Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

NESC 2570 > krueger 3 > Flashcards

krueger 3 Flashcards

1
Q

transmitter exocytosis needs? (2)

A

priming

calcium dependent fusion of SV

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what happens during priming (exocytosis)

  • requires?
  • main purpose
A
  • SV membrane and plasma membrane are brought close in proximity through interactions of SV membrane proteins and plasma membrane proteins
  • required 3-4 SNARE proteins
  • NSF and SNAP involved (regulate SNARE)
  • main purpose: organize SNARE proteins into correct conformation for membrane fusion
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Ca dependent fusion of SV

A
  • cooperative Ca binding to the Ca sensory leads to the fusion of closely apposed membranes
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what is involved to bring SV and plasma membrane close in proximity

A
  • membrane fusion involves SNARE proteins

- 3.4 SNARE, on 2 fusing membranes form SNARE complex

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

what is a SNARE protein

A

SNAP receptors

  • do not bind Ca
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what is a SNARE complex, how is it assembled?

A

assembly of SNARE motifs into parallel 4-helical bundle

  • assembled by formation of helical bundle of
    one SNARE motif of each synaptobrevin and syntaxin
    and 2 SNARE motifs of SNAP-25
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

SV exocytosis is mediated by… (3 proteins)

A

1) SNAREs synataxin (plasma membrane)
2) SNAP-25 (plasma membrane)
3) Synaptobrevin (SV membrane)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

what inhibits neurotransmitter release by cleaving SNARE proteins?

A

Clostridial neurotoxins
- responsible for tetanus and botulism (proteases), block neurotransmitter release by cleaving SNAREs

  • this inhibition of neurotransmitter release shows the SNARE complex formation is required for synaptic vesicles
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

potential role of the SNARE complex in membrane fusion

A
  • free energy gained during formation of SNARE complex may be utilized for energetically unfavourable membrane fusion
  • SNARE complex may be “minimal machinery” sufficient for membrane fusion
  • SNARE complex, is not sufficient for kinetically efficient membrane fusion
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

roles for accessory proteins in aiding SNARE complex in fusion (2)

A

1) keep in position favourable for membrane fusion

2) structurally organize several SNARE complexes that have to act cooperatively

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Munc18

  • 2 functions
A
  • essential for neurotransmitter release
  • genetic ablation causes inhibition of both Ca-dependent and spontaneous neurotransmitter release

1) bind to syntaxin in “closed” conformation (inhibiting SNARE complex formation)
- may regulate syntaxin recruitment into SNARE complexes

2) binds tightly to syntaxin complexes
- enhances fusion of synaptobrevin, syntaxin and SNAP-25
- essential part in fusion of machinery
- facilitating SNARE complex mediated fusion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

disassembly of SNARE complex

A
  • need to be dissasembled after fusion to allow reuse of individual SNAREs
  • SNARE complexes are very stable– a chaparone is needed to dissociate it
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what is a chaparone?

A

protein folding enzyme

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is the chaparone that dissociates SNARE complexes?

  • how does it dissociate?
A

NSF

  • binds to complex via an adaptor protein (a-SNAP)
  • unwinds the a-helical bundle of SNARE comples, using ATP as energy source
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

2 proteins that unwind SNARE comples

A

NSF and a-SNAP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Synaptotagmin is made up of

A
  • Ca and phospholipid binding protein

composed of
- short N-terminus intravesicular sequence
- single transmembrane region
2 cytoplasma C2 domains

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

role of synaptotagmin (3)

A
  • both C2 domains bind Ca (Ca binding is cooperative)
  • C2 domains also bind to phospholipid membranes in Ca-dependent and Ca in a phospholipid-dependent manner
  • also binds to SNARE complexes (in partly Ca-dependent manner)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

evidence that synaptotagmin is the Ca sensory responsible for Ca-dependent fast transmitter release

A
  • destroy gene, no synaptotagmin left, eliminated fast release, no fusion of SV to plasma membrane
  • in synaptotagmin knockout mice, fast transmitter release is absent
  • spontaneous AP and Ca-independent release is unchanged— vesicle fusion not affected
  • asynchronous transmitter release unaffected by synaptotagmin, it is caused by SV fusions that are triggered in Ca-dependent manner, with some delay, has to rely on other Ca sensory (not synaptotagmin)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

how synaptotagmin allows for SV fusion (3)

A

1) synaptotagmin binds to SNARE complex

3) energetically unfavourable, promoting membrane fusion (lipid layers fuse, and have fusion pore)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

endocytosis at presynaptic cell is…

A

clathrin mediated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

clathrin mediated endocytosis steps (4)

A

1) nucleation- assembly of clathrin lattice on patches of plasma membrane to be endocytosed
2) Membrane invagination- generation of clathrin-coated pits through changes in membrane curvature
3) fission- of clathrin coated membrane invagination to create clathrin-coated vesicles
4) uncoating- disassembly of clathrin coat

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

what is nucleation (endocytosis)

A
  • begins with recruitment of adaptor protein AP-2 to plasma membrane region to be endocytosed
  • synaptic vesicle proteins containing tyrosine-based AP-2 binding motif recruiting them

AP180 (neuron specific protein), binds AP-2 and clathrin
- required for assembly of clathrin coats

  • patches of AP-2 and AP180 recruit clathrin (triskelion structure), oligomerize into lattice with characteristic ultrastructure
23
Q

lattice of clathrin made up of.. (2)

  • structure name
A

heavy chain

light chain

  • triskelion structure (3-legged appearance)
24
Q

2 things involved in fission of clathrin-coated vesicles from plasma membrane

A

Dynamin and Actin

25
Q

role of dynamin in membrane fission

A

dynamin= GTPase

  • absence causes arrest of SV endocytosis at stage of deeply invaginated pits
  • with endophilin and amphiphysin, dynamin oligomerizes into stacks of rings around the membrane stalk of coated pits
  • pinches off synaptic vesicle
26
Q

role of actin in membrane fission

A
  • perturbation of actin function inhibits endocytosis, leading to accumulation of coated puts with a wide neck
  • dynamin interacts with proteins that participate in regulation of actin cytoskeleton= syndapin
  • actin is localized to coated pits immediately following the recruitment of dynamin
  • helps dynamin pinch off vesicle
  • exist in 2 forms
    1) g-actin
    2) f-actin - generated to force to pinch off SV
27
Q

process of uncoating of clathrin

  • 2 things required
A
  • uncoating follows fission of vesicles from plasma membrane

Required: chaparone (unfolding protein)= Hsc70 and cofactor auxilin

  • auxilin binds clathrin and AP-2 and targets Hsc70 to clathrin coat
  • subsequent removal of the adaptor proteins is Hsc70-independent
28
Q

regulation of endocytosis at presynaptic terminals

A
  • endocytosis in neurons is highly regulated and coupled to exocytoss
  • phosphatidylinositides (photpholipids containing inositol head groups), may represent the regulative link between endocytosis and exocytosis
29
Q

PI(4,5)P2

A

phosphatidylinositol-4,5-bisphosphate

  • plasma membrane is enriched with it
  • generated by PIPK1y
  • PIPK1y activated by neuronal stimulation
  • PIP(4,5)P2 dephosphorylated by synaptojanin
  • synaptojanin= phosphatase localized to clathrin- coated membranes
30
Q

why generation and degradation of PIP(4,5) P2 is important for endocytosis (2 reasons)

A

1) absence of PIPK1y leads to delayed endocytosis
2) presynaptic terminals deficient in synaptojanin show large increase in number of coated vesicles and decrease in SV available for exocytosis

31
Q

PI(4,5)P2 role in clathrin-mediated endocytosis

  • in the 4 steps
A

1) nucleation: AP-2 needs PIP2 to bind to SV proteins in plasma membrane, AP180 also recruited to plasma membrane by PIP2
2) invagination: epsin recruitment and activity in inducing membrane bending is dependent on PIP2
3) fission: dynamin binds PIP2, which may contribute to its recruitment to coated pits

4) uncoating: clathrin-coated vesicles are only coated in absence of PIP2 in vesicle membrane
- clathrin only dissociated from vesicle membrane protein if PIP2 levels are low due to metabolization by synaptojanin

32
Q

are recently endocytosed synaptic vesicles actively transported back to the active zone?

A

SV only have to travel short distances from sites of endocytosis to nearby active zone

  • but passive diffusion may be to ineffective for fast recycling
33
Q

are there mechanisms to ensure that endocytosed SV are targeted to the active zone?

A

many vesicular transport processes in nonneuronal and neuronal cells are directional and regulatory mechanisms exist that target transport vesicles to the correct acceptor membrane

34
Q

is the transport of recycled vesicles back to the active zone regulated, influencing their availability to release?

A
  • the presence of different SV pools that vary in their availability for release suggests that there may be regulatory mechanisms that prevent many recycling vesicles from reaching active zone
35
Q

the actin cytoskeleton may be involved in transport of SV

A
  • F-actin may serve as cytoskeletal tracks for transport of vesicles between endocytotic sites and SV cluster
  • F-actin may facilitate the transport between SV pool and active zone
36
Q

actin may provide a barrier between synaptic vesicle cluster and active zones

A
  • actin cytoskeleton surring the SV cluster may prevent vesicles in reserve pool from reaching active zone (might hold back SV, preventing reaching plasma membrane)
  • actin barrier may break down in an activity-dependent manner, allowing vesicles from reserve pool to participate in release after prolonged stimulation
37
Q

synapsins

  • regulate
  • abundance of..
A
  • regulate the availability of SV for release
  • they are abundant peripheral membrane proteins on synaptic vesicles
  • oligomerize and bind to actin, may anchor SV in reserve pool to actin cytoskeleton
  • connection between actin and vesicles can be modulated by phosphorylation causes dissociation of synapsins from SV
38
Q

function of synapsins

A

maintenance of reserve pool of SV, making reserve pool available for release in response to sustained activity

39
Q

Rab3 is..

involved in…

associates with…

required for..

A

GTP-binding proteins

  • involved in vesicular transport throughout cell
  • serve as “identifying tags” of specific transport vesicle, interacting with specific effectors on target membrane (zipcode)
  • associated with synaptic vesicle membrane via lipid anchor
  • required for targeting SV to active zone
40
Q

Rab3 effectors (2)

A

1) Rabphilin- cytosolic protein, unknown function

2) RIM- protein resident in active zone

41
Q

active zone ultrastructure

A
  • docked SV surrounded by filamentous material (CAZ= cytoskeletal matrix at active zones)
  • CAZ at neuromuscular junctions, and possibly CNS synapses has modular structure
42
Q

CAZ function

3 things it does

A
  • limits calcium-dependent neurotransmitter release to sites that are apposed to postsynaptic specializations by..
    1) provide docking sites for SV, facilitate priming
    2) recruit voltage-gated Ca channels to release sites (close proximity necessary)
    3) recruit cell adhesion proteins, mediate contact to postsynaptic specialization (trans-synaptic contact between 2 neurons)
43
Q

CAZ constituents

  • proteins involved with scaffolding and cell adhesion of active zone
A
  • active zone of cytomatix consists scaffolding proteins, that have multiple, strong protein interactions with other CAZ proteins, SV proteins, voltage-gated Ca channels, and synaptic cell adhesion proteins

proteins

  • munc13
  • RIM and RIM-BP
  • a-liprins
  • CASK
  • neurexins
  • protein tyrosine phosphatase receptors

(Proteins such as syntaxin, SNAP-21 and munc18 are not restricted to active zone, but present throughout axonal plasma membrane, not considered CAZ components)

44
Q

role of Mun13

A
  • regulator of vesicle priming
  • essential for neurotransmitter release
  • release of glutamate and GABA completely inhibited
  • proteins bind to syntaxin (stabilize it, facilitatin SNARE complex formation)
45
Q

Munc13 regulated by 2 things

  • isoforms bind..?
A

1) diacylglycerol- binding of its 2nd messenger to central C1 domain in enhanced neurotransmitter release
2) calmodulin- Ca/calmodulin binding facilitates priming
- isoforms bind RIM (scaffolding protein)

46
Q

RIM function

A
  • recruits voltge-gated Ca channels to the CAZ
  • interacts with RIM-binding protein (RIM-BP)
  • facilitates docking of SV to plasma membrane (via interactions with Rab3)
  • interact with Munc113- regulation of priming and Ca-dependent fusion
47
Q

transsynaptic cell adhesion

A
  • Cell adhesion molecules (CAM) meduate contact between pre and post synaptic plasma membrane
  • presynaptic neurexins bind to postsynaptic neuroligins and LRRTMS
  • LAR family protein tyrosine phosphatase receptors bind to NGL-3
  • all the CAM are recruited by scaffolding proteins
48
Q

examples of how scaffolding proteins anchor/bind CAMs in CAZ

A
  • in active zone cytomatirx, scaffolding protens bind to presynaptic CAMs
  • a-liprins (scaffolding protein) bind to one of the phosphatase domains of LAR family PTPR (tyrosune phosphatase receptor)
  • also bind to other CAZ scaffolding proteins (ex: RIM, anchoring PTPR in the CAZ)
  • CASK- membrane associated guanylate kinase, contains PDZ domain that binds to neurexins, also bind to a-liprins
  • post synaptic CAMS are likewise anchored in the postsynaptic specialization via postsynaptic scaffolding proteins such as PSD-95
49
Q

What is the function of synaptobrevin, syntaxin, and SNAP25 in NT release? What is the role
of Munc18, NSF and α-SNAP?

A

Synaptobrevin,
- a SNARE protein on the synaptic vesicle membrane as well as syntaxin and SNAP25, SNARE proteins on the plasma membrane, form a protein complex during synaptic vesicle priming.
- Complex formation is energetically favourable and supplies the energy needed to bring the negatively
charged synaptic vesicle and plasma membranes into close contact.

Munc-18
- may have a dual role. It binds and stabilizes syntaxin and may control the spatially correct assembly of core complexes for SNARE-dependent fusion.

NSF

  • is an ATPase (ATP consuming enzyme) that disassembles the SNARE complex following synaptic vesicle fusion, making its components available for another round of synaptic vesicle exocytosis.
  • NSF requires α-SNAP to bind to the SNARE complex.
50
Q

Which protein triggers synaptic vesicle fusion in response to action potential-evoked calcium
influx and does it work mechanistically?

Is this protein required for all forms of NT release?

A

The protein triggering synaptic vesicle fusion in response to action potential-evoked calcium influx is synaptotagmin. - Synaptotagmin-1 is a membrane protein in synaptic vesicles that binds to the SNARE complex and is thus in close proximity to both synaptic vesicle membrane and plasma membrane.
- Elevation of the calcium concentration leads to binding of 4 or 5 calcium ions to two C2 domains in synaptotagmin.
- This increases the affinity of synaptotagmin for phospholipids present in synaptic vesicle and plasma
membrane.
- Increased membrane interactions of synaptotagmin may bring synaptic vesicle membrane and plasma membrane into even closer contact, thus leading to their fusion.

51
Q

What role do clathrin, dynamin, Hsc70, auxilin, PIP kinase 1γ, and synaptojanin have in endocytosis?

How is SV endocytosis coupled to SV exocytosis?

A

Clathrin
— A protein that oligomerizes to form a polyhedral lattice that forms over membrane undergoing endocytosis.
- Clathrin may contribute to membrane invagination.

Dynamin
— GTPase (GTP consuming enzyme) that causes the fission of newly formed synaptic vesicles from the plasma membrane.

Hsc70
— ATPase functioning in the disassembly of the clathrin coat after endocytosis of a vesicle.

Auxilin
— An adaptor protein aiding Hsc70 in the disassembly of the clathrin coat.

PIP kinase 1γ
— Generates Phosphatidylinositol-4,5-bisphosphate (PIP2) in response to membrane depolarization.
- PIP2 facilitates clathrin-mediated endocytosis.

Synaptojanin
–– Enzyme that dephosphorylates PIP2.
- Required to initiate uncoating of endocytosis synaptic vesicles.

52
Q

Which proteins are responsible for targeting SVs to the active zone?

Which proteins regulate SV recycling to the active zone?

A

F-actin
— may be involved in transport of vesicles between endocytotic site, synaptic vesicle cluster, and active zone.
- Alternatively, could prevent synaptic vesicles from reaching active zone.

Synapsin
— Protein associating with synaptic vesicle membranes and F-actin. Links synaptic vesicles to actin cytoskeleton, potentially keeping them from reaching active zone.
- Phosphorylation of synapsin leads to dissociation from synaptic vesicles; may provide additional synaptic vesicles after intense synaptic stimulation.

Rab3:

  • G protein that associates with synaptic vesicle membranes when in GTP bound state.
  • Binds to active zone cytomatrix protein RIM, thus targeting synaptic vesicles to active zone cytomatrix.
53
Q

What is the function of the active zone cytomatrix?

What role do Munc13, RIM, α- liprins,CASK, neurexins and LAR family protein tyrosine phosphatase receptors have?

A

Active zone cytomatrix

(a) provides docking sites for synaptic vesicles and facilitates priming
(b) highly enriched in voltage-gated calcium channels
(c) enriched in cell adhesion proteins that provide contact to postsynaptic specialization

Munc-13
- facilitates synaptic vesicle priming.
- Binds to syntaxin and stabilizes it in open conformation,
available for binding with synaptobrevin and SNAP-25.
- Regulated by calcium and diacylglycerol (second
messenger).

RIM:
(a) Recruits voltage-gated calcium channels
(b) interacts with Rab3 and thus facilitates targeting
of synaptic vesicles to active zone cytomatrix
(c) interacts with Munc13 and has role in priming

Neurexins and protein tyrosine phosphatase receptors:
- Presynaptic cell adhesion molecules that mediate transsynaptic contacts

α-liprins and CASK:
- Presynaptic scaffolding proteins that bind to neurexins (CASK) and protein tyrosine phosphatase receptors (α-liprins)

NESC 2570 (5 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions