Module 9 - polarity Flashcards
(218 cards)
1
Q
what is cell polarity?
A
Some cells have distinct environments on either side of the cell (apical vs basolateral)
2
Q
name an example of a cell that has distinct environment on either side of it (polarized cell)
A
kidney cells
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
4
Q
give an example of what can make the outside world faced by the apical side hostile
A
stomach acids
5
Q
name some examples of proteins destined for a specific environment (apical/basolateral)
A
ion channels, transporters, lipids, other PM and secreted proteins
6
Q
what kind of junctions define interface between apical and basolateral sides
A
tight junctions
7
Q
what are tight junction’s function?
A
- prevent leakage through epithelial layer
- prevents membrane proteins diffusing back and forth between 2 sides
8
Q
what are called the 2 general solutions to cell polarity?
A
- vectorial sorting
- selective retention
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
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
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.
12
Q
what do “sorting” endosomes do?
A
many sorting decisions are made in sorting endosomes! and not in TNG
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
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
15
Q
where are basolateral signals found?
A
in cytoplasm (except exception)
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
17
Q
what can O be in Tyrosine-based motif (YXXO)?
A
bulky hydrophobic group such as phenylalanine or tryptophan
18
Q
what do APs bind to again?
A
bind proteins on membrane and clathrin coat
19
Q
what coat protein mostly mediates basolateral sorting?
A
clathrin (Similar signals as endosome and endocytosis)
20
Q
where are AP1 and AP2 found?
A
cytoplasm
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
22
Q
what does m subunit of AP1B bind to?
A
tyrosine based motifs
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)
24
Q
what motif is AP1B only important for?
A
tyrosine-based motif (does not affect dileucine based signals)
25
do all basolateral proteins bind AP1B?
no
26
AP1B is specific for what kind of cells?
polarized cells
27
how does apical sorting work?
no specific direct signal for apical membrane!
- directly through transport vesicles
- sorting through endosomes
- sorting through basolateral membranes and then transcytosis through endosomes
28
where are apical signals found?
in the membrane or the lumen
29
name apical signals
- Lipid linkage (GPI)
- Transmembrane domains
- Glycosylation signals (O-glycosylation, N-glycans); aggregation by co-factors
- Segregation into rafts by aggregation of proteins
30
what and where is the exceptions sorting signal?
Syntaxin-III; in cytoplasmic exceptionally
31
where are basolateral vs apical sorting signals found?
basolateral = outside vesicle
apical = inside vesicle
32
how does raft clustering and domain-induced budding work?
- apical-targeted proteins are aggregated by a lumenal adaptor to the membrane
- lipid (rafts) segregation and aggregation
33
why can apical vesicles have weird shapes?
there is no known coat on apical-transported structure
34
what type of sorting is most complicated? what is a key component of that mechanism?
apical; aggregation
35
what are the 2 types of APICAL sorting that happen in endosomes and exit vesicles from TNG?
- protein aggregates with annexin in lipid rafts
- protein aggregates with a leptin, galectin 3
36
where are the aggregation molecules annexin, leptin, galectin 3 found?
in the lumen of the vesicle
37
which type of transport was blocked by tetanus toxin? how?
basolateral transport is blocked because tetanus cleaves V-SNARES
38
why is apical sorting not sensitive to tetanus toxin?
because the V-SNARE used, TI-VAMP, is not sensitive to toxin
39
what is Cellubrevin implicated in?
AP1B vesicle fusion
40
what is blocked by TI-VAMP ko?
direct route of apical sorting, but not indirect route!
41
what protein is used for the indirect route of apical sorting?
VAMP8
42
where are t-SNAREs syntaxin 3 vs 4 found?
Syntaxin3 found in apical membrane, Syntaxin4 found on basal membrane
43
what type of cells have the most polarity?
neurons
44
in neurons, what is a modification of apical (axon) vs basal (dendrite) polarity?
axon vs dendrite differentiation
45
what is Axon Hillock?
site of AP generation in a neuron
46
where does vesicular sorting happen in a neuron?
cell body (same spot as protein synthesis)
47
what is axon determination?
which process of a developing neuron will become the axon
48
what are the axon determination rules?
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
49
how is the longest neurite hypothesized to be determined?
May be determined by last cell division by placement of centrosome and golgi apparatus
50
what is cytochalasin D and what does it do?
it's a drug that destabilizes actin filaments and allows multiple axons to grow
51
more specifically what does cytochalasin D do when applied locally to neurites?
it can make any neuron become an axon
52
what can we hypothesize from the fact that cytochalasin D causes multiple axons / axon formation?
cytochalasin D induces actin instability; actin is involved in axon formation
53
what does cutting axon and allowing another neurite to become a axon demonstrates?
it demonstrates plasticity in axon determination
54
what motor proteins are important for sorting vesicles down axons and dendrites?
kinesin
55
kinesin moves proteins down axon and dendrites via what structure?
microtubules
56
what specificity can isoforms of kinesin have?
only transports vesicles down axons, not dendrites
57
this kinesin isoform was used for what?
used as a MARKER of and a MECHANISM for axonal determination
58
describe axon determination
dynamic process; kinesin kind of goes down each neurites until some positive / negative feedback wins
59
what signaling enzyme at tip of process is necessary and sufficient to determine the axon?
PI-3 kinase (makes PI-3,4,5-P3)
60
what happens if you block PIP3 signaling? and if you over activate it?
block = no neurite becomes the axon
over activate = get more than one axon
61
what is found downstream of PI-3K signaling?
Actin and microtubule cytoskeletal dynamics
62
how are positive and negative feedback important of axon determination?
many redundant positive feedback necessary for axon formation, many redundant negative feedback necessary to keep the other neurites from becoming axons
63
what type of microtubules does kinesin prefer?
detyrosinated post-translational modifications of microtubules (tubulin)
64
what happens if you remove enzymes that put on tyrosine on microtubules?
multiple axons form
65
what tubulin modification help determine the different between axon and dendrites?
not just DETYROSINATION! also glutamylation, deacetylation, glycylation, deglycylation, phosphorylation,
66
Tubulins can lose their carboxy-terminal tyrosine. This can be replaced by what enzyme?
tubulin tyrosine ligase TTI
67
kinesins that select for axons prefer what kind of tubulin?
tubulin without the tyrosine
68
what forms at the axon hillock soon after axon determination?
ankyrin G and actin filaments
69
what do ankyrin G and actin filaments at the axon hillock do?
generate a diffusion barrier between axon and soma: only some motor proteins can make it through, limiting the transport down the axon
70
what channels are very present at axon hillock?
sodium channels
71
how do sodium channels transmembrane proteins get sorted at the axon hillock?
selective retention
72
how does selective retention of sodium channels work?
Na channels are made of 4 repeats with hooks between each;
cytoplasmic region between segment II and III is sufficient for axon sorting
73
describe the cytoplasmic region between segment II and III
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?)
74
how did they show that cytoplasmic region between segment II and III of Na channel is sufficient for axon sorting?
putting the "loop" (segment) is sufficient to localize CD4, a soma protein, to the axon hillock (marked by AnkG)
75
what is aa1010-1030 vs as1098-111?
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
76
remember what is CD4?
protein localized at the soma; soma marker
77
protein sorting to dendrite is similar to what kind of targeting?
basolateral targeting in MDCK cells
78
how many different compartments are in dendrites?
8 or 9! very complex, lots of sorting
79
what is the main sorting mechanism of dendrites?
vectorial sorting
80
what is different between microtubules in axons vs dendrites?
axon = 1 direction, stronger (they just have a + end)
dendrites = both direction (+ AND - end)
81
since microtubules are orientated in both directions in dendrites, what kind of motors are good to get to dendrites?
retrograde motors (kinesin or dyenin)
82
why would selective endocytosis from axon would be bad for dendritic targeting?
because the axon is very long
83
what does having 2 directions dendrites mean for speed?
slower; ok cus dendrites don't need to be as fast
84
what happens to baso/apical sorting if you loose microtubules?
loose basolateral sorting because it is the farthest
85
what are the use of synaptic vesicles vs dense core vesicles DCVs in regulated secretion?
synaptic vesicles = Classical Neurotransmitters
DCVs = neuropeptides
86
what is the MAIN difference between synaptic vesicles and DCVs?
synaptic vesicles can be reformed and refilled locally at the synapses;
DCVs must go back to the soma
87
what is the definition of regulated secretion?
When fusion of vesicle to plasma membrane requires a specific stimulus (opposed to constitutive secretion)
88
name other types of regulated secretion
- 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
where are DCVs located?
not at the active zone of the presynaptic terminal
90
what is the dense core?
aggregated crystal of the peptide transmitter stored inside of it (crystal formed from aggregation; DENSE)
91
what are the 2 types of receptors we talked about at post-synaptic spine?
- neurotransmitter ligand gated channel: for FAST transmission; located next to active zone
- g-protein gated receptors; slower transmission
92
what are the major difference between classical vs peptide transmitters in neurons due to?
due to how they are made and how they get into vesicles
93
where are classical transmitters synthesized? how do they get into synaptic vesicles?
synthesized in the cytoplasm and packaged into vesicles by transporters
94
where are peptide transmitters synthesized? how do they get into synaptic vesicles?
synthesized by translation, then processed through ER and Golgi and packaged into regulated secretory vesicles at the TGN
95
classical vs neuropeptide transmitters can do fast or slow neurotransmission?
- classical transmitters can do both (fast via ligand-gated ion channels, slow via g-protein linked receptors).
- neuropeptides only mediate slow neurotransmission
96
why do neuropeptide only do slow neurotransmission?
because they are released from outside the active zone
97
what kind of signal are triggers by ligand gated ion channels vs g-protein linked receptor?
ligand gated ion channels = electrical signal
g-protein linked receptor = second messenger causes chemical signal
98
How are synaptic vesicles made?
- 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
how does sorting of synaptic vesicles happen?
via endocytosis
100
What are regulated secretory vesicles/ dense core vesicles/secretory granules?
vesicles that are only released after a specific stimulus
101
give examples of a stimulus for regulated secretory vesicle / DCVs release
often is calcium entry in neurons; sometimes cyclic nucleotides in some endocrine cells
102
how do proteins get in regulated secretory vesicles / DCVs?
proteins are sorted at the TGN into a special type of vesicle
103
can regulated secretory vesicles / DCVs be recycled?
no; Once peptides are release, cannot be recycled. Need new vesicle from TGN.
104
in what cells can DCVs be found?
neurons and endocrine cells
105
what is special about DCVs fusion?
they can partially fuse to release biogenic amines
106
can neuropeptides be stored in synaptic vesicles?
no! no way for them to get in synaptic vesicles
107
can classical transmitter be stored in DCVs?
yes! DCVs can store both classic and neuropeptides
108
what does partial fusion / release of DCVs allow for?
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
what can not be released by partial fusion (release) of DCVs?
neuropeptides
110
how are neuropeptides sorted into DCVs/regulated secretory vesicles?
via aggregation (formation of the dense core) at the TNG; there is no real cue for sorting
111
what do protein aggregates bind to to get in DCVs?
receptor or lipid rafts
112
what is facilitated aggregation?
proteins meant for DCVs bind to chromogranin or secretogranin
113
name the 5 mechanisms involved in sorting of proteins in DCVs by entry
- aggregation
- facilitated aggregation
- sorting receptor
- association with lipid rafts
114
what are sorting receptor for protein sorting by entry into DCVs?
Carboxypeptidase E binding
Di-basic residue binding prohormone convertase
115
what is sorting of proteins by retention? (DCV sorting)
Remove all other proteins from TGN as immature secretory granules are still a sorting compartment
116
why do you see clathrin at DCVs?
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
what are chromogranins?
acidic proteins that aggregate in the acidic environment of the TGN
118
why do neuropeptides need other proteins to co-aggregate with?
because neurons don't make a lot of neuropeptide
119
what happens if you remove chromogranin A (CGA) using siRNA?
the number of DCVs decrease
120
what happens if you overexpress CGA (chromogranin A)?
DCVs are formed in cells lacking a regulated secretory system
121
what happens if you KO CGA?
not complete loss of secretory granules! aka other proteins help with aggregation
122
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?
probably not because were missing snares and tether proteins for fusion
123
what is the neuropeptide precursor structure?
signal sequence, neuropeptide A and B with cleavage sites around both sides, glycine motif for amidation
124
why do neuropeptide precursor need a signal sequence?
because the only way to get in secretory vesicles is via the ER like all other secretory proteins
125
how many peptides are there per neuropeptide precursor?
1 to 50
126
what is the glycine for on the neuropeptide precursor?
for amidation: prevents degradation of the protein in the ECM
127
briefly explain the processing steps of neuropeptides
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
what is furin?
enzyme that does the first cleavage of neuropeptide precursor
129
where and what does furin cleave?
cleaves furin site (BXBB) (basic amino acid: lysine or arginine) at the pH of TGN
130
131
what are PC1 and PC2?
enzymes that cleave BB (di-basic residues) in immature secretory granules (more acidic than TGN; cleaves after furin)
132
what characteristic of cleavage is important for sorting of neuropeptides?
the timing of cleavage
133
what does the furin cleavage do in egg laying precursor?
separates the precursor into two sides
134
what happens to the 2 sides of the egg laying hormone precursor after furin cleavage?
The two sides are sorted into different DCVs at the TGN
135
what happens to egg laying precursor in cells with less furin?
precursor is not cleaved and peptides are co-stored in same vesicle
136
what did prof Sossin discover?
that there is differential sorting of different peptides from the same precursor in different DCVs (Via gold immuno thing)
137
The fact that different aggregates form separate granules suggest that ...
aggregation is sufficient for DCVs entry sorting
138
are TM proteins also sorted to DCVs via aggregation?
probably not.. specific cytoplasmic signals have been identified in VMAT2, Phogrin, and the PMAT enzyme
139
what do the specific cytoplasmic signals for transmembrane proteins do?
converts glycines into N-terminal amides required for sorting into DCVs
140
where does transmembrane proteins sorting into DCVs happen?
not in the TGN! sorted later in endosomes
141
what happens to the endosomes with TM proteins meant for DCVs?
they fuse with immature DCVs
142
name 3 proteins involved in endosomal trafficking that have been identified in genetic screens for neuropeptide secretion
- Rab2 and its interactors
- A specific Vamp-like protein (Vtia)
- EARP complex (tethering proteins important for fusion)
143
what event is critical for DCV maturation?
fusion
144
remember: where does sorting of TM proteins into DCVs happen?
NOT at the TGN! in endosomes
145
remember: how are neurotransmitters packaged in DCVs?
through specific vesicular transporters
146
where are classical transmitters synthesized?
cytoplasm, synthesized by enzymes
147
are the vesicular transporters that specific?
not really.. many neurotransmitters share the same transporter
148
are regulated secretory vesicles or synaptic vesicles oldest (evolutionary)?
regulated secretory vesicles appeared before
149
what can we hypothesize for the evolution of ATP and glutamate?
regulated secretion of ATP and glutamate probably started from DCVs and were ready for the ‘invention’ of synaptic vesicles
150
are transporters only in synaptic vesicles?
no, they are also in DCVs! nothings stops them from being sorted into DCVs like other TM proteins
151
can neuropeptides get into synaptic vesicles?
no
152
name differences between vesicular transporter vs plasma membrane transporterer
- vesicular: not neurotransmitter specific; transports from cyto to vesicle lumen
- PM: neurotransmitter selective; transports out AND back in the neuron
153
what energy do vesicular transporter use?
H+ from inside vesicle lumen that comes from vesicular protein ATPase
154
what does extra H+ from ATPase in synaptic vesicle lumen cause?
acidification of the vesicle
155
What determines which transmitter a neuron uses? neuropeptide vs classical?
- neuropeptides: the expression of the peptide itself, perhaps with a specific Prohormone convertase
- classical transmitter: specific synthetic enzymes to make the transmitter
156
what is an exception to classical transmitters being determined with the presence of its specific synthetic enzymes?
there is no specific enzymes for glutamate and glycine which are present in all cells
157
again(i think), how are classical transmitters uptaken into vesicles?
via specific vesicular transporters
158
what is dale's hypothesis? is it true?
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
what does it mean that GABA and glycine can be released from some neurons from the same vesicles?
share a transporter: prove Gale's hypothesis to be wrong
160
what's special about biogenic amines?
all use the same transporter, but are differentiated by the synthetic enzymes present in each cell
161
where is glutamate found?
in all cells! many cell types have glutamate transporters
162
AGAIN; compared to DCVs, synaptic vesicles can be ...
re-used many times! DCVs must be reformed from TGN
163
what systems did they use in the paper?
Hippocampal neurons and drosophila neuromuscular junction
164
what system did they use for imaging in the paper?
Human IPSCs in which glutaminergic neurons were induced by grow factors cocultured with astrocytes to form synapses
165
what is the marker for synaptic vesicles?
FP-tagged synaptophysin, an SV protein
166
what does overexpression of synaptophysin lead to? how did they overcome this problem?
leads to mistargeting of most proteins.
they used crispr to add the eGFP coding region before the stop codon of endogenous synaptophysin
167
what is SNAP live imaging?
add a 20 kD segment of an alkyltransferase optimized to react specifically and rapidle with benzylguanine derivative -> protein fluorescence only after addition
168
what is the benzylguanine derivative used for?
it is fluorescent and cell permeable
169
why is crispr used for tagging?
for endogenous tagging; adding tag to endogenous locus
170
SNAP- and HALO- tags can be linked in- …
vivo!
171
when did they image the IPSC derived neuron (iN neurons)? why?
at day 12-14 after differentiation, in the middle of the synapse formation
172
how were axon initial segments identified?
1. using extracellular antibody to neurfascin, or
2. expressing ankyrinG-mCherry
173
what is Munc13-1 tagged for?
ectosome protein also part of the vesicle (not a TM protein)
174
if something is moving, what will you see in a kymograph? what does the direction and slope mean?
a diagonal line;
direction of the line = moving forward or back.
slope of the line = speed of movement
175
what is a straight line going down on a kymograph?
stationary
176
this paper looks at what type of puncta?
anterograde puncta; most manipulations don’t affect retrograde or stationary puncta
177
kymograph allow to measure what?
% of anterograde cotransport with SYP-mRFP (or other marker)
178
figure 1 showed that anterograde synaptophysin containing vesicles have what other proteins in them?
synaptic vesicle and active zone AZ proteins: Bassoons and Unc13
179
what happened to synaptophysin vesicle proteins when protein synthesis was blocked for 24h?
see less anterograde vesicles, aka the anterograde vesicles are newly synthesized.
doesn’t affect number of stationarry and retrograde vesicles
180
what did they use to block protein synthesis?
24-hour incubation with DMSO, CGX, or anisomycin
181
what is the question of the paper?
how are the components of presynaptic neurotransmission are transported and assembled?
182
what is PI(3,5)P2 ?
rare late endosomal signaling lipid
183
what are precursor vesicles?
vesicles transporting SV proteins to the presynapse
184
how are precursor vesicles (PVs) transported from the soma to the synapse?
via the kinesin KIF1A
185
what is VAMP2?
an SV protein used as an SV marker
186
what are Bassoon and MUNC13-1 markers for?
active zone (they are active zone proteins)
187
they localized synaptophysin with a ton of other markers and found co-localization with which ones?
late endosomes/lysosome proteins (LAMP1, CD63, ARL8B)
188
Lamp was showed to colocalize with what? in figure 1
with Synaptophysin (syp) and synaptotagmin (syt), a synaptic vesicle marker
189
The LAMP1+ vesicles that dont colocalize with Syp have _______ in them, but the LAMP1+ vesicles that colocalize with Syp don't.
cathepsin B (degradative lysosomal enzyme)
190
how did they show that the syyp+ presynaptic vesicles are distinct from lysosomes? 3 ways
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
FIB-SEM and 3D reconstruction revealed the presence of a population of vesicles and tubules in direct contact with what? why?
mitochondrial surface because they linked Synaptophysin to FKBP, a mitochondria rapamycin binding domain of mTOR
192
why did they use FKBP and rapamycin?
to localize synaptic vesicles and active zone markers to mitochondria with rapamycin addition
193
whats a weakness of the study?
not all syp+ vesicles are anterogradely moving
194
the FIB-SEM + FKBP and rapamycin experiment showed what? how?
that the synaptophysin vesicles are not synaptic vesicles! because they have different size and shapes than SVs
195
what did they test for after finding out that the syp+ vesicles are not synaptic vesicles SVs?
test if they are required for transport of SVs and active zone formation
196
how did they test if syp+ vesicles are required for transport of SVs and active zone formation? (3 things)
- 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
what did ARL8A and B KO cause?
impaired anterograde axonal transport of syp+ vesicles
no change in stationary and retrograde Cyp+ vesicles
198
what does it mean that all the proteins immunoprecipitated with the GTPase ARL8B?
the proteins of interest are associated with ARL8B
199
what happens when they KO KIF1A?
impaired anterograde transport of syp+ vesicles
200
why test KIF1A KO in hippocampal neurons?
to confirm their findings in a different system
201
what happens when they rescue KIF1A with a normal or a mutant (rigor) KIF1A?
normal = rescue anterograde transport defect
mutant = does not rescue
202
so do Syp+ vesicles required ARL8 and KIF1A?
yes
203
how did they test for the functional effect of reducing syp+ vesicles?
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
what did they see when localizing Arl8?
colocalisation with Syp(endo)
205
what were bassoon and homer markers for?
bassoon = presynaptic protein
homer = postsynaptic protein
206
what is the intensity measurement they did in figure 3 after ARL8 and KIF1A KO?
intensity of the protein fluorescence
207
what happens to the number of puncta when they KO ARL8?
decreases at synapse, decrease of bassoon conc, but not homer conc.
208
what does it mean that the intensity of CAV2 does not decrease after ARL8KO?
some presynaptic proteins, like CAV2, are not affected by ARL8 KO
209
how did they measure transmission after ARL8 and KIF1A KO? what results did they get?
measured the release, the transmission, the strength of the synapse;
transmission decreased, as expected, correlatingly with loss of presynaptic proteins
210
figure 3 results: are syp+ vesicles needed for presynaptic terminal formation?
yes: ARL8 or KIF1A KO = decrease presynaptic protein conc (bassoon), synaptic organizer Unc13 conc, Syp conc itself at the presynaptic active zone
211
how does PI(3,5)P2 come into play?
- 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
what happens if you inhibit PI(3)P formation?
decrease anterograde SYp+ vesicles
213
what happens if you KO PIKFYVE kinase? why?
decrease anterograde SYp+ vesicles; makes sense because PIKFYVE makes PI(3)P into PI(3,5)P2
214
what is KIF1A pH domain?
domain that binds PI(3,5)P2 and binds an enzyme that makes it localize to the vesicle
215
what happens if you KO the pH domain of KIF1A?
same as KIF1A KO: decrease anterograde Syp+ vesicles
216
what happens if you add apilimod? (drug that block production of PI(3,5)P2
inhibits colocalization of Syp-mRFP vesicles and PI(3,5)P2
-> they are really looking at the lipid, not some other interaction
217
in drosophila, what happens if you decrease the enzyme that makes PI(3,5)P2?
decrease glutamate at the synapse -> decrease presynaptic vesicles i guess
218
if KIF1A does not bind PI(3,5)P2, ....
there is no anterograde transport of vesicles
