified proteins and reconstrucuting sytem in vitro?
- non physiological
- artificial conditions
- making proteins can be hard
- need to know what components to mix
- protiens may interact that don’t usually mix in cell
Name uses of using purified proteins and reconstrucuting sytem in vitro?
Has been succesful in phosphorylation studies, enzyme activity assays and protein/ protein interactions
How are isolated cells used to investigate signal transduction systems?
- primary cell culture: cells derived from animal or human tissue/ organ samples
- cell lines: cells originally derived from human or animal tissues and then immortalised
What are pros of using isolated cells to investigate signal transduction systems?
- can be more physiological
- cells relatively easy to grow, maintain, manipulate
- data can be clean as cell conditions are easy to control
- possible to get alot of data from single prep of cells
What are cons of using isolated cells to investigate signal transduction systems?
- cultures easily infected & need to be regularly fed
- still not truly physiological
- method is expensive in terms of time and consumables (reagents)
- cell preps may not be pure
- cells can change during extended periods of culture
How are whole animals used to investigate signal transduction systems?
- via whole animal studies e.g. drug trials
- or by organ/tissue specific studies e.g. isolation of organs/tissue for cell culture or in vitro
Name pros of using whole animals used to investigate signal transduction systems?
- truly physiological
- cheap and relatively easy
- models of specific disease states/ conditions available
Name pros of using whole animals used to investigate signal transduction systems?
- data can be noisy due to competing mechanisms, so there’s variation animal to animal
- ethical consideratinos & reproducibility
- primary cultured cells can rapidly change
- cells preps can contain multile cell types (not pure)
How are humans used to investigate signal transduction systems?
Need IC and there are concerns over data protection but restricted to non invasive techniques
Name pros of using humans to investigate signal transduction systems?
- truly physiological
- great feedback, subject can say what’s occuring
Name cons of using humans to investigate signal transduction systems?
- data can be noisy due to competing mechanisms and compensation
- ethical and risk consideratinos: few exps per human (reproducibility)
- primary cultured cells can rapidly change
- cell preps from organs/ tissue can contain mulitple cell types
- shortage of material
What are the 3 main animal models used diabetes/ studies?
- Streptozotocin: injected into rats, destroys their pancreatic beta cells
- Ob/Ob mouse: fat and diabetic (type II) and has been used for years
- Zucker rat: fat and diabetic (type II)
Explain why specific mouse models are used in diabetes studies
- Ob/Ob mouse fails to make leptin which regulates appetite so becomes obeses and develops diabetes
- Zucker rat has a defective hypothalamic receptor for leptip so doesn’t stop eating
Leptin deficiency/ failure has been identified in humans making these good models to use for type II diabetes
What methods can be used with mouse models?
- inhibitors/ antagonists to block receptor or signalling cascade
- radiolabels e.g. radioactive markers to determine the phosphporylation of proteins
- reporter genes/ knockouts e.g.g to look at pathways & levels of expression
- FRET
What is the main problem with studying cell signalling?
The cell will try to counter the change made and correct it
What chemicals may be used when working with cAMP systems?
- 3 isobutyl methylxanthine (IBMX), caffeine & threophylline: all block PDEs converting cAMP to AMP
- Forskolin: activates cAMP, giving max stimulation of system
- Non hydrosable analogues of cAMP
- Cholera & pertussis toxins: activate/ deactivate key proteins in the cAMP generation
Explain cell conversion of IP3
Cell will convert IP3 to either IP2 or IP4 then back around to inositol and then to IP2
- Li blocks number of enzymes in psthway e.g. myo-inositol-1-phosphate and the net result is that cycle stops
- IP pool can be maintained and measured: if we label IP pool w tritum we can see buildup of breakdown products of IP3 telling us IP3 system has been activated
How is cAMP typically measured?
In a sample using a binding asay where cAMP in sample competes for binding of known amount of radioactive baleed cAMP
- FRET can be used fpr measuring localised cAMP by fluorescence
- binding tells us how much cAMP was in sample
How is IP3 measured?
Cells preloaded with radio labelled or tritiated inositol to block recycling with Li
- purify the IP products (which are now radiolablled)
- measure levls to determine that the pathway has been turned on
How can receptor studies be used?
Agonists and antagonists can be used to pick apart pathways
- agonist activates recepto, antagonist blocks receptor
- if we suspect certain agonist involved can add it to culture medium and to show it’s that agonist activating system, add an antagonist to see effect
