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BMS2002: Cell Bio and Disease > L17: Investigating Intra-Cellular Signalling > Flashcards

L17: Investigating Intra-Cellular Signalling Flashcards

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1
Q

What parts does signal transduction typically consist of?

A
  1. a mesasge outside the cell
  2. some sort of receptor for the message
  3. some sort of response/ change inside the cell
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2
Q

What ways are there to examine a signal transduction system?

A
  • 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
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3
Q

What are the main approaches for examining signal transuction system?

A
  • work w purified proteins and try to reconstruct system in vitro
  • use isolated cells from tissue/ cell culture
  • use whole animals
  • use humans
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4
Q

Name pros of using purified proteins and reconstrucuting sytem in vitro?

A
  • ease of manipulation
  • easy to control conditions
  • reproducible
  • molecular level analysis
  • multiple data points
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5
Q

Name cons of using purified proteins and reconstrucuting sytem in vitro?

A
  • 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
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6
Q

Name uses of using purified proteins and reconstrucuting sytem in vitro?

A

Has been succesful in phosphorylation studies, enzyme activity assays and protein/ protein interactions

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7
Q

How are isolated cells used to investigate signal transduction systems?

A
  • 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
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8
Q

What are pros of using isolated cells to investigate signal transduction systems?

A
  • 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
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9
Q

What are cons of using isolated cells to investigate signal transduction systems?

A
  • 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
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10
Q

How are whole animals used to investigate signal transduction systems?

A
  • 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

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11
Q

Name pros of using whole animals used to investigate signal transduction systems?

A
  • truly physiological
  • cheap and relatively easy
  • models of specific disease states/ conditions available
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12
Q

Name pros of using whole animals used to investigate signal transduction systems?

A
  • 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)
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13
Q

How are humans used to investigate signal transduction systems?

A

Need IC and there are concerns over data protection but restricted to non invasive techniques

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14
Q

Name pros of using humans to investigate signal transduction systems?

A
  • truly physiological

- great feedback, subject can say what’s occuring

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15
Q

Name cons of using humans to investigate signal transduction systems?

A
  • 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
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16
Q

What are the 3 main animal models used diabetes/ studies?

A
  • 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)
17
Q

Explain why specific mouse models are used in diabetes studies

A
  • 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
18
Q

What methods can be used with mouse models?

A
  • 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
19
Q

What is the main problem with studying cell signalling?

A

The cell will try to counter the change made and correct it

20
Q

What chemicals may be used when working with cAMP systems?

A
  • 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
21
Q

Explain cell conversion of IP3

A

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
22
Q

How is cAMP typically measured?

A

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
23
Q

How is IP3 measured?

A

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
24
Q

How can receptor studies be used?

A

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
25
Q

How is FRET used to measure cAMP

A

Preloaf cells with fret molecule then cAMP binds it, changing fluorescence properties of fret molecules causing colour change
- in absence of cAMP a conformational change occurs and the molecule’s components move apart

26
Q

Explain the structure of fret molecules?

A

They have 2 fluorescent components, GFP and YF

27
Q

At resting what signal does FRET produce?

A

YFP produces strong signal at 540m bc of FRET (transfer CFP), CFP signal low at 480nm

28
Q

What happens to FRET signal when cAMP binds?

A

YFP and CFP move apart, YFP signal drops as no longer getting FRET from CFP & CFP singal increases from 480nm as no longer giving energy to YFP

29
Q

How can phosphorylation studies be used to look at signalling pathways?

A

Can preload cells with ‘hot’ radioactive P so ATP pool is ‘hot’ or use cell free system so we can add P to gamma position on ATP and then transferred over to protein that’s being phosphorylated
- can see protein on gel, so run it on gel, dry down gel, expose to photographic film, develop film and then see s band where RA protein is

30
Q

Name activators of kinase

A

Phorbol esters that activate PKC & kinas inhibitors e.g. staurosporine
- with these is important to inhibit dephosphorylation as otherwise unable to capture data

31
Q

Give exampls of specific kinase inhibitors

A
  • wortmanin: inhibits PI3 kinases
  • rapamycin: P70 56 kinase
  • PD98059: inhibits MAP kinase
  • substrate of kinase is likely ot be specific for that kinase
32
Q

Explain a probelm with phosphorylation studies

A

As fast as P group is added, cell will use a P to remove it
- adds phosphate inhibitors e.g. akadaic acid that inhibits protein serine & vanodate which inhibits protein tyrosine phosphate

33
Q

What effect does protein phosphorylation have?

A
  • increase MW so band will be slightly higher on gel
34
Q

Name an alternative method to detect phosphorylated proteins?

A
  • Mass spec and do nuetral ion loss and peptide sequencing
  • Reporter genes: taking gene of interest and couple to reporter gene e.g. lucierase or GFP, then stimulate pathway and measure changes in levels of our reporter observed
35
Q

How can IP3 and cAMP be picked apart?

A

Using a range of compounds that either inhibit or stimulate different parts of the pathway

36
Q

Explain genetic approaches to investigate signalling pathways

A
  • introduce mutated (dominant -ve) protein that still interacts w pathway but isn’t functional
  • use modified proteins to change function/ location
  • using constitutively active portein tht is permanently active

BMS2002: Cell Bio and Disease (16 decks)

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