endocytosis and synaptic vesicle trafficking

vesicle mobilization

golgi app. makes vesicles and moved to axon terminal through anterograde axonal transport/recycled, then chaperoned to axon terminal throguh anterograde axonal transport, chaperoned to terminal with scaffolding proteins (piccolo/bassoon) interacts with Rab on synaptic vescile, and tethering proteins at active zone, PRA1, rabhphilin and RIM move vesicles for docking

Docking

vesicles are chaperoned by Munc-13 between tethering proteins and core complex. Munc-18 keeps syntaxin folded until the vesicle is close enough when Munc - 13 and Munc-18 interact to dissociate Munc-18 from syntaxin so it can form the CORE complex is formed by the coiling together of alpha helices of syntaxin, SNAP 25, and syanptobrevin (VAMP). Together, the CORE complex proteins = SNARE proteins, making a binding site for complexin

priming

complexin coils with core complex to prevent SNARE proteins from completing their coiling. Synaptotagmin also binds to CORE complex to act as a calcium sensor for a NT release

calcium sensing and vesicle fusion

calcium binds to synaptotagmin on its C2 domains which neutralizes the negative charge on the C2 domains causing a conformational change in synaptotagmin, release. hold on complexin allowing coiling of the complex, and synaptic vesicle to fuse to membrane, SNARE proteins uncoiled by a-snap and NSF. forms a cap that uses atp hydrolysis to uncoil core complex

what is endocytosis used for

recycle synaptic vesicles and recycle neurotransmitter, and takes longer than exocytosis and therefore if its continuously run for a long period time, NT pools can be depleted

what can be used to study endocytosis

patch clamp experiments to measure changes in capacitance, structural imaging with rapid-freeze electron microscopy, as well as fluorescent dyes and high resolution microscopy

what is a FM dye

fluorescent molecules that merge with lipid membranes with an increase in fluorescence intensity when bound to a membrane

pH-Sensitive fluorophores

Engineered green fluorescent protein (GFP - or another fluorescent protein which only glows at certain pH) as synaptic vesicles are more acidic than the cytoplasm and extracellular space

how are synaptic vesicles created

synaptogmin is a recognition site for endocytosis, and Ca binds to synaptotagmin causing it to bind to PIP2 (phospholipid), and and it recruits AP2 which creates a binding site for clathrin, and link together to coat the vesicle makin a curvature budding it off, and dynamin used to pinch off budded vesicle

describe the process of endocytosis of vesicles

1. NT uptake of the synaptic vesicles

2. synaptic vesicle docks onto the membrane

3. then atp binds to synaptic vesicle

4. then priming prefusion of synaptic vesicle with Ca2+

5. this causes the synaptic vesicle to undergo exocytosis

6. then vesicle uncoats and translocates and then endosome forms with proton

7. endosome fuses with the membrane

8. then endosome buds off restarting process

readily releasable pool

vesicles are docked and ready to fuse (1-2%)

recycling pool

vesicles that are ready to be docked as soon as docking sites are available (10-20%)

resting pool

sequestered vesicles bound in cytoskeletal matrix that are released as needed to replenish the other pools (80-90%)

kiss and run endocytosis

common when exocytosis is infrequent, and synaptic vesicle makes tiny openings (1-2s)

clathrin-mediated

common after high frequency bursts of neural activity - recycling (15-20s)

clathrin-mediated endocytosis

clathrin = triskeleton complex made up of 6 proteins, 3 heavy chains, 3 light chains, binds its triskeleton complex to AP2 proteins on vesicle membrane, link together to coat the vesicle creating a cage

what makes the vesicular membrane curve

the shape of the clathrin molecules linked together and form budding vesicle shape

dynamin role in vesicle formation

a gtpase that uses energy form gto to pinch off the coated vesicle which pinches off the vesicle needs to be dephosphorylated by calcium-dependent phosphatase calcineurin

what is the role of endophilin

accumulates aroudn the neck of the budding vesicle for dynamin to bind

what proteins uncoat the vesicle

hsc70, auxilin, synaptoganin, and stoned B

what is kiss and run endocytosis

increases in intracellular calcium concentration trigger more instances of kiss and run, and rate limiting step is reacidification and refilling of neurotransmitter

what is the difference between the two snare proteins

synaptic vesicles - v-SNARE, cell membrane - t-SNARE, they both play a role in determining the membrane segment to recycle for a synaptic vesicle

explain what is bulk endocytosis

intense stimulation of synaptic vesicles released and even depleted while also increase PM area, and vesicles must be replenished rapidly, also retrieves a large portion of the membrane to shrink its area while creating an infolding of membrane to produce new vesicles (4-6s)

describe alzheimer’s disease

ad a neurodegen disease, impaired memory and cognitive function, APP produced by glycoprotein and cleaved by secretase proteins to form amyloid beta, but endocytosis and sorting = determines how APP processed and if amyloid produced, endocytosis of synaptic vesicles after stimulation inc app and inc amt of amyloid produced