L8 - Critcal Paper Review

Question 1

Author and date

Answer 1

Bilan et al. 2004

Question 2

CFTR interacts with protein which contain what domain

Answer 2

PDZ

Question 3

What general class of proteins are involved in vesicle fusion and trafficking

Answer 3

SNARe proteins

Question 4

Syntaxin belongs to which family of proteins

Answer 4

SNARE

Question 5

What is the effect of interaction of syntaxin 1A and CFTR

Answer 5

Inhibition of channel activity

Question 6

What cell lines were produced

Answer 6

cDNA Syn8, Syn1-A, Syn3 cell lines
cDNA CFTR lines
Mutant syn8 delta TM

Question 7

What was mutated in Syn8deltaTM

Answer 7

Lacked the transmembrane anchor - so was unable to anchor into the cell membrane

Question 8

What was CFTR fused with

Answer 8

GFP

Question 9

How many forms of CFTR, why?

Answer 9

Two

One mature, the other lack glycosylation

Question 10

What is Fsk, what was its effect

Answer 10

Forskolin

Activation of CFTR

Question 11

What were the methods for the Iodide eflux experiemnt

Answer 11

Load cells with radioactive iodide

Look at how quickly it leaves the cells - out through CFTR

Question 12

What were the results and conclusions from the iodide eflux

Answer 12

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

Question 13

In the immunoflourescence experiement where was
CFTR
Syn8
Syn8deltaTM

Answer 13

Membrane
Membrane
Cytosol

Question 14

What were the conclusions from this immunoflourescence experiement

Answer 14

Syn8 inhibits the response of CFTR to FSK but this can only be achieved if Syn8 is in the membrane

Question 15

What information does a whole cell patch clamp give

Answer 15

Total currents due to ion channel function

Question 16

In a whole cell patch clamp the further a value is away from 0 the …

Answer 16

Larger the ion channel function

Question 17

What were the results of the whole cell patch clamp - what was the effect of syn8

Answer 17

Decreased CFTR magnitude

Question 18

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

Answer 18

Inhibits
No effect
Immunoflourescence

Question 19

Lysosome markers

Answer 19

Lamp1

Question 20

Was their co-localisation of CFTR and Syn8 in the lysosomes

Answer 20

No

Question 21

Endocytic recycling marker

Answer 21

Transferrin

Question 22

Was their co-localisation of CFTR and Syn8 in endocytic recylcing vesicles

Answer 22

Yes, a little

Question 23

Recycling/post golgi marker

Answer 23

Rab11

Question 24

Was there co-localisation of CFTR and Syn8 in Recycling/post golgi

Answer 24

Yes

Question 25

What experiement was carried out to test for physical interaction

Answer 25

Immunoprecipitation

Question 26

What happens in a Immunoprecipitation

Answer 26

An antibody is used to pull out target protein, any other proteins that are attached are also pulled out

Question 27

What happened in the Immunoprecipitation when a Syn8 ab was used

Answer 27

Syn8 AB pulled out Syn8 and CFTR

Question 28

What happened in the Immunoprecipitation when a CFTR ab was used

Answer 28

CFTR AB pulled out CFTR and Syn8

Question 29

What was the second experiment used to look for physical interaction

Answer 29

In vitro binding assay

Question 30

Explain how an in vitro binding assay works

Answer 30

Bead bound GST fusion protein incubated in cell lysate

Protein that binds to the beads examined by Western blot

Question 31

What were the results of the in vitro bidning assay and what conclusions were drawn from this

Answer 31

Syn1A strong binding
Syn8 (all forms) - weaker binding
Suggests an intermediate protein

Question 32

Syn8 inhibits macroscopic CFTR function

WHERE IS THE EVIDENCE FOR THIS

Answer 32

Patch clamp and I efflux

Question 33

Syn8 affects the trafficking of CFTR

WHERE IS THE EVIDENCE FOR THIS

Answer 33

Colocalisation experiments

Question 34

Syn8 must be anchored to the PM to have any effect on CFTR function
WHERE IS THE EVIDENCE FOR THIS

Answer 34

Colocalisation experiemnts - Syn8deltaTM lacks the effect

Question 35

Syn8 and CFTR go to recycline endosomes at TGN membranes

WHERE IS THE EVIDENCE FOR THIS

Answer 35

Colocalisation with Rab11 and transferrin

Question 36

Direct interaction between CFTR and Syn8

WHERE IS THE EVIDENCE FOR THIS

Answer 36

Immunoprecipitation and in vitro binding assay

Question 37

What would a minor critisism of the paper be

Answer 37

No evidence for change in channel activity (Po)

Single channel studies would be required