L8 - Critcal Paper Review Flashcards
Author and date
Bilan et al. 2004
CFTR interacts with protein which contain what domain
PDZ
What general class of proteins are involved in vesicle fusion and trafficking
SNARe proteins
Syntaxin belongs to which family of proteins
SNARE
What is the effect of interaction of syntaxin 1A and CFTR
Inhibition of channel activity
What cell lines were produced
cDNA Syn8, Syn1-A, Syn3 cell lines
cDNA CFTR lines
Mutant syn8 delta TM
What was mutated in Syn8deltaTM
Lacked the transmembrane anchor - so was unable to anchor into the cell membrane
What was CFTR fused with
GFP
How many forms of CFTR, why?
Two
One mature, the other lack glycosylation
What is Fsk, what was its effect
Forskolin
Activation of CFTR
What were the methods for the Iodide eflux experiemnt
Load cells with radioactive iodide
Look at how quickly it leaves the cells - out through CFTR
What were the results and conclusions from the iodide eflux
With Syn8 rate of I loss was less therefore due to either a change in number of CFTR channels at the membrane or change in Po
In the immunoflourescence experiement where was
CFTR
Syn8
Syn8deltaTM
Membrane
Membrane
Cytosol
What were the conclusions from this immunoflourescence experiement
Syn8 inhibits the response of CFTR to FSK but this can only be achieved if Syn8 is in the membrane
What information does a whole cell patch clamp give
Total currents due to ion channel function
In a whole cell patch clamp the further a value is away from 0 the …
Larger the ion channel function
What were the results of the whole cell patch clamp - what was the effect of syn8
Decreased CFTR magnitude
What is the effect of Syn8 on the trafficking of CFTR
What is the effect of Syn8deltaTM on the trafficking of CFTR
What experiement was this seen in
Inhibits
No effect
Immunoflourescence
Lysosome markers
Lamp1
Was their co-localisation of CFTR and Syn8 in the lysosomes
No
Endocytic recycling marker
Transferrin
Was their co-localisation of CFTR and Syn8 in endocytic recylcing vesicles
Yes, a little
Recycling/post golgi marker
Rab11
Was there co-localisation of CFTR and Syn8 in Recycling/post golgi
Yes
What experiement was carried out to test for physical interaction
Immunoprecipitation
What happens in a Immunoprecipitation
An antibody is used to pull out target protein, any other proteins that are attached are also pulled out
What happened in the Immunoprecipitation when a Syn8 ab was used
Syn8 AB pulled out Syn8 and CFTR
What happened in the Immunoprecipitation when a CFTR ab was used
CFTR AB pulled out CFTR and Syn8
What was the second experiment used to look for physical interaction
In vitro binding assay
Explain how an in vitro binding assay works
Bead bound GST fusion protein incubated in cell lysate
Protein that binds to the beads examined by Western blot
What were the results of the in vitro bidning assay and what conclusions were drawn from this
Syn1A strong binding
Syn8 (all forms) - weaker binding
Suggests an intermediate protein
Syn8 inhibits macroscopic CFTR function
WHERE IS THE EVIDENCE FOR THIS
Patch clamp and I efflux
Syn8 affects the trafficking of CFTR
WHERE IS THE EVIDENCE FOR THIS
Colocalisation experiments
Syn8 must be anchored to the PM to have any effect on CFTR function
WHERE IS THE EVIDENCE FOR THIS
Colocalisation experiemnts - Syn8deltaTM lacks the effect
Syn8 and CFTR go to recycline endosomes at TGN membranes
WHERE IS THE EVIDENCE FOR THIS
Colocalisation with Rab11 and transferrin
Direct interaction between CFTR and Syn8
WHERE IS THE EVIDENCE FOR THIS
Immunoprecipitation and in vitro binding assay
What would a minor critisism of the paper be
No evidence for change in channel activity (Po)
Single channel studies would be required
