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ANAT365 > Module 4 > Flashcards

Module 4 Flashcards

1
Q

what are the 3 kinds of autophagy?

A

macroautophagy, microautophagy and chaperone-mediated autophagy

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2
Q

what is the goal of autophagy?

A

ensuring the removal of damaged cellular content and recycling cellular components, amino acids, lipids, ions

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3
Q

what kind of diseases does autophagy prevents?

A

prevents cancers, neurodegeneration, etc. Errors in autophagy are linked to many diseases, and even lifespan

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4
Q

what are autophagic bodies and when do they appear?

A

they are structures in vacuoles that appear after starvation

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5
Q

how was Osumi able to do a genetic screen of autophagic bodies?

A

by generating a yeast strain with mutations in vacuole proteases that stabilize the structures

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6
Q

why would osumi not have been able to do the screen of autophagic bodies in mammalian system?

A

because we don’t have big vacuoles, we have lysosomes, and yeast don’t.

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7
Q

what are the 3 stages of autophagy that we dissected?

A

initiation, elongation and degradation

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8
Q

name 3 things that can be done by autophagy to protect the cell from cancer?

A

stop genomic instability, reduce oxidative stress, stop necrosis-dependent inflammation

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9
Q

what more specifically activates autophagy?

A

low nutrients (aa) or energy (ATP)

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10
Q

what is the first thing that shuts down when nutrients or atp is low?

A

protein translation

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11
Q

what is AMPK?

A

primary cellular sensor of nutrient status

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12
Q

how is AMPK activated?

A

2 conditions:
1. adenosine monophosphate (AMP). high AMP reflects the low of ATP
2. LKB1 must phosphorylate AMPK.

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13
Q

what is briefly the downstream effect of AMPK substrates?

A
  1. stop energy storage and utilization
    - downregulate protein, glycogen, sterol, and fatty acid synthesis
  2. promote nutrient uptake and recycling
    - upregulate glycose uptake, glycolysis, fatty acid oxidation, autophagy
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14
Q

what are the 2 required conditions for the initiation of autophagy?

A

high AMP and LKB1 phosphorylation

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15
Q

how does LKB1 work?

A

it’s a kinase upstream of AMPK that phosphorylates AMPK to activate it.

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16
Q

what is mTOR?

A

(Target Of Rapamycin) a highly conserved protein kinase

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17
Q

what activates TOR?

A

hormone binding and high amino acid levels activate mTOR, a multisubunit complex.
really complex signaling pathway follows.

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18
Q

what are the effect of TOR activation?

A
  • promotes cell growth, metabolism, protein translation and division
  • inhibits cell death and autophagy
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19
Q

what is Rheb? where is it found?

A

(Ras homologue enriched in brain) GTPase that regulates TORC1.
There is a lot of Rheb localized at the lysosome.

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20
Q

how does TORC1 get inactivated?

A
  1. AMPK phosphorylates TSC1/2 (TSC1/2 is the Rheb GAP)
  2. TSC1/2 is activated and transforms active GTP-bound Rheb in inactive GDP-bound Rheb.
  3. Inactive GDP-bound Rheb shuts down TORC1
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21
Q

how does Rheb activate TORC1?

A
  1. in the GTP-bound form, Rheb binds mTORC1, recruiting it to the lysosome.
  2. It also leads to increased PA which promotes TORC1 activity via lysosome.
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22
Q

what can activates vs inhibits TSC1/2?

A

AMPK activates it by phosphorylation.
Growth factors inhibit it.

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23
Q

hat does inactive TSC1/2 do?

A

inhibited TSC1/2 favours active GTP-bound Rheb which can then activate TORC1

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24
Q

do we know the Rheb GEF?

A

no it is unclear

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25
Q

what is PRAK? what does it do.

A

kinase that phosphorylates inactive GDP-bound Rheb to stabilize its inactive form and the inactivation of TORC1

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26
Q

remember: small GTPases are usually associated with …

A

the membrane

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27
Q

what happens to mTORC1 in growth conditions?

A

(growth conditions = presence of aa)
mTORC is ACTIVATED and recruited to the lysosome

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28
Q

what happens to mTORC1 when amino acid levels are low?

A

it is turned off and primarily cytosolic (inactive)

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29
Q

how can mTORC1 sense amino acids?

A

There are amino acid binding proteins both within lysosomes, and in cytosol that signal to this machinery

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30
Q

name a lysosomal amino acid binding protein that senses aas

A

Ragulator complex that can recruit RagD and RagB GTPases

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31
Q

what is different about ragD and RagB compared to other GTPases?

A

they are unusual GTPases since they are obligate dimers, and they don’t have any lipid modifications at the C-terminus to mediate membrane binding. they must be recruited by the Ragulator

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32
Q

name 2 characteristics of the ragulator?

A

it is membrane-bound at the lysosome and acts as a GEF for RagD and RagB (modulates exchanges)

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33
Q

what does it mean that RagD and RagB can be in different nucleotide states?

A

one can be GTP-bound, the other can be GDP-bound

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34
Q

what is the condition for Ragulator to recruit TORC1?

A

Ragulator senses aa levels:
In low amino acids, RagB is in the GDP-bound form, and TORC1 does not bind (is inactive).
In high amino acids, Ragulator acts as GEF, RagB is in GTP-state, TORC1 is recruited and active from the lysosome

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35
Q

briefly mention the combination of events (3) necessary for mTOR activation

A
  1. Rheb GTPase active (GTP-bound) on the lysosome
    - therefore TCS1/2 (Rheb GAP) must be inactive
  2. Rheb effectors increase phospholipase D and PA.
  3. Ragulator acts as a GEF for RagD and RagB and actives them (GTP-bound)
    -> TORC1 recruited to lysosome
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36
Q

remember what does mTORC1 do once recruited to the lysosome?

A

activates translation, cell growth and proliferation

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37
Q

what does inactivation of mTOR lead to?

A

block in translation, growth and active autophagy

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38
Q

what is the first trigger of mTOR inactivation? explain the cascade after that

A

AMPK senses low energy and phosphorylates TCS1/2 -> activated TCS1/2 (GAP) inactivates Rheb -> shuts down and release mTORC1 from lysosome

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39
Q

remember: what else can affect (mostly inactivate) TSC1/2?

A

growth factors

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40
Q

how does inactive mTORC1 trigger autophagy?

A

it releases the inhibition that active mTORC1 has on the autophagy initiating complex

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41
Q

name 3 members of the autophagy initiating complex that is controlled by mTORC1?

A

ATG13, ULK1, FIP200

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42
Q

how does active mTORC1 inhibit autophagy machinery?

A

via direct phosphorylation of different players

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43
Q

what is another way that mTORC1 is inhibited (and therefore activates autophagy)?

A

rapamycin! is binds and inhibits mTORC

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44
Q

what does mTORC1 phosphorylation of ATG13, ULK1/2 do?

A

it inhibits their activity, stopping autophagy in high nutrient conditions

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45
Q

we know that active AMPK inhibits TORC1 via TSC1/2 and Rheb inactivity, but how else can it also inhibit TORC1?

A

by direct phosphorylation of mTORC1!

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46
Q

AMPK, like mTORC1, can also phosphorylation ATG13 and ULK1/2, How is its effect different?

A

mTORC1 phosphorylation of ATG13 and ULK1/2 INACTIVATES them, but AMPK phosphorylation ACTIVATES them and activates autophagy

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47
Q

where is the active AMPK-phosphorylated form of the Ulk1/2 complex recruited to?

A

to the initiating pre-autophagosomal
structure (PAS) on a membrane

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48
Q

what does active ULK1/2 complex do once recruited to the PAS?

A

phosphorylates a complex
that includes a PI(3)Kinase called Vps34

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49
Q

how to you start to make the autophagosome membrane?

A

the membrane generally comes from the ER

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50
Q

how does the PAS membrane start to be form

A

vps34 complex starts to make PI(3)P at ER membrane (with help of ULK1, ATG13, FIP200)

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51
Q

where is PI(3)P usually limited to?

A

EARLY ENDOSOME

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52
Q

what genes are involved in autophagosome membrane formation?

A

over 30 different Atg genes

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53
Q

ATG12/7/10/5 are essential for what? (with ULK1, Beclin, Vps34)

A

formation of the autophagosome membrane and its wrapping event

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54
Q

what regulates VPS34? what else does this protein regulate?

A

ULK1 complex regulates AMBRA/Beclin/VPS34

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55
Q

what regulates the membrane recruitment of ULK1 and Beclin complexes to the PAS in the ER membrane?

A

chain of phosphorylation via AMPK

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56
Q

the elongation of the autophagosome membrane machinery involves what?

A

a ubiquitin-like conjugation

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57
Q

what autophagy protein is like a small ubiquitin-like protein? why?

A

Atg12: has a c-terminal diglycine motif like ubiquitin

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58
Q

what is Atg12 conjugated to?

A

ATG5 via an E1 (Atg7) and and E2 (Atg10)

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59
Q

what is “missing” to the ubiquitin-like conjugation, compared to a normal ubiquitin mechanism?

A

an E3! no necessary here because Atg10 (E2) directly conjugate Atg12 (Ub-like) to Atg5

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60
Q

what does the Atg12-Atg5 conjugation trigger?

A

an oligomeric assembly that includes another Atg protein: Atg16

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61
Q

what can happen once the oligomer made of Atg12, Atg5, and Atg16 forms?

A

the autophagosome membrane can elongate around the cargo!

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62
Q

reformulate the Atg conjugation that leads to membrane elongation

A

Atg12 (Ub-like) -> Atg7(E1) + Atg12 -> Atg10(E2) + Atg12 -> conjugates Atg12 to Atg5 -> starts elongating membrane with Atg16 = oligomer made of Atg12, 5 and 16

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63
Q

what is LC3?

A

a specific lipid conjugated protein required for cargo recruitment to the autophagosome

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64
Q

how is LC3 made?

A

Atg4 cleaves proLC3

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65
Q

how is the lipid conjugated to LC3 to form the membrane?

A

via Atg7 (same E1 as before) and Atg3 (E2)

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66
Q

what is LC3-II?

A

lipid modified form of LC3 that gets recruited to the already elongating autophagosomal membrane

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67
Q

what happens to LC3-II once the autophagosome seals away?

A

LC3-II on the internal membrane of the autophagosome is degraded, and the ones on the external face are cleaved and RECYCLED BY ATG4

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68
Q

“the ATG5 oligomeric complex resembles a “coat” type structure, but on the ____ of the phagophore”

A

inside

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69
Q

how are cargos for autophagosomes chosen?

A

via ubiquitination

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70
Q

“Soluble, ubiquitinated cargo is degraded by the _________, but when proteins begin to aggregate, they are selected by the
_________”

A

proteasome ; autophagosome

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71
Q

compared to proteasomes, autophagosomes can selectively degrade bigger things such as

A

protein aggregates, damaged mitochondria, bacteria

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72
Q

what links the autophagosome membrane to its cargo?

A

autophagy receptors that recognize LC3 and ubiquitinated cargo via Ub interacting domain

73
Q

what is LIR on autophagy receptors?

A

LC3 interacting motif

74
Q

what is the most commonly studied autophagy receptor?

A

p62

75
Q

briefly explain mitophagy

A

dysfunctional mito -> loss of electrocheminal potential needed for PINK1 import
-> PINK1 accumulates
-> E3 ligase Parkin is recruited
-> PINK1 phosphorylates Parkin on its UBL & phosphorylates Ub
-> Parkin ubiquitinates surface proteins
-> p62 is recruited
-> mitophagy

76
Q

what is ESCRTIII role in autophagy???

A

sealing to autophagosome

77
Q

how is ESCRTIII recruited to the autophagosome?

A

Vps21 (Rab5 in mammals) drives recruitment of ESCRTIII protein Snf7 to Atg17

78
Q

what happens to Atg17 once the autophagosome is sealed by ESCRTIII?

A

it dissociates and leaves

79
Q

what happens to the autophagosome once it is finally sealed and mature?

A

fuses with late endosome before finally fusing with the lysosome -> amphysome

80
Q

what proteins are needed for autophagosome fusion with late endosome?

A

Rab7 and some SNAREs

81
Q

what is an amphisome?

A

hybrid of MVB (late endosome) fused with autophagosome

82
Q

what is the final step of autophagy!!!!

A

complete degradation in the AUTOLYSOSOME

83
Q

there are hints that unlike macroautophagy that we just described, micro and chaperone-mediated autophagy dont require what?

A

the core Atg machinery

84
Q

what are exosomes?

A

the intraluminal vesicles of the MVBs when they are released from the cell

85
Q

what size are exosomes?

A

30-200nm

86
Q

what are ectosomes? what is their size?

A

microvesicles that shed directly from the plasma membrane. are generally bigger than exosomes (1000nm)

87
Q

how were exosomes discovered?

A

Rose Johnstone followed RBCs transferrin receptor as it stopped recycling and went to MVBs and then was secreted from the cell

88
Q

do ectosomes or exosomes have more diverse function?

A

ectosomes have more funtcions.
exosomes are simpler, they come from MVBs

89
Q

what makes it difficult to purify and understand the function of the vesicles with certainty?

A

heterogeneity of the cell

90
Q

if we know that ESCRT3 is involved in exosomes, why can’t we just KO ESCRT3 and study them?

A

because this would completely block MVBs formation and disrupt other pathways

91
Q

name some core hallmark features of exosomes

A
  • Tetraspanins: 4 TMD proteins,
  • microRNA, odd RNA species and DNA: Some microRNAs can be enriched 1000X over the cytosolic concentration!
  • Lipid content is unique with ceramide, cholesterol
  • can carry MHC1 or MHCII: to present antigens and activate T-cells in
    circulation
92
Q

what tetraspanin is a core marker of EXOSOMES?

A

CD63

93
Q

where does the DNA in exosome is thought to come from?

A

from mitochondria

94
Q

what does ceramide and cholesterol content provide to exosomes?

A

stabilize protein enrichment in ecto and exosomes

95
Q

what could be the role of microRNAs in exosomes?

A

regulate the translation / rewire protein expression in another cell

96
Q

an internalized receptor destined for exosome goes through what path?

A

early sorting endosome -> MVB ILV (intraluminal vesicles) -> MVB fuse with PM -> exosome

97
Q

how does MVB get to the PM to fuse with it?

A

via microtubule transport

98
Q

what is the role of SNAREs in exosomes?

A

they must be maintained on the limiting membrane of the MVB to allow fusion with PM

99
Q

what did they surprisingly found about vacuole while doing a reconstitution of yeast SNARE pairs in 2000?

A

vacuolar V-SNARE could drive liposome fusion with PM T-SNARES -> hint of the existence of exosomes (not yet discovered)

100
Q

formation of ectosomes step

A
  1. membrane cargo clustering via tetraspanins, ESCRTI. cholesterol and ceramide help stabilize.
  2. recruitment of additional cytosolic cargoes (ex RNA).
    - flippases, floppases and scramblases are involved
  3. actin and myosin mediate contraction; ESCRT3 and Vps4 mediate final fission. GTPases are involved
101
Q

how is the outward curvature of the ectosome allowed via lipid flipping?

A

the lipid flipping contributes to the remodeling of the actin cytoskeleton

102
Q

what small GTPases regulate actin and myosin function in budding of ectosomes?

A

Arf6, Rho, and CDC42

103
Q

how are calcium spikes involved in ectosome budding?

A

involved in activation of lipid flipping, activation of myosin contraction, activation of an acidic sphingomyelinase to generate ceramide, and more

104
Q

can exosome still be formed in absence of ESCRTI?

A

yes. indicates and ESRTI-independent pathway

105
Q

what is CD63 role?

A

it acts to cluster cargo

106
Q

what is syntenin and its function?

A

adaptor that links a variety of receptor to ALIX, which adapt to ESCRTIII.
It replaces the actions of Ub and PI3P binding functions in normal ESCRT pathway

107
Q

syntenin allows for the bypassing of what for intralumenal vesicle cargo incorporation?

A

ubiquitin requirement

108
Q

what else is enriched in syntenin platforms for stability?

A

ceramide

109
Q

this whole syntenin thing is for ectosomes or exosomes?

A

exosomes! because its for intralumenal vesicles in the MVB

110
Q

re explain the steps of ESCRTI-independent generation of intralumenal vesicles

A
  1. cargo is clustered onto syntenin and tetraspanin platform
  2. syntenin links caargo to ALIX
  3. ALIX binds to ESCRTIII
111
Q

the ESCRT-independent mechanism of ILV formation is specific to what?

A

MVBs that will be secreted

112
Q

the syntenin (ESCRT-independt) pathway can bring what type of cargo in ILVs?

A

membrane receptors, selectively recruited RNAs, other cytosolic proteins, metabolites, cytoskeletal cargoes

113
Q

do exosomes only form in ESCRT-independent manner?

A

no, there is also the normal ESCRT-dependent MVB pathway involving clathrin

114
Q

what ESCRT complex members are involved in ubiquitinated receptor cargo enrichment in intralumenal vesicles?

A

ESCRT I, II, III, and VPS4

115
Q

does the ESCRT-dependent path use syntenin?

A

no, because it doesn’t need alix either, because it has the whole ESCRT machinery

116
Q

ESCRT-dependent pathway of ILV formation can bring what cargoes in ILVs?

A

ubiquitinated receptors, random RNA and cytosolic proteins that can randomly piggy back their way in

117
Q

what are the 3 options of how MVBs can be choosen for secretion?

A
  1. ILVs are generated via the syntenin and the ESCRT pathway at the same time and the content of MVBs is mixed
  2. early endosome decides to generate either syntenin derived ILVs OR ESCRT mediated ILVs
    3.syntenin vs ESCRT sorting event could be refined in a polarized (apical/basolateral) manner
118
Q

how do ectosomes and exosomes get to their target once they exit the cell?

A
  • can have adhesion proteins on their surface that bind target cell
  • can get internalized by target cell and get recycled or back-fuse
  • fuse with target cell PM and release content in cytosol
119
Q

what does it mean if a ecto/exosome gets “recycled” by the target cell?

A

they get re-secreted, directly from early endosome or they stay until MVBs

120
Q

what does it mean if a ecto/exosome “back fuses” in the target cell?

A

it is internalized into early then late endosome and secreted into the cytosol

121
Q

what can microRNAs in exo/ectosomes do to the target cell?

A

they can reprogram the whole cell!

122
Q

example: what can stem cell-derived extracellular vesicles do in kidney injury?

A

stop apoptosis, inflammation, fibrosis.
increase mito protection, renal oxygenation, expansion of immune cells

123
Q

how can extracellular vesicles “help” cancer?

A

they can reprogram their niche to drive blood vessel formation, remodeling the extracellular matrix, promote proliferation, remodel the immune system, etc

124
Q

how are researchers looking to use exosomes as therapeutic delivery system?

A

culture cell that may express “payloads” that incorporate into exosomes and inject the exosomes into a diseased tissue/tumour or into circulation

125
Q

what could the “payloads: of therapeutic vesicles be?

A

microRNAs, antibodies against cancer cells, siRNAs, mRNA, DNA, anti-sense oligonucleotides, chemotherapeutics

126
Q

more information is needed about what part of EVs to develop therapies?

A

on the specificity of entry into the target cell and on the delivery out of endosomes

127
Q

what are different ways of entry in intact exosomes into target cell?

A

receptor mediated, direct binding, clathrin-coated pit, lipid raft, phagocytosis, caveola, macroponocytosis, direct fusion

128
Q

how could exosomes target the tumour?

A

having content that stimulate APC to generate T-cells against the tumor, or that directly kills the tumour cell with chemicals or chemotherapeutics

129
Q

the clinical interest in EVs is interesting for therapies and for ??

A

for biomarkers of diseases

130
Q

remember: what is PINK1?

A

mitochondrial protein that accumulate when mito is dysfunctional to signal mitophagy

131
Q

what is xCT?

A

cystine-glutamate antiporter that allows cancer cells to survive in metabolically stressful microenvironments

132
Q

how does xCT promote cancer invasive behaviors?

A

it leads to increased extracellular glutamate which activates mGluR3, which activates Rab27-dependent release of EVs, via which invasive characteristics are transferred to other cells

133
Q

how is glutamate formed in cancer cells?

A

glutaminolysis: glutamine -> glutamate

134
Q

Rab27 activates EV release which leads to invasiveness, but also promote invasiveness in another way

A

directly increase Mt1-MMP release to the PM

135
Q

what is MT1-MMP? function?

A

Membrane Type-1 Matrix Metalloproteinase: promotes disruption of BM which promotes invasion

136
Q

what is glutaminolysis and why is it enhanced in cancer cells?

A

glutamine -> glutamate reaction.
cancer cells love glutamate

137
Q

what is glutathione (GSH?

A

anti-oxidant built from cystine; can be made from excess glutamate at PM via exchange with cystine; important for cancer cells because they are so stressed

138
Q

what is LY95? what did it do to EVs when injected in the cancer cells?

A

mGluR3 antagonist; reduced the number of EVs by more than half

139
Q

what did they use as an EV marker?

A

CD63 (and CD9 and flotillin)

140
Q

what did he see on the SDS-PAGE gel in cells and EVs before vs after LY95 addition?

A

reduced CD63, CD9, and flotillin in EVs after LY95 (after mGluR3 inhibition).
(no change in cellular levels)

141
Q

they tested the dose response of EVs number after glutamate addition. what were the results?

A

increased nb of EVs after addition of glutamate

142
Q

the increase in EVs caused by glutamate addition proves what?

A

that the glutamate receptor acts as a driver of MVB fusion with PM

143
Q

what happened to CD63, CD9 and flotillin levels after glutamate addition?

A

increased

144
Q

what did the EM analysis of secreted vesicles show after LY95 addition?

A

conjugated gold particle to CD63 antibodies and saw CD63 labelling: conclude that what they see are exosomes.
Nb of labelled particles is reduced when mGluR3 is inhibited.

145
Q

they did western blotting and qPCR found what kind of proteins in the EVs?

A

mitochondrial components (VDAC, Cyclphilin D, GLUD1, COX1, CYTB, ND1) and mitochondrial chromosome and genes

146
Q

addition of LY95 did what to the quantity of mitochondrial proteins associated with EV preparations?

A

reduced; it shows that mito contents in EVs is linked to the glu signaling pathway

147
Q

how did LY95 affect OCR (oxygen consumption rate) (mito respiration function)

A

LY95 decreased respiration

148
Q

silencing Rab27 and CD63 also decreased respiration. but what is wack about it?

A

Manipulating the endosomal pathways could lead to very indirect effects on mitochondria

149
Q

why do they add a DNAse to the cancer cells?

A

to isolate EV content because the DNAse will degrade all the DNA (mtDNA and nuclear) that is not protected by a membrane, to make sure that what he sees in his results is not JUST noise.
turns out there is a lot of noise but wtv

150
Q

what is beta-globin?

A

nuclear DNA/chromosome

151
Q

PCR reaction after DNAse addition show what?

A

that there is still amplification of mtDNA in EVs compared to b-globin

152
Q

how did he test if mtDNA (COX1) incorporation in EVs is regulated by mGluR3?

A

adding LY95, silencing Rab27, silencing CD63 -> it all reduced COX1 levels

153
Q

what did they use the sucrose density gradient centrifugation for?

A

to confirm that mtDNA (ND1) was associated with CD63+EVs.
result: ND1 and CD63 peaked in the same fraction

154
Q

why did jim turn to PINK1 when looking at what regulates mtDNA entry in MVBs?

A

because it is known to regulate delivery into MVB via mitochondrial derived vesicles or into the autophagosome via mitophagy

155
Q

did jim actually test mitochondrial vesicle transport?

A

no they only look at contact sites… they don’t test MVBs

156
Q

what happened when jim silenced PINK1?

A

decrease in EVs and in CD63+ EVs

157
Q

what did the results of sucrose gradient of CD63 after siPINK1 show?

A

much reduced CD63

158
Q

what happens when he rescues PINK1 is siPINK1 cancer cells?

A

CCCP induced Ub-phosphorylation is rescued

159
Q

what is PINK1KD?

A

kinase dead PNI1 (basically defective PINK1 that can’t phosphorylate)

160
Q

why is PINK1KD used?

A

just as a control to show that he actually silence PINK1 because silencing isn’t always precise

161
Q

what happened to EVs levels when he rescued PINK1 in siPINK1 cells?

A

rescued, and even got MORE EVs.

162
Q

what happened to EVs levels when he rescued kdPINK1 in siPINK1 cells?

A

rescued, and even got MORE EVs…. this means that PINK1 kinase activity was not necessary for EVs formation.. show that PINK1 function in EVs is unrelated to its function in mitophagy

163
Q

what results did they get from PINK1-YFP overexpression?

A

gets more CD63+EVs, and gets more mtDNA

164
Q

in the supplement they show that loss of the core autophagy machinery ….

A

does not alter PINK1 driven EVs

165
Q

how did they study the relationship between mito and lysosome?

A

looked at videos of mito and lysosome interactions after cccp addition (triggers mito fragmentation)

166
Q

how did loss of PINK1 affect mito and lysosome contact?

A

reduced the contacts a lot

167
Q

what happen to mito-lysosome contact when they silence PINK1?

A

reduction in contact.. and they assume this means that there is less mtDNA EVs

168
Q

how did he test if the mtDNA in EVs has an impact on the cancer invasiveness?

A

by adding the mtDNA+ EVs into recipient cells that were glutamate deprived (no MMP on surface) and looking at MT1MMP recruitment

169
Q

what did they get from adding the EVs to glutamate deprived cells?

A

increase in MT1MMP recruitment

170
Q

then he looked at the collagen degradation and invasion activity of the recipient cells after EVs addition. results?

A

saw more degradation!
no degradation when he blocked LY95 and siRab27 (EVs cant be secreted and allow invasion)

171
Q

they also measured the length of the cell protrusions of the recipient cells as an index of ….?

A

the cells migrating away

172
Q

how did they test if the mtDNA was actually important for the metastatic features?

A

they made cells with no mtDNA: Rho0

173
Q

what did they find in exosomes in Rho0 cells vs CTRL?

A

they still secrete exosomes but can no longer amplify mtDNA from them

174
Q

how was Rho0 cell degradation?

A

can’t degrade as much: loss of function

175
Q

they activated TLR9, a DNA binding receptor in the endosome and found what?

A

that it promoted EVs action of driving collagen degradation and protrusion length

176
Q

they inhibited TLR9, a DNA binding receptor in the endosome and found what?

A

reduced protrusion length

177
Q

last experiment was they took the mtDNA and made fake exosomes and added them to starved cells. results?

A

promotion of MT1MMP recruitment and or protrusion length

178
Q

what did TLR antagonist do when they added the fake mt-DNA exosomes?

A

decrease the effects that were promoted by the mtDNA -> shows again that it is a TLR9 dependent process

179
Q

Dr McBridge said the only conclusion we can get is

A

that mtDNA is important in signaling

ANAT365 (10 decks)

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