Week 6 (synaptic vesicle endocytosis) physiology Flashcards
basic concept of endocytosis
regenerating synaptic vesicles
endocytosis compared to exocytosis
- calcium-dependent (neuronal activity), requires less Ca2+.
- Building a synaptic vesicle from scratch - more complexity
- much slower than exocytosis (due to complexity)
is Ca2+ trigger for endocytosis the same as exocytosis?
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
calcineurin
As a protein phosphatase: activated by Ca2+ influx
Dephosphorylates the 8 essential proteins (Dephosphins) for endocytosis
endocytosis blockers
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.
invagination - coated pits
- starts to deform and bend the plasma membrane
- select the cargo (recycle the released proteins used in exocytosis) to internalise in the vesicle
coated pits - proteins
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.
AP-2 structure:
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.
AP180 structure
- N terminus: AP180 N-terminal homology domain - binds to PIP2
- middle section (DLL): 11 sites of clathrin binding
- C terminus: binds to AP2 (appendage domains) - enhances clathrin assembly (synergist)
PIP2
locally produced at the site of endocytosis: recruits AP-2 and binds to AP180
protein cargo - signal for endocytosis (examples)
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.
AP-2 and synaptotagmin effect
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.
AP-2 as a bridge
mu2 binds to synaptotagmin - plasma membrane
appendage domains bind to Clathrin and AP180
endophilin
modular: lipid modifying - invagination
binds to lipids on one end (N-BAR), binds to (Dynamin I and synaptojanin) via SH3 domain.
antibody effect on endophilin (imaging)
acutely halt endophilin action:
stimulation of endocytosis: At the coated pits, mini swelling of plasma membrane but cannot proceed to be fully invaginated.
dynamin I (GTPase)
synaptic vesicle fission
one end: GTPase
other end: Proline rich domain (PRD) - binds to amphiphysin
dynamin I fission mechanism
- wraps around the neck of invaginated vesicle.
- coil formation will stimulate GTPase activity
- GTP hydrolysis will generate energy and expand to push the vesicle away from membrane
dynamin I role in fission (experiment - mammalian)
artificially puts in non-hydrolysable analogue of GTP: cuts off energy source for dynamin I.
Cannot execute fission without GTP’s energy.
dynamin I role in fission (experiment - fly)
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.
dynamin I vesiculating lipids
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.
better mimic of lipid tubules (dynamin I)
Using natural proportions of lipids and fatty-acid galactoceramides (structural role in making the lipids a rigid tubule structure)
pinchase - dynamin I
GTP hydrolysis action of dynamin I constricts the lipid tubule (shorter diameter)
complication of pinchase experiment
the lipid tubules are too structurally-fragile, thus doesn’t accurately simulate the conditions of in situ dynamin I action.
poppase - dynamin I
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.
complication of poppase experiment
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.
(accepted) twistase - dynamin I
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.
twistase bead experiment
tubule-bound bead twisting as GTP is added, showing twisting action.
dynamin I locating vesicle neck - protein involved
amphiphysin I, II recruits dynamin to invaginated coated pits
amphiphysin
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
Excessive amphiphysin-SH3 injection into nerve terminal
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?
uncoating - proteins
synaptojanin: nerve terminal lipid phosphatase
auxilin: chaperone
hsc70: ATPase (chaperone)
uncoating process
Final result: clathrin and AP-2 are all stripped off.
- auxilin and auxilin-bound hsc70 is recruited to the coating since auxilin can bind to clathrin and AP-2 (C-terminus of auxilin)
- Hsc70 hydrolyses ATP - strips off clathrin coat.
- 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.
synaptojanin
dephosphorylate membrane lipid (PIP2)
PIP2: locally produced lipid that concentrates at endocytosis site.
synaptojanin KO mice
Nerve terminals had 10x more clathrin-coated vesicles than wild type mice.
auxilin - hsc70
auxilin c terminus: binds to hsc70 (ATPase)
auxilin binds to clathrin and AP-2 (like amphiphysin)
hsc70 hydrolyses ATP for energy to uncoat auxilin.
nerve terminal injected with mutant hsc70-unbindable auxilin (squid)
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.
does endocytosis occur at a defined region?
Anywhere but the active zone: shown by the fact that intersectin (an endocytosis protein) is absent in the active zone regions.
lamprey nerve terminal high freq/long duration stimulation
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.
where does SV endocytosis occur?
just outside of active zone - periactive zone
why does SV endocytosis occur only around the active zone?
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.
