JC4 paper

Question 1

what is Appl1?

Answer 1

• an endosomal protein that interacts with TMRs and Akt
• a rab effector
• required for cell survival (as regulate AKT)

Question 2

what is rab5?

Answer 2

• a small GTPase
• regulate early endosome fusion and sorting
• localise to early endosome?

Question 3

what are the roles of rab5 and its effectors?

Answer 3

have multiple roles in the early endocytotic pathway regulate:

• cargo sorting
• early endosome fusion
• actin+microtubule dependent motility of vesicles

-some - regulate PI synthesis and turnover

Question 4

what is the main hypothesis of this paper?

Answer 4

-spatial distribution of signalling proteins may contribute to signalling specificity

Question 5

where does the rab 5 effector Appl1 localise?

Answer 5

-localises to endosomes

Question 6

what does Appl1 bind to and how was this shown?

Answer 6

• Appl1 binds specifically to Rab5-GTP (active form)
• this was shown using a GST pulldown - Rab5-GTP and APPl1 immunoprecipitated toegther - shown on a western blott using immunoblotting

(in zebra fish)

Question 7

where are App1 proteins expressed during zebrafish development?

Answer 7

• expressed early and widely

- enriched in pronephros, neural tube and forebrain during embryogenesis

Question 8

what does Appl1 KD cause?

Answer 8

-apoptosis of cells
(high apoptosis in areas where Appl1 was expressed highly - e.g. pronephros and telencephalon
-undeveloped yolk extension, swelling and bent body aixs

Question 9

How can apoptotic cells be identified?

Answer 9

• acridine orange stain labels apoptotic cells

Question 10

what co-localizes with Appl1 on endosomes?

Answer 10

• Akt and gsk-3B

not TSC2)(appl1) independent

Question 11

what does Appl1 regulate to do with Akt?

Answer 11

-Appl1 regulates Akt activity, substrate specificity and Akt-dependent survival in zebrafish

Question 12

Is endosomal localisation essential for Appl1 function?

Answer 12

YES

-endosomal localisation is required for Appl1 mediated activation of Akt and cell survival singalling

Question 13

how was it shown that endosomal localisation was necessary for Appl1?

Answer 13

GOF studies(thick yolk extension, swolen telencephalon)

• when Appl1 excluded from endosome (by triM) there was no negative effect of GOF
• but when was allowed to enter endosome - GOF phenotype