JC3 paper

Question 1

what enzyme is shown to control endosomal to lysosomal sorting of EGFR?

Answer 1

type I gamma PIP5-kinase (PIPKIγi5)

synthesises PtdIns4,5P2

Question 2

What does EGFR stand for?

Answer 2

epidermal growth factor receptor

Question 3

what plays a role in controlling the signaling of EGFR?

Answer 3

-trafficking and degradation of EGFR

Question 4

what does PIPKγi5 interact with?

Answer 4

• interacts with SNX5 (sorting nexin 5)

- SNX5 binds to PtdIns4,5P2 and other phosphoinositides

Question 5

where do SNX5 and PIPKIγi5 localise to?

Answer 5

-localise to endosomes

Question 6

What happens when there is loss of either SNX5 or PIPKIγi5?

Answer 6

• EGFR trafficking form the early endosome into the ILVs of a MVB is BLOCKED
• loss of ILV sorting prolongs EGFR signalling

Question 7

How do SNX5 and PIPKIγi5 facilitate ILV sorting?

Answer 7

• they prevent ubiquitination of hrs

- this allows hrs to bind to EGFR which is required for ILV sorting

Question 8

what does EGFR govern/what is its function?

Answer 8

Governs growth and differentiation of cells during development and adult homeostasis

Question 9

what can happen to EGFR once it has been internalised?

Answer 9

IT undergoes trafficking to decide whether it:

• gets recycled back to PM
• is degraded by lysosome
• translocation to the nucleus

Question 10

what does PtdIns4,5p2 do?

Answer 10

regulates endosomal trafficking

phosphoinositide

Question 11

How long does EGFR continue to signal in the cell once being endocytosed?

Answer 11

• signals until agonist is separated from receptor

- signals until trafficked into ILVs of the MVB

Question 12

give principles of immunoprecipitation

Answer 12

-precipitating a particular protein (or complex)out of a solution using antibodies for that protein

• sepharose beads are used which can be sedimented by centrifugation
• Antibody for protein (e.g.SNX5) attached to bead.
• add bead to solution - will bind to protein/SNX5 if it present and pull down SNX5 complex when centrifuged
• wash and remove unbound proteins
• separate proteins and run on gel/immunoblot for SNX5 and PIPKIγi5
• shows if there is an interaction between SNX5 and PIP5 (there is)

Question 13

Give the principles of a GST pull down

Answer 13

• Gluthionine bound to sepharose bead
• protein of interest has a fusion tag (e.g. GST)
• GST tag on protien of interest will bind to gluthionine (affinity ligand) which is attached to the sepharose bead.
• when centrifuged the beads with centrifuge with protein of interest attached

Question 14

what happens when PIPKIγi5 siRNA is used?

Answer 14

• no PIPKIγi5 produced
• there is no trafficking to the lysosome of EGFR (stays in EE)
• EGFR is more abundant and activated

Question 15

give an early endosome marker

Answer 15

EEA1

Question 16

give a lysosomal marker

Answer 16

LAMP1

Question 17

how can you tell that PIPKIγi5 siRNA prevents EGFR trafficking to the lysosome?

Answer 17

• Use immuno-fluorescence
• Stain cells with EGFR and LAMP1 antibodies- see overlap between the 2 (colocalization)

-when PIPKIγi5 siRNA is used there is less/no colocalising between LAMP1 and EGFR

Question 18

what is actin used for in blot?

Answer 18

• a loading control

- shows all lanes have equal loaded amount of protein/ cells

Question 19

Why was EGFR labelled with gold?

Answer 19

as heavy metal so shows up when using electron microscopy

Question 20

what is BSA and why is it used?

Answer 20

bovine serum albumin

- used as it prevents non-specific binding

Question 21

why was ubiquitin modified to have histadine molecules?

Answer 21

• histadine has a high affinity to nickel ions
• has high affinity to nickel matrix (nickel agarose)
• using the nickel argarose and histadine labelled ubiquitin allows us to identify ubiquitinated proteins

(non-ubiquitinated proteins will be washed off of the gel)