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ANAT365 > Module 9 - polarity > Flashcards

Module 9 - polarity Flashcards

1
Q

what is cell polarity?

A

Some cells have distinct environments on either side of the cell (apical vs basolateral)

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2
Q

name an example of a cell that has distinct environment on either side of it (polarized cell)

A

kidney cells

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3
Q

describe apical vs basolateral side of a cell

A
  • Apical side faces the outside world; often hostile environments
  • Basolateral side faces the inside world; neighboring cells and internal tissues
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4
Q

give an example of what can make the outside world faced by the apical side hostile

A

stomach acids

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5
Q

name some examples of proteins destined for a specific environment (apical/basolateral)

A

ion channels, transporters, lipids, other PM and secreted proteins

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6
Q

what kind of junctions define interface between apical and basolateral sides

A

tight junctions

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7
Q

what are tight junction’s function?

A
  • prevent leakage through epithelial layer
  • prevents membrane proteins diffusing back and forth between 2 sides
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8
Q

what are called the 2 general solutions to cell polarity?

A
  • vectorial sorting
  • selective retention
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9
Q

explain vectorial sorting

A

components of apical and basolateral
membranes are sorted into distinct vesicles in the TGN or in endosomes that then fuse specifically to their target membranes

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10
Q

explain selective retention

A

After fusion, proteins are endocytosed and reinserted if they are in the wrong compartment/membrane until they reach the right one

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11
Q

what determines what polarity sorting mechanism is used?

A

both can happen at the same time, sometimes just one.
depends on cell type and on the protein itself.

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12
Q

what do “sorting” endosomes do?

A

many sorting decisions are made in sorting endosomes! and not in TNG

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13
Q

how do proteins who are mistargeted get to the right membrane?

A

they are endocytosed in early endosomes and go into recycling endosomes, and then to their good membrane

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14
Q

how does basolateral sorting happen?

A
  • Direct sorting in TGN into vesicle
  • Sorting to common endosome, then vesicles
  • Non-specific sorting and then recycling
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15
Q

where are basolateral signals found?

A

in cytoplasm (except exception)

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16
Q

what are the 2 basolateral signals and what do they bind to?

A
  • tyrosine-based motif (YXXO) binds mu subunit of AP1 and AP2
  • Di-leucine motif (D/ExxxLL or RxxxLL or LLxxxD/ED/E) binds beta subunit of AP1 and AP2
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17
Q

what can O be in Tyrosine-based motif (YXXO)?

A

bulky hydrophobic group such as phenylalanine or tryptophan

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18
Q

what do APs bind to again?

A

bind proteins on membrane and clathrin coat

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19
Q

what coat protein mostly mediates basolateral sorting?

A

clathrin (Similar signals as endosome and endocytosis)

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20
Q

where are AP1 and AP2 found?

A

cytoplasm

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21
Q

what is AP1B?

A

Special subunit of AP-1 only found in polarized cells contains different m subunit (m1B) that binds to cargo

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22
Q

what does m subunit of AP1B bind to?

A

tyrosine based motifs

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23
Q

where is AP1B found vs AP1?

A

they do not colocalize: AP1B is at recycling endosome, AP1 is at the TNG
(they work in step)

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24
Q

what motif is AP1B only important for?

A

tyrosine-based motif (does not affect dileucine based signals)

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25
Q

do all basolateral proteins bind AP1B?

A

no

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26
Q

AP1B is specific for what kind of cells?

A

polarized cells

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27
Q

how does apical sorting work?

A

no specific direct signal for apical membrane!
- directly through transport vesicles
- sorting through endosomes
- sorting through basolateral membranes and then transcytosis through endosomes

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28
Q

where are apical signals found?

A

in the membrane or the lumen

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29
Q

name apical signals

A
  • Lipid linkage (GPI)
  • Transmembrane domains
  • Glycosylation signals (O-glycosylation, N-glycans); aggregation by co-factors
  • Segregation into rafts by aggregation of proteins
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30
Q

what and where is the exceptions sorting signal?

A

Syntaxin-III; in cytoplasmic exceptionally

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31
Q

where are basolateral vs apical sorting signals found?

A

basolateral = outside vesicle
apical = inside vesicle

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32
Q

how does raft clustering and domain-induced budding work?

A
  • apical-targeted proteins are aggregated by a lumenal adaptor to the membrane
  • lipid (rafts) segregation and aggregation
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33
Q

why can apical vesicles have weird shapes?

A

there is no known coat on apical-transported structure

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34
Q

what type of sorting is most complicated? what is a key component of that mechanism?

A

apical; aggregation

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35
Q

what are the 2 types of APICAL sorting that happen in endosomes and exit vesicles from TNG?

A
  • protein aggregates with annexin in lipid rafts
  • protein aggregates with a leptin, galectin 3
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36
Q

where are the aggregation molecules annexin, leptin, galectin 3 found?

A

in the lumen of the vesicle

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37
Q

which type of transport was blocked by tetanus toxin? how?

A

basolateral transport is blocked because tetanus cleaves V-SNARES

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38
Q

why is apical sorting not sensitive to tetanus toxin?

A

because the V-SNARE used, TI-VAMP, is not sensitive to toxin

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39
Q

what is Cellubrevin implicated in?

A

AP1B vesicle fusion

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40
Q

what is blocked by TI-VAMP ko?

A

direct route of apical sorting, but not indirect route!

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41
Q

what protein is used for the indirect route of apical sorting?

A

VAMP8

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42
Q

where are t-SNAREs syntaxin 3 vs 4 found?

A

Syntaxin3 found in apical membrane, Syntaxin4 found on basal membrane

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43
Q

what type of cells have the most polarity?

A

neurons

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44
Q

in neurons, what is a modification of apical (axon) vs basal (dendrite) polarity?

A

axon vs dendrite differentiation

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45
Q

what is Axon Hillock?

A

site of AP generation in a neuron

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46
Q

where does vesicular sorting happen in a neuron?

A

cell body (same spot as protein synthesis)

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47
Q

what is axon determination?

A

which process of a developing neuron will become the axon

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48
Q

what are the axon determination rules?

A
  1. longest neurite becomes axon
  2. neurite with greatest amounts of dynamic actin becomes axon
  3. cutting an axon shorter than another neurite allows other neurite to become axon
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49
Q

how is the longest neurite hypothesized to be determined?

A

May be determined by last cell division by placement of centrosome and golgi apparatus

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50
Q

what is cytochalasin D and what does it do?

A

it’s a drug that destabilizes actin filaments and allows multiple axons to grow

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51
Q

more specifically what does cytochalasin D do when applied locally to neurites?

A

it can make any neuron become an axon

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52
Q

what can we hypothesize from the fact that cytochalasin D causes multiple axons / axon formation?

A

cytochalasin D induces actin instability; actin is involved in axon formation

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53
Q

what does cutting axon and allowing another neurite to become a axon demonstrates?

A

it demonstrates plasticity in axon determination

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54
Q

what motor proteins are important for sorting vesicles down axons and dendrites?

A

kinesin

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55
Q

kinesin moves proteins down axon and dendrites via what structure?

A

microtubules

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56
Q

what specificity can isoforms of kinesin have?

A

only transports vesicles down axons, not dendrites

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57
Q

this kinesin isoform was used for what?

A

used as a MARKER of and a MECHANISM for axonal determination

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58
Q

describe axon determination

A

dynamic process; kinesin kind of goes down each neurites until some positive / negative feedback wins

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59
Q

what signaling enzyme at tip of process is necessary and sufficient to determine the axon?

A

PI-3 kinase (makes PI-3,4,5-P3)

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60
Q

what happens if you block PIP3 signaling? and if you over activate it?

A

block = no neurite becomes the axon
over activate = get more than one axon

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61
Q

what is found downstream of PI-3K signaling?

A

Actin and microtubule cytoskeletal dynamics

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62
Q

how are positive and negative feedback important of axon determination?

A

many redundant positive feedback necessary for axon formation, many redundant negative feedback necessary to keep the other neurites from becoming axons

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63
Q

what type of microtubules does kinesin prefer?

A

detyrosinated post-translational modifications of microtubules (tubulin)

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64
Q

what happens if you remove enzymes that put on tyrosine on microtubules?

A

multiple axons form

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65
Q

what tubulin modification help determine the different between axon and dendrites?

A

not just DETYROSINATION! also glutamylation, deacetylation, glycylation, deglycylation, phosphorylation,

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66
Q

Tubulins can lose their carboxy-terminal tyrosine. This can be replaced by what enzyme?

A

tubulin tyrosine ligase TTI

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67
Q

kinesins that select for axons prefer what kind of tubulin?

A

tubulin without the tyrosine

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68
Q

what forms at the axon hillock soon after axon determination?

A

ankyrin G and actin filaments

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69
Q

what do ankyrin G and actin filaments at the axon hillock do?

A

generate a diffusion barrier between axon and soma: only some motor proteins can make it through, limiting the transport down the axon

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70
Q

what channels are very present at axon hillock?

A

sodium channels

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71
Q

how do sodium channels transmembrane proteins get sorted at the axon hillock?

A

selective retention

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72
Q

how does selective retention of sodium channels work?

A

Na channels are made of 4 repeats with hooks between each;
cytoplasmic region between segment II and III is sufficient for axon sorting

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73
Q

describe the cytoplasmic region between segment II and III

A

2 motifs (both required for full sorting):
- one required for association with ankyrin-G, leading to trapping of protein
- one gets endocytosed everywhere else (neg feedback?)

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74
Q

how did they show that cytoplasmic region between segment II and III of Na channel is sufficient for axon sorting?

A

putting the “loop” (segment) is sufficient to localize CD4, a soma protein, to the axon hillock (marked by AnkG)

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75
Q

what is aa1010-1030 vs as1098-111?

A

regions of the Na channel “loop”:
- aa1010-1030 = required for endocytosis, but not for interaction with ankyrin-G.
- as1098-111 = required for association with ankyrin-G but not endocytosis

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76
Q

remember what is CD4?

A

protein localized at the soma; soma marker

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77
Q

protein sorting to dendrite is similar to what kind of targeting?

A

basolateral targeting in MDCK cells

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78
Q

how many different compartments are in dendrites?

A

8 or 9! very complex, lots of sorting

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79
Q

what is the main sorting mechanism of dendrites?

A

vectorial sorting

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80
Q

what is different between microtubules in axons vs dendrites?

A

axon = 1 direction, stronger (they just have a + end)
dendrites = both direction (+ AND - end)

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81
Q

since microtubules are orientated in both directions in dendrites, what kind of motors are good to get to dendrites?

A

retrograde motors (kinesin or dyenin)

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82
Q

why would selective endocytosis from axon would be bad for dendritic targeting?

A

because the axon is very long

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83
Q

what does having 2 directions dendrites mean for speed?

A

slower; ok cus dendrites don’t need to be as fast

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84
Q

what happens to baso/apical sorting if you loose microtubules?

A

loose basolateral sorting because it is the farthest

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85
Q

what are the use of synaptic vesicles vs dense core vesicles DCVs in regulated secretion?

A

synaptic vesicles = Classical Neurotransmitters
DCVs = neuropeptides

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86
Q

what is the MAIN difference between synaptic vesicles and DCVs?

A

synaptic vesicles can be reformed and refilled locally at the synapses;
DCVs must go back to the soma

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87
Q

what is the definition of regulated secretion?

A

When fusion of vesicle to plasma membrane requires a specific stimulus (opposed to constitutive secretion)

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88
Q

name other types of regulated secretion

A
  • synaptic vesicle
  • regulated release of vesicle from endosomes (ex AMPA receptor insertion)
  • Regulated secretory vesicles
  • Lysosome or lysosome-related organelle (e.g. Melanophore) secretion
89
Q

where are DCVs located?

A

not at the active zone of the presynaptic terminal

90
Q

what is the dense core?

A

aggregated crystal of the peptide transmitter stored inside of it (crystal formed from aggregation; DENSE)

91
Q

what are the 2 types of receptors we talked about at post-synaptic spine?

A
  • neurotransmitter ligand gated channel: for FAST transmission; located next to active zone
  • g-protein gated receptors; slower transmission
92
Q

what are the major difference between classical vs peptide transmitters in neurons due to?

A

due to how they are made and how they get into vesicles

93
Q

where are classical transmitters synthesized? how do they get into synaptic vesicles?

A

synthesized in the cytoplasm and packaged into vesicles by transporters

94
Q

where are peptide transmitters synthesized? how do they get into synaptic vesicles?

A

synthesized by translation, then processed through ER and Golgi and packaged into regulated secretory vesicles at the TGN

95
Q

classical vs neuropeptide transmitters can do fast or slow neurotransmission?

A
  • classical transmitters can do both (fast via ligand-gated ion channels, slow via g-protein linked receptors).
  • neuropeptides only mediate slow neurotransmission
96
Q

why do neuropeptide only do slow neurotransmission?

A

because they are released from outside the active zone

97
Q

what kind of signal are triggers by ligand gated ion channels vs g-protein linked receptor?

A

ligand gated ion channels = electrical signal
g-protein linked receptor = second messenger causes chemical signal

98
Q

How are synaptic vesicles made?

A
  • Synaptic vesicles do not bud de-novo from TGN, but instead SYNAPTIC VESICLES PROTEINS are transported in AXONAL TRANSPORT VESICLES
  • they form after ENDOCYTOSIS from plasma membrane OR from ENDOSOMES through budding of clathrin-coated vesicles (same for de novo AND recycling)
99
Q

how does sorting of synaptic vesicles happen?

A

via endocytosis

100
Q

What are regulated secretory vesicles/ dense core vesicles/secretory granules?

A

vesicles that are only released after a specific stimulus

101
Q

give examples of a stimulus for regulated secretory vesicle / DCVs release

A

often is calcium entry in neurons; sometimes cyclic nucleotides in some endocrine cells

102
Q

how do proteins get in regulated secretory vesicles / DCVs?

A

proteins are sorted at the TGN into a special type of vesicle

103
Q

can regulated secretory vesicles / DCVs be recycled?

A

no; Once peptides are release, cannot be recycled. Need new vesicle from TGN.

104
Q

in what cells can DCVs be found?

A

neurons and endocrine cells

105
Q

what is special about DCVs fusion?

A

they can partially fuse to release biogenic amines

106
Q

can neuropeptides be stored in synaptic vesicles?

A

no! no way for them to get in synaptic vesicles

107
Q

can classical transmitter be stored in DCVs?

A

yes! DCVs can store both classic and neuropeptides

108
Q

what does partial fusion / release of DCVs allow for?

A

allows them to act partially like synaptic vesicles and release small transmitters like biogenic amines
(but it does not allow for fast neurotransmission, not involved at active zone; these require synaptic vesicles)

109
Q

what can not be released by partial fusion (release) of DCVs?

A

neuropeptides

110
Q

how are neuropeptides sorted into DCVs/regulated secretory vesicles?

A

via aggregation (formation of the dense core) at the TNG; there is no real cue for sorting

111
Q

what do protein aggregates bind to to get in DCVs?

A

receptor or lipid rafts

112
Q

what is facilitated aggregation?

A

proteins meant for DCVs bind to chromogranin or secretogranin

113
Q

name the 5 mechanisms involved in sorting of proteins in DCVs by entry

A
  • aggregation
  • facilitated aggregation
  • sorting receptor
  • association with lipid rafts
114
Q

what are sorting receptor for protein sorting by entry into DCVs?

A

Carboxypeptidase E binding
Di-basic residue binding prohormone convertase

115
Q

what is sorting of proteins by retention? (DCV sorting)

A

Remove all other proteins from TGN as immature secretory granules are still a sorting compartment

116
Q

why do you see clathrin at DCVs?

A

because there is budding of endosomes taking what’s not supposed to be in DCVs out; not important for sorting
IN DCVs, important for sorting OUT

117
Q

what are chromogranins?

A

acidic proteins that aggregate in the acidic environment of the TGN

118
Q

why do neuropeptides need other proteins to co-aggregate with?

A

because neurons don’t make a lot of neuropeptide

119
Q

what happens if you remove chromogranin A (CGA) using siRNA?

A

the number of DCVs decrease

120
Q

what happens if you overexpress CGA (chromogranin A)?

A

DCVs are formed in cells lacking a regulated secretory system

121
Q

what happens if you KO CGA?

A

not complete loss of secretory granules! aka other proteins help with aggregation

122
Q

if you overexpress chromogranin in cell that does not make secretory granules, you see dense core vesicles. Is that sufficient to make regulated secretory granules?

A

probably not because were missing snares and tether proteins for fusion

123
Q

what is the neuropeptide precursor structure?

A

signal sequence, neuropeptide A and B with cleavage sites around both sides, glycine motif for amidation

124
Q

why do neuropeptide precursor need a signal sequence?

A

because the only way to get in secretory vesicles is via the ER like all other secretory proteins

125
Q

how many peptides are there per neuropeptide precursor?

A

1 to 50

126
Q

what is the glycine for on the neuropeptide precursor?

A

for amidation: prevents degradation of the protein in the ECM

127
Q

briefly explain the processing steps of neuropeptides

A
  1. signa; sequence cleavage in the ER
  2. endoproteolytic cleavage at furin site in the TNG
  3. carboxypeptidase cleavage of cleavage sites in the secretory vesicles
  4. peptidyl-glycine-a-amidation event converts glycine in amide group in the vesicles
  5. amide addition
128
Q

what is furin?

A

enzyme that does the first cleavage of neuropeptide precursor

129
Q

where and what does furin cleave?

A

cleaves furin site (BXBB) (basic amino acid: lysine or arginine) at the pH of TGN

130
Q
A
131
Q

what are PC1 and PC2?

A

enzymes that cleave BB (di-basic residues) in immature secretory granules (more acidic than TGN; cleaves after furin)

132
Q

what characteristic of cleavage is important for sorting of neuropeptides?

A

the timing of cleavage

133
Q

what does the furin cleavage do in egg laying precursor?

A

separates the precursor into two sides

134
Q

what happens to the 2 sides of the egg laying hormone precursor after furin cleavage?

A

The two sides are sorted into different DCVs at the TGN

135
Q

what happens to egg laying precursor in cells with less furin?

A

precursor is not cleaved and peptides are co-stored in same vesicle

136
Q

what did prof Sossin discover?

A

that there is differential sorting of different peptides from the same precursor in different DCVs (Via gold immuno thing)

137
Q

The fact that different aggregates form separate granules suggest that …

A

aggregation is sufficient for DCVs entry sorting

138
Q

are TM proteins also sorted to DCVs via aggregation?

A

probably not.. specific cytoplasmic signals have been identified in VMAT2, Phogrin, and the PMAT enzyme

139
Q

what do the specific cytoplasmic signals for transmembrane proteins do?

A

converts glycines into N-terminal amides required for sorting into DCVs

140
Q

where does transmembrane proteins sorting into DCVs happen?

A

not in the TGN! sorted later in endosomes

141
Q

what happens to the endosomes with TM proteins meant for DCVs?

A

they fuse with immature DCVs

142
Q

name 3 proteins involved in endosomal trafficking that have been identified in genetic screens for neuropeptide secretion

A
  • Rab2 and its interactors
  • A specific Vamp-like protein (Vtia)
  • EARP complex (tethering proteins important for fusion)
143
Q

what event is critical for DCV maturation?

A

fusion

144
Q

remember: where does sorting of TM proteins into DCVs happen?

A

NOT at the TGN! in endosomes

145
Q

remember: how are neurotransmitters packaged in DCVs?

A

through specific vesicular transporters

146
Q

where are classical transmitters synthesized?

A

cytoplasm, synthesized by enzymes

147
Q

are the vesicular transporters that specific?

A

not really.. many neurotransmitters share the same transporter

148
Q

are regulated secretory vesicles or synaptic vesicles oldest (evolutionary)?

A

regulated secretory vesicles appeared before

149
Q

what can we hypothesize for the evolution of ATP and glutamate?

A

regulated secretion of ATP and glutamate probably started from DCVs and were ready for the ‘invention’ of synaptic vesicles

150
Q

are transporters only in synaptic vesicles?

A

no, they are also in DCVs! nothings stops them from being sorted into DCVs like other TM proteins

151
Q

can neuropeptides get into synaptic vesicles?

A

no

152
Q

name differences between vesicular transporter vs plasma membrane transporterer

A
  • vesicular: not neurotransmitter specific; transports from cyto to vesicle lumen
  • PM: neurotransmitter selective; transports out AND back in the neuron
153
Q

what energy do vesicular transporter use?

A

H+ from inside vesicle lumen that comes from vesicular protein ATPase

154
Q

what does extra H+ from ATPase in synaptic vesicle lumen cause?

A

acidification of the vesicle

155
Q

What determines which transmitter a neuron uses? neuropeptide vs classical?

A
  • neuropeptides: the expression of the peptide itself, perhaps with a specific Prohormone convertase
  • classical transmitter: specific synthetic enzymes to make the transmitter
156
Q

what is an exception to classical transmitters being determined with the presence of its specific synthetic enzymes?

A

there is no specific enzymes for glutamate and glycine which are present in all cells

157
Q

again(i think), how are classical transmitters uptaken into vesicles?

A

via specific vesicular transporters

158
Q

what is dale’s hypothesis? is it true?

A

neurons use only one transmitter: true for classical transmitters only; neuropeptides are co-transmitters in many cells; however GABA and glycine have been shown to be released from the same vesicles

159
Q

what does it mean that GABA and glycine can be released from some neurons from the same vesicles?

A

share a transporter: prove Gale’s hypothesis to be wrong

160
Q

what’s special about biogenic amines?

A

all use the same transporter, but are differentiated by the synthetic enzymes present in each cell

161
Q

where is glutamate found?

A

in all cells! many cell types have glutamate transporters

162
Q

AGAIN; compared to DCVs, synaptic vesicles can be …

A

re-used many times! DCVs must be reformed from TGN

163
Q

what systems did they use in the paper?

A

Hippocampal neurons and drosophila neuromuscular junction

164
Q

what system did they use for imaging in the paper?

A

Human IPSCs in which glutaminergic neurons were induced by grow factors cocultured with astrocytes to form synapses

165
Q

what is the marker for synaptic vesicles?

A

FP-tagged synaptophysin, an SV protein

166
Q

what does overexpression of synaptophysin lead to? how did they overcome this problem?

A

leads to mistargeting of most proteins.
they used crispr to add the eGFP coding region before the stop codon of endogenous synaptophysin

167
Q

what is SNAP live imaging?

A

add a 20 kD segment of an alkyltransferase optimized to react specifically and rapidle with benzylguanine derivative -> protein fluorescence only after addition

168
Q

what is the benzylguanine derivative used for?

A

it is fluorescent and cell permeable

169
Q

why is crispr used for tagging?

A

for endogenous tagging; adding tag to endogenous locus

170
Q

SNAP- and HALO- tags can be linked in- …

A

vivo!

171
Q

when did they image the IPSC derived neuron (iN neurons)? why?

A

at day 12-14 after differentiation, in the middle of the synapse formation

172
Q

how were axon initial segments identified?

A
  1. using extracellular antibody to neurfascin, or
  2. expressing ankyrinG-mCherry
173
Q

what is Munc13-1 tagged for?

A

ectosome protein also part of the vesicle (not a TM protein)

174
Q

if something is moving, what will you see in a kymograph? what does the direction and slope mean?

A

a diagonal line;
direction of the line = moving forward or back.
slope of the line = speed of movement

175
Q

what is a straight line going down on a kymograph?

A

stationary

176
Q

this paper looks at what type of puncta?

A

anterograde puncta; most manipulations don’t affect retrograde or stationary puncta

177
Q

kymograph allow to measure what?

A

% of anterograde cotransport with SYP-mRFP (or other marker)

178
Q

figure 1 showed that anterograde synaptophysin containing vesicles have what other proteins in them?

A

synaptic vesicle and active zone AZ proteins: Bassoons and Unc13

179
Q

what happened to synaptophysin vesicle proteins when protein synthesis was blocked for 24h?

A

see less anterograde vesicles, aka the anterograde vesicles are newly synthesized.
doesn’t affect number of stationarry and retrograde vesicles

180
Q

what did they use to block protein synthesis?

A

24-hour incubation with DMSO, CGX, or anisomycin

181
Q

what is the question of the paper?

A

how are the components of presynaptic neurotransmission are transported and assembled?

182
Q

what is PI(3,5)P2 ?

A

rare late endosomal signaling lipid

183
Q

what are precursor vesicles?

A

vesicles transporting SV proteins to the presynapse

184
Q

how are precursor vesicles (PVs) transported from the soma to the synapse?

A

via the kinesin KIF1A

185
Q

what is VAMP2?

A

an SV protein used as an SV marker

186
Q

what are Bassoon and MUNC13-1 markers for?

A

active zone (they are active zone proteins)

187
Q

they localized synaptophysin with a ton of other markers and found co-localization with which ones?

A

late endosomes/lysosome proteins (LAMP1, CD63, ARL8B)

188
Q

Lamp was showed to colocalize with what? in figure 1

A

with Synaptophysin (syp) and synaptotagmin (syt), a synaptic vesicle marker

189
Q

The LAMP1+ vesicles that dont colocalize with Syp have _______ in them, but the LAMP1+ vesicles that colocalize with Syp don’t.

A

cathepsin B (degradative lysosomal enzyme)

190
Q

how did they show that the syyp+ presynaptic vesicles are distinct from lysosomes? 3 ways

A
  1. they did not colocalize with lysosomal cathepsin B
  2. they are not acidic
  3. most Syp(endo) colocalized with LAMP1-eGFP; but just a little bit of LAMP1-eGFP colocalizes with Syp(endo)
191
Q

FIB-SEM and 3D reconstruction revealed the presence of a population of vesicles and tubules in direct contact with what? why?

A

mitochondrial surface because they linked Synaptophysin to FKBP, a mitochondria rapamycin binding domain of mTOR

192
Q

why did they use FKBP and rapamycin?

A

to localize synaptic vesicles and active zone markers to mitochondria with rapamycin addition

193
Q

whats a weakness of the study?

A

not all syp+ vesicles are anterogradely moving

194
Q

the FIB-SEM + FKBP and rapamycin experiment showed what? how?

A

that the synaptophysin vesicles are not synaptic vesicles! because they have different size and shapes than SVs

195
Q

what did they test for after finding out that the syp+ vesicles are not synaptic vesicles SVs?

A

test if they are required for transport of SVs and active zone formation

196
Q

how did they test if syp+ vesicles are required for transport of SVs and active zone formation? (3 things)

A
  • KO ARL8 (a GTPase regulating anterograde lysosome motility along microtubules and involved in presynaptic biogenesis/presynaptic vesicle precursors)
  • KO KIF1A (a kinesin implicated in axonal transport of SV proteins)
  • localize ARL8
197
Q

what did ARL8A and B KO cause?

A

impaired anterograde axonal transport of syp+ vesicles
no change in stationary and retrograde Cyp+ vesicles

198
Q

what does it mean that all the proteins immunoprecipitated with the GTPase ARL8B?

A

the proteins of interest are associated with ARL8B

199
Q

what happens when they KO KIF1A?

A

impaired anterograde transport of syp+ vesicles

200
Q

why test KIF1A KO in hippocampal neurons?

A

to confirm their findings in a different system

201
Q

what happens when they rescue KIF1A with a normal or a mutant (rigor) KIF1A?

A

normal = rescue anterograde transport defect
mutant = does not rescue

202
Q

so do Syp+ vesicles required ARL8 and KIF1A?

A

yes

203
Q

how did they test for the functional effect of reducing syp+ vesicles?

A

looked at the accumulation of proteins at synapses ( nb and intensity of puncta (synapses) marked by postsynaptic puncta) when KO Arl8 and KIF1A (figure 3)

204
Q

what did they see when localizing Arl8?

A

colocalisation with Syp(endo)

205
Q

what were bassoon and homer markers for?

A

bassoon = presynaptic protein
homer = postsynaptic protein

206
Q

what is the intensity measurement they did in figure 3 after ARL8 and KIF1A KO?

A

intensity of the protein fluorescence

207
Q

what happens to the number of puncta when they KO ARL8?

A

decreases at synapse, decrease of bassoon conc, but not homer conc.

208
Q

what does it mean that the intensity of CAV2 does not decrease after ARL8KO?

A

some presynaptic proteins, like CAV2, are not affected by ARL8 KO

209
Q

how did they measure transmission after ARL8 and KIF1A KO? what results did they get?

A

measured the release, the transmission, the strength of the synapse;
transmission decreased, as expected, correlatingly with loss of presynaptic proteins

210
Q

figure 3 results: are syp+ vesicles needed for presynaptic terminal formation?

A

yes: ARL8 or KIF1A KO = decrease presynaptic protein conc (bassoon), synaptic organizer Unc13 conc, Syp conc itself at the presynaptic active zone

211
Q

how does PI(3,5)P2 come into play?

A
  • deleting BORC, a complex that activates ARL8, rescues loss of ARL8…
  • it also increase PI(3,5)P2 levels…
  • can PI(3,5)P2 rescue ARL8?
212
Q

what happens if you inhibit PI(3)P formation?

A

decrease anterograde SYp+ vesicles

213
Q

what happens if you KO PIKFYVE kinase? why?

A

decrease anterograde SYp+ vesicles; makes sense because PIKFYVE makes PI(3)P into PI(3,5)P2

214
Q

what is KIF1A pH domain?

A

domain that binds PI(3,5)P2 and binds an enzyme that makes it localize to the vesicle

215
Q

what happens if you KO the pH domain of KIF1A?

A

same as KIF1A KO: decrease anterograde Syp+ vesicles

216
Q

what happens if you add apilimod? (drug that block production of PI(3,5)P2

A

inhibits colocalization of Syp-mRFP vesicles and PI(3,5)P2
-> they are really looking at the lipid, not some other interaction

217
Q

in drosophila, what happens if you decrease the enzyme that makes PI(3,5)P2?

A

decrease glutamate at the synapse -> decrease presynaptic vesicles i guess

218
Q

if KIF1A does not bind PI(3,5)P2, ….

A

there is no anterograde transport of vesicles

ANAT365 (10 decks)

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