Synaptic vesicles and synaptic vesicle recycling

Question 1

What process allows vesicles to be recycled from the synaptic cleft? Or even to be formed de novo?

Answer 1

Clathrin-mediated endocytosis.

Question 2

Where are found v-SNAREs? t-SNAREs?

Answer 2

v-SNAREs are found on the SV, t-SNAREs are found on the membrane in active zones

Question 3

Explain how NSF, SNAPs and SNARE interact.

Answer 3

NSF is an ATPase that triggers the fusion of the vesicle membrane with the plasma membrane (?)

SNAPs (soluble NSF attachment proteins) allow NSF to bind

SNARE proteins are SNAP receptors (SNAp REceptors), interaction between v-SNARE and t-SNARE creates a binding site for SNAPS, where NSF can be recruited

Question 4

What forces must be surmounted for membrane fusion to occur at active sites? What aspect of the SNARE complex structure allows to surmount those forces?

Answer 4

1. V and P membranes are negatively charged
2. Protein density

The coiled-coil interactions between the helices of the v-SNARE and t-SNARE store a lot of energy, enough to bring membranes together.

Question 5

Is synaptotagmin a SNARE protein?

Answer 5

No.

Question 6

On which membrane are the following SNARE proteins located?

Answer 6

Vesicle membrane: synaptobrevin
Plasma membrane: syntaxin, SNAP-25

Question 7

What is the calcium sensor for the SNARE complex to carry fusion?

Answer 7

Synaptotagmin

Question 8

What sites of synaptotagmin bind Ca++?

Answer 8

C2 domains

Question 9

True or false: calcium influx allows the SNARE complex to assemble and carry fusion.

Answer 9

False: the SNARE complex is already assembled and ready for fusion, only awaiting for Ca++ influx to trigger the fusion

Question 10

Is it clathrin itself that forces curvature of vesicles endocytosed?

Answer 10

No, it is thought that it is rather other proteins that bind on the scaffolds formed by clathrin that interact with the lipid membrane to force curvature of the invaginated membrane

Question 11

Does clathrin nonspecifically induce endocytosis?

Answer 11

No, they mediate the endocytosis of specific plasma membrane cargos such as receptors

Question 12

What recruits the clathrin triskelia to the plasma membrane? And how can clathrin identify the cargo to endocytose into a vesicle?

Answer 12

Occurs through adaptor proteins, like AP-2

Question 13

How does AP-2 get recruited to the plasma membrane?

Answer 13

Through interactions with cell membrane phospholipid component PtdIns(4,5)P2

Question 14

What protein nucleates the formation of the clathrin-coated pit?

Answer 14

PtdIns(4,5)P2

Question 15

What is the interaction site between AP-2 and clathrin for the recruitment of the latter?

Answer 15

Beta-hinge

Question 16

Which receptors are endocytosed by clathrin?

Answer 16

Those bound to AP-2

Question 17

When does PtdIns(4,5)P2 get inserted in the membrane?

Answer 17

When the SV first fuses for exocytosis

Question 18

Apart from AP-2, what other protein is thought to orient clathrin to its proper location on the plasma membrane?

Answer 18

Synaptotagmin, which was first present on the SV before fusing with the plasma membrane

Question 19

Why does the clathrin triskelia have an inherent ability to assemble into cages?

Answer 19

Clathrin triskelia itself is convex

Question 20

What protein is responsible to severe the SV formed on the cytosolic side by its clathrin coat?

Answer 20

Dynamin (GTPase): mechano-chemical enzyme