Week 6 (synaptic vesicle endocytosis) physiology

Question 1

basic concept of endocytosis

Answer 1

regenerating synaptic vesicles

Question 2

endocytosis compared to exocytosis

Answer 2

1. calcium-dependent (neuronal activity), requires less Ca2+.
2. Building a synaptic vesicle from scratch - more complexity
3. much slower than exocytosis (due to complexity)

Question 3

is Ca2+ trigger for endocytosis the same as exocytosis?

Answer 3

Not synaptotagmin:
1. high affinity for Ca2+ influx (requires low concentration)
2. Ba2+ is a similar compound as Ca2+, it could bind to synaptotagmin and activate exocytosis but not endocytosis

Question 4

calcineurin

Answer 4

As a protein phosphatase: activated by Ca2+ influx
Dephosphorylates the 8 essential proteins (Dephosphins) for endocytosis

Question 5

endocytosis blockers

Answer 5

cyclosporin A and FK506 (very selective blockers of endocytosis):
experimental evidence:
- inhibit uptake of a fluorescent dye (FM1-43)
- cyclosporin A will deplete the nerve terminal of vesicles but the nerve terminal will have a larger surface area.

Question 6

invagination - coated pits

Answer 6

• starts to deform and bend the plasma membrane
• select the cargo (recycle the released proteins used in exocytosis) to internalise in the vesicle

Question 7

coated pits - proteins

Answer 7

Clathrin (2 subunits): assembles to triskelia in a stable state (light chain in the middle, heavy chain outside), easy to build and form triskelia.
Clathrin defines the structure and size of the vesicle.
AP-2: helps assembly of clathrin cages, act as a bridge between clathrin and synaptic vesicle proteins, links the essential proteins for endocytosis to the coated pits.
AP180: main helper of clathrin cage assembly, regulator that ensures the homogenous assembly of clathrin cages.

Question 8

AP-2 structure:

Answer 8

4 polypeptide subunits - adaptins (2 big ones: alpha2 and beta2)

PIP2 and protein cargo - eg. synaptotagmin: binds to mu2

HINGE REGION and APPENDAGE DOMAIN: alpha2 and beta 2 respectively has an extended arm with the start called the hinge region and an end called the appendage domain.

CLATHRIN: Clathrin will bind to beta2’s hinge region and appendage domain.

AP 180: AP180 will bind to the 2 appendage domains

OTHER PROTEINS (amphiphysin and auxilin) will bind to alpha2’s appendage domain.

Question 9

AP180 structure

Answer 9

1. N terminus: AP180 N-terminal homology domain - binds to PIP2
2. middle section (DLL): 11 sites of clathrin binding
3. C terminus: binds to AP2 (appendage domains) - enhances clathrin assembly (synergist)

Question 10

PIP2

Answer 10

locally produced at the site of endocytosis: recruits AP-2 and binds to AP180

Question 11

protein cargo - signal for endocytosis (examples)

Answer 11

signal sequences that are tyrosine based, or dileucine (LL) based.
- synaptotagmin - binds AP-2 via C2B domain
- Many proteins including synaptophysin have tyrosine based signal sequences.

Question 12

AP-2 and synaptotagmin effect

Answer 12

synaptotagmin is integral of plasma membrane, its C2B domain will bind to AP-2 in a calcium-indep. manner (mu2 subunit).

Other vesicle proteins will increase affinity to AP-2 and a cluster will form after synaptotagmin binds to AP-2.

Question 13

AP-2 as a bridge

Answer 13

mu2 binds to synaptotagmin - plasma membrane
appendage domains bind to Clathrin and AP180

Question 14

endophilin

Answer 14

modular: lipid modifying - invagination
binds to lipids on one end (N-BAR), binds to (Dynamin I and synaptojanin) via SH3 domain.

Question 15

antibody effect on endophilin (imaging)

Answer 15

acutely halt endophilin action:
stimulation of endocytosis: At the coated pits, mini swelling of plasma membrane but cannot proceed to be fully invaginated.

Question 16

dynamin I (GTPase)

Answer 16

synaptic vesicle fission
one end: GTPase
other end: Proline rich domain (PRD) - binds to amphiphysin

Question 17

dynamin I fission mechanism

Answer 17

1. wraps around the neck of invaginated vesicle.
2. coil formation will stimulate GTPase activity
3. GTP hydrolysis will generate energy and expand to push the vesicle away from membrane

Question 18

dynamin I role in fission (experiment - mammalian)

Answer 18

artificially puts in non-hydrolysable analogue of GTP: cuts off energy source for dynamin I.

Cannot execute fission without GTP’s energy.

Question 19

dynamin I role in fission (experiment - fly)

Answer 19

shibire mutation that led to temperature sensitive defect:
restricted dynamin from binding to GTP or hydrolysing it

flies are paralysed when temperature raises beyond normal range.

Question 20

dynamin I vesiculating lipids

Answer 20

turns lipid tubules to liposomes
liposomes consist of mostly phosphatidylserine (making the tubules fragile), which doesn’t completely mimic the active zone plasma membrane which has other types of lipids.

Question 21

better mimic of lipid tubules (dynamin I)

Answer 21

Using natural proportions of lipids and fatty-acid galactoceramides (structural role in making the lipids a rigid tubule structure)

Question 22

pinchase - dynamin I

Answer 22

GTP hydrolysis action of dynamin I constricts the lipid tubule (shorter diameter)

Question 23

complication of pinchase experiment

Answer 23

the lipid tubules are too structurally-fragile, thus doesn’t accurately simulate the conditions of in situ dynamin I action.

Question 24

poppase - dynamin I

Answer 24

Using the rigid nanotubules, the pitch (space between the dynamin helices) is larger in the hydrolysed tubules compared to the non-hydrolysed.
During the process, there will be a mix of different pitches, but will eventually reach the largest pitch.

Showing that dynamin expanded.

Question 25

complication of poppase experiment

Answer 25

the nanotubule's structure is too rigid thus can only show that GTPase activity of dynamin I expands the tubules and has a looser pitch.

Question 26

(accepted) twistase - dynamin I

Answer 26

during the twisting action: the length of tubules doesn't change, supercoiling of the tubules may occur. Fission will occur as the twisting force overcomes the maximum tension that the tubule can take.

Question 27

twistase bead experiment

Answer 27

tubule-bound bead **twisting** as GTP is added, showing twisting action.

Question 28

dynamin I locating vesicle neck - protein involved

Answer 28

amphiphysin I, II recruits dynamin to invaginated coated pits

Question 29

amphiphysin

Answer 29

In the middle, the **CLAP** region binds to **clathrin and AP-2** amphiphysin binds to **AP-2**'s appendage domain, and AP-2's mu2 subunit binds to the membrane-bound receptor. Thus amphiphysin **recruits dynamin** to AP-2 which is near the invagination site. C terminus: SH3 binds to **dynamin I**

Question 30

Excessive amphiphysin-SH3 injection into nerve terminal

Answer 30

Excessive injected SH3 domain will competitively bind to dynamin I. Under stimulation: Blocks endocytosis and stuck due to absence of dynamin recruitment and its twisting action. Added: could add free fatty tubules in the same environment to see if they are vesiculated?

Question 31

uncoating - proteins

Answer 31

synaptojanin: nerve terminal **lipid phosphatase** auxilin: chaperone hsc70: ATPase (chaperone)

Question 32

uncoating process

Answer 32

Final result: clathrin and AP-2 are all stripped off. 1. **auxilin** and auxilin-bound hsc70 is recruited to the coating since auxilin can bind to clathrin and AP-2 (C-terminus of auxilin) 2. **Hsc70** hydrolyses ATP - **strips off clathrin coat**. 3. **synaptojanin** destroys PIP2 (**weakens AP-2** interaction with vesicle membrane - as **AP-2 is bound to PIP2, a membrane phospholipid**) weakens interaction of other endocytosis proteins.

Question 33

synaptojanin

Answer 33

dephosphorylate membrane lipid (PIP2) PIP2: locally produced lipid that concentrates at endocytosis site.

Question 34

synaptojanin KO mice

Answer 34

Nerve terminals had 10x more clathrin-coated vesicles than wild type mice.

Question 35

auxilin - hsc70

Answer 35

auxilin c terminus: binds to **hsc70** (ATPase) auxilin binds to **clathrin and AP-2** (like amphiphysin) **hsc70 hydrolyses ATP** for energy to uncoat auxilin.

Question 36

nerve terminal injected with mutant **hsc70-unbindable auxilin** (squid)

Answer 36

this mutant auxilin competes with normal auxilin, reducing the function of normal auxilin clathrin-coated vesicle in the nerve terminal interaction of auxilin/hsc70 is crucial for uncoating.

Question 37

does endocytosis occur at a defined region?

Answer 37

Anywhere but the active zone: shown by the fact that **intersectin** (an endocytosis protein) is absent in the active zone regions.

Question 38

lamprey nerve terminal high freq/long duration stimulation

Answer 38

convert as much vesicle membrane as possible to the plasma membrane. A great proportion of the membrane is waiting to endocytose (reverse priming) No proceeding without calcium (calcium-dependent) Calcium stimulation restores normal condition.

Question 39

where does SV endocytosis occur?

Answer 39

just outside of active zone - periactive zone

Question 40

why does SV endocytosis occur only around the active zone?

Answer 40

**local calcium influx** (caged Ca2+ experiment): as Ca2+ level gets above 250nM: endocytosis is reduced. above 1microM: ceased endocytosis **Ca2+ inhibits dynamin I GTPase** activity (reduced vesiculation). But Ca2+ is also needed for calcineurin activity to activate endocytosis There's a **defined ring range** (not too close for inhibition or too far from triggering calcineurin) around the active zone for endocytosis to occur.