W14

Question 1

Which cytoskeletal structure underpins the secretory and endocytic pathways?

Answer 1

Microtubules

Question 2

How are motor proteins coupled to membranes?

Answer 2

Through Rab GTPases and phospholipids in a coincidence detection mechanism

Question 3

Name the domains of KIF16B.

Answer 3

N-terminal KISc kinesin motor domain, forkhead homology (FHA) domain for protein interactions, C-terminal PHOX-homology (PX) domain for phosphoinositide binding (preference for 3’-phosphorylated lipids i.e. PI3P)

Question 4

Describe the structure of the early embryo.

Answer 4

The endoderm and ectoderm are formed within the inner cell mass (ICM). The primitive endoderm secretes the basement membrane which supports the differentiation of primitive ectoderm

Question 5

Describe the effect of knockout of the kinesin KIF16B on formation of the primitive ectoderm.

Answer 5

FGFR2 no longer transported to the plasma membrane hence fibroblast growth factor (FGF) can no longer bind, ERK and Akt kinases are no longer phosphorylated (activated) so do not phosphorylate two key transcription factors necessary for ectoderm development

Question 6

Name the three routes of entry into the endocytic pathway.

Answer 6

Phagocytosis, macropinocytosis, and pinocytosis via clathrin/caveolin mediation

Question 7

Describe the process of phagocytosis.

Answer 7

Receptor-driven actin-dependent process, macrophage Fc receptors are activated by bacterial antibodies, signalling cascade by Rac and Cdc42 GTPases which induce actin polymerisation to form cell surface extensions that engulf the bacterium into a phagosome

Question 8

Describe the process of macropinocytosis.

Answer 8

Non-selective internalisation of soluble material and membrane in response to growth factor stimulation, small GTPases (e.g. Rho family Rac1) and actin form a ruffle that protrudes from the membrane and folds back on itself to form a macropinosome

Question 9

Describe the process of pinocytosis via clathrin mediation.

Answer 9

Clathrin-coated pits cause invagination of the plasma membrane to form a triskelion comprised of three clathrin light chain and three clathrin heavy chain, dynamin scission releases the pinosome into the cell

Question 10

Describe the process of pinocytosis via caveolin mediation.

Answer 10

Caveolins form a cavin complex which causes invagination to internalise membrane enriched in lipid rafts/microdomains, dynamin scission releases the pinosome into the cell

Question 11

What two molecules are LDL particles mainly comprised of?

Answer 11

Apolipoprotein B and cholesterol

Question 12

How does familial hypercholesterolemia arise?

Answer 12

Either defects in LDL binding to the extracellular domain of the LDL receptor or defects in the cytoplasmic LDL receptor domain binding to the AP-2 clathrin adaptor complex due to a mutation in the sorting sequence

Question 13

What are the pH values at each point of the endocytic pathway?

Answer 13

At plasma membrane pH7, at early endosome pH5.9-6.0, at late endosome pH5.0-6.0, at lysosome pH5.0-5.5

Question 14

How is pH maintained at each compartment of the endocytic pathway?

Answer 14

Vacuolar ATPase pumps protons from the cytosol across the respective membrane

Question 15

Describe the release of cholesterol from LDL from the point of internalisation.

Answer 15

LDL is internalised via clathrin-mediated pinocytosis, once in the cell the clathrin triskelion uncoats and the pinosome fuses with an early endosome, as the pH drops LDL dissociates from receptor and the receptor is returned to the plasma membrane, LDL is eventually degraded by the endolysosome to release cholesterol

Question 16

Describe the internalisation of iron from transferrin.

Answer 16

Apotransferrin binds iron to become transferrin, binds receptor and is internalised, iron dissociates at low pH while apotransferrin and receptor are recycled back to the plasma membrane

Question 17

Describe how an early endosome becomes a late endosome.

Answer 17

Rab5 is recruited to the early endosome by the GEF Rabex-5/Rabaptin-5, Rab5 recruits the Rab7 GEF Mon1/Ccz1, the Rab7 effector HOPS multisubunit tethering complex fuses the late endosome with lysosome to form an endolysosome