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ANAT365 > Module 3 - Mitochondria > Flashcards

Module 3 - Mitochondria Flashcards

1
Q

name a few roles of the mitochondria

A

ATP production, beta-oxidation of fatty acids, lipid synthesis, steroidogenesis, adaptive thermogenesis, aging, apoptosis, calcium homeostasis. innate immune response

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2
Q

what origins does mitochondria and chloroplasts come from?

A

cyanobacterial origin (came from host/parasite symbiosis)

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3
Q

when did the host/parasite symbiosis of the cyanobacterial origin happen?

A

2100 million years ago

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4
Q

how much of mitochondria’s own DNA is in the matrix?

A

16kB

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5
Q

how many proteins are encoded in the mitochondria? what proteins?

A

mtDNA: - 13 proteins (OXPHOS oxidative-phosphorylation complexes - 2% of mitochondrial proteins)
- 22 tRNA
- 2 rRNA
- no introns in humans

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6
Q

what % of mito proteins are encoded in the nucleus? how do they get to the mito?

A

98%. they are then imported post-translationally

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7
Q

how can a phenotype from a mutation appear?

A

if the mutation takes over 80% of the copies of the mutated DNA (mutation is dose-dependent, not mendelian)

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8
Q

are mito diseases only coming from mutations in mito DNA?

A

no, can also come from nuclear encoded proteins (mendelian)

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9
Q

how many people get mitochondrial diseases? is there a cure?

A

1 / 5 000 people.
no cure.

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10
Q

where is the mtDNA nucleoid proteome located?

A

anchored in the inner face of the IMM

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11
Q

mtDNA is assembled in what?

A

in nucleoids

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12
Q

do we know what regulates mtDNA replication and transcription?

A

no;
hypothesis that PGC-1 nuclear encoded factor might regulate transcription

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13
Q

did they find a correlation between mtDNA copies and cell tissue properties?

A

no;
No correlation between a tissues OX
capacity and mtDNA copy number or between proliferation of mitos and mtDNA copy number.

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14
Q

how many proteins are encoded in the nuclear genome and posttranslationally imported into the mitochondria?

A

1500 proteins (99%) (only 13 proteins are made in the mitochondria)

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15
Q

how do nucleus and mito speak different languages?

A

their genetic code had drifted, they use different codons; any new mito gene translocation would become a pseudogene

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16
Q

what acts as a mitochondrial signals?

A

positively charged helices and amphipathic alpha helices

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17
Q

what are TOMs and TIMs? what is their function?

A

Translocase of the outer membrane and Translocase of the inner membrane.
They help route protein precursors to the outer, intermembrane, inner membrane, matrix compartments…

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18
Q

by the mid 1990s, what was known about mitochondria?

A

Kreb’s cycle and that mito activity depended on metabolite concentrations

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19
Q

what is Fzo1?

A

the first GTPase for mitochondria fusion to be discovered by Margaret Adaline Reed Lewis in 1914. sparked up the field of mito

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20
Q

what was weird about the mitochondria transport?

A

unlike other transport mechanisms, there is no polarity/directionality in the movement

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21
Q

what happened in yeast mating assay?

A

they mixed yeast containing red vs green mitochondria (tagged via Fzo1) and induced fusion with alpha-factor

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22
Q

what happened to mitochondria when Fzo1 was lost via temperature incubation of Fzo1-temperature-sensitive mutant strain?

A

it lead to fragmented mitochondria that could not fuse

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23
Q

what happened when they fused mitochondria from heterokaryons expressing distinct mtDNA mutations?

A

the mutations complemented eachother and only then could the mitochondria build the proper ETC complexes

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24
Q

how do mtDNA mutations compliment eachother?

A

mitos lower their mutations %, allowing mitochondria to function properly and lower the risk of mito disease

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25
Q

what are mitofusin1 and 2 ?

A

genes that are 60% similar, located in the outer membrane. Orthologue of Fzo1 in yeast.

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26
Q

what part of mitofusin genes is exposed in the intermembrane space?

A

HR2 coiled-coil domain

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27
Q

in what condition is Mfn2 mutated?

A

in Charcott Marie Tooth Type 2A

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28
Q

why are mitofusins and OPA1 necessary for mitochondrial fusion?

A

because they are GTPases, and GTP hydrolysis is required to drive fusion

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29
Q

how do mitofusins get “primed” to drive fusion?

A

stress (oxidative stress) causes disulfide bonds formation within the intermembrane, which primes mfns to bind in trans and drive fusion

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30
Q

mitofusins priming is a more ancient version of what?

A

of the NSF/SNAP priming of the SNARES

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31
Q

compared to mitofusins proteins, where is OPA1 anchored? what part of it is exposed in the intermembrane space?

A

in the inner membrane. HR domains are in intermembrane space.

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32
Q

what is different about mitofusins and Opa1 HR domains?

A

mfns have 1 HR2 domain each, Opa1 has 2 HR domains

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33
Q

what are HR domains?

A

HR2 (in mitofusins) and HR (in OPA1) are coiled-coil domains in the intermembrane space

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34
Q

what are Mtns1 and 2, and Opa1 proteins considered to be because they are so big?

A

they are commonly considered to be like dynamins, and are sometimes all called DRPs (dynamin related proteins)

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35
Q

what does mitochondrial fusion protect against?

A

protect against cell death; mixes content to rescue failing organelle and distribute genomes

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36
Q

does yeast have Mfns genes?

A

non juste Fzo1

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37
Q

where does the ETC form supercomplexes?

A

in the IMM

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38
Q

what is the complex called MICOS required for?

A

anchoring the IMM cristae (holds the junction together)

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39
Q

what other protein (other than MICOS complex) is required for cristae anchoring? how does it work? what else is this protein regulating?

A

Opa1; it forms dynamin-like oligomers that hold the cristae together.
Also regulates mitochondrial fusion.

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40
Q

what do Opa1 oligomers do during cell death?

A

they get broken, release cytochrome c, allowing it to exit through pro-apoptotic Bax pores formed specifically during death.

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41
Q

when are Bax/Bak pores formed?

A

specifically during death

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42
Q

what does cytochrome c being released during cell death do?

A

drives assembly of the cellular death machine (apoptosome)

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43
Q

what protein mediates mitochondrial fission?

A

DRP1 (it’s a dynamin)

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44
Q

how does Drp1 work?

A

it couples fission with cell signaling

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45
Q

what does DRP1 lack compared to other dynamins? how does it get recruited without that?

A

lacks a PH domain.
it has Mff, a membrane anchored adaptor for Drp1 recruitment

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46
Q

what is mitochondrial fission required for?

A

required for biogenesis, to isolate organelles for mitophagy, and to remodel cristae during cell death

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47
Q

name the membrane anchored adaptor necessary for Drp1 recruitment (since DRP1 doesn’t have a PH (lipid binding) domain)

A

Mff

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48
Q

name 2 additional adaptors to Mff that help recruit Drp1 for mitochondrial fission?

A

Fis1 and MIDs

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49
Q

is the mitochondrial fusion reversible? why?

A

yes. mitochondrial plasticity is critical during cellular stress

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50
Q

briefly explain what happens once the mito encounters stress or starvation

A

mitos will FUSE: PKA activates, DRP1 is blocked, Mfns and Opa1 activate

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51
Q

what happens to mitochondria under chronic stress?

A

functional loss, mito gets fragmented, cytochrome c is released, leading to apoptosis

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52
Q

what happens when a mito just receives an apoptotic signal?

A

fusion is blocked, Bax active (pores), DRP1 is recruited for fission, cristae remodeling, cytochrome c, apoptosis

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53
Q

what is nrf2 involved is?

A

rescuing the mito after stress once the nutrients are back

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54
Q

what is protected by SLP2 during mitochondrial fusion?

A

Opa1

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55
Q

name organelles with which mitos are in intimate contact

A

ER, peroxisome, early and late endosome (late = only in yeast), lipid droplet

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56
Q

what is the mitochondrial primary function?

A

iron sulfur clusters, involved in electron transfer

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57
Q

yeast high pressure frozen EM tomography revealed what organelle to have a function in mito division? (first one discovered)

A

ER; it wraps around the mitochondria (it was later shown in mammalian cells too)

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58
Q

where does the ER contact sites with mito form?

A

at sites of mtDNA replication

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59
Q

what does mitochondria-ER contact trigger to start fission?

A

actin machinery recruitment for extra force, calcium flux to remodel cristae, initiates mitochondrial constriction

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60
Q

what other organelles (appart from ER) are necessary for mito fission?

A

lysosome and golgi vesicles

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61
Q

why are lysosomes required for mito division?

A

Mitochondrial receptors regulate lysosomal Rab7 GTP hydrolysis in trans.

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62
Q

what does DRP1 do once lysosome binds to mito in the process of fission?

A

it oligomerizes and constricts mito to a small tubule

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63
Q

what is golgi vesicle’s role in mito division?

A

they are essential for the final scission of the membrane

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64
Q

summarize the steps of mito division

A
  1. ER contact at mtDNA replication site
  2. initiation of mito constriction: actin recruitment, calcium fluxes
  3. lysosome contact
  4. DRP1 oligomerization, constriction of mito
  5. PI4P-containing TGN vesicle recruitment
  6. final scission via golgi contact
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65
Q

what is something important that happens to DRP1 for it to constrict mito to a small tubule?

A

oligomerization

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66
Q

mitochondrial plasticity orchestrates _______ _________ in response to ________

A

metabolic transitions; signaling

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67
Q

how many biochemical reactions happens in mitochondria?

A

~1000

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68
Q

what is the current holy grail of mitochondrial dynamics research?

A

how is mitochondria coupled to signaling and how the signals regulate metabolism

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69
Q

name diseases associated with mitochondrial malfunction (6)

A
  • Alzheimer’s: Drp1-Nitrosylated, PS2 in ER/mito contacts, Abeta on mitochondria
  • Huntington’s: Drp1 activated
  • Cerebellar degeneration: loss of Mfn2
  • Charcot-Marie-Tooth: Mfn2 mutation
  • Parkinson’s: Pink1 and Parkin malfunction
  • Dominant Optic Atrophy optic nerve: Opa1 mutation
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70
Q

after what discoveries did mitochondrial research take off?

A

once core mechanisms and machinery were established

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71
Q

interaction with what part of the cell position the mito?

A

with the cytoskeleton

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72
Q

why is mito fission vs fusion required?

A

fission is required to maintain mitochondrial numbers and for apoptosis.
fusion protects against cell death.

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73
Q

what machinery overlaps with the mitochondrial fusion machinery?

A

cristae assembly machinery

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74
Q

"”mitochondria are central in all signaling pathways to link ….”

A

metabolic transition to cell fate decisions

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75
Q

why were MDVs not a suprise to discover?

A

Alpha-proteobacteria (and all bacteria) shed vesicles

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76
Q

what are some functions of bacterial membrane vesicles?

A

good: protection, delivering antibiotic resistance factors, DNA transfer within the colony, signal nutrient status
bad: toxins to competitor bacteria; shed phage receptors to avoid infection and fuse with competitors to make them vulnerable

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77
Q

how do bacteria use vesicles to infect humans?

A

via vesicles that target the gut and resist pH change, depolarizing epithelial cells to drive infection

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78
Q

what is TOM20?

A

outer membrane receptor of TOM import complex. found in some MDVs.

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79
Q

what is PDH?

A

Pyruvate dehydrogenase is a multisubunit enzyme complex in the mitochondrial matrix. Found in some MDVS where TOM20 is not found.

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80
Q

what did TOM20 and PDH being founf in separate MDVs tell us?

A

that there are distinct classes of MDVs

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81
Q

what happens to MDV generation when respiration increases?

A

MDV generation increases

82
Q

how did they find that respiration increases MDV production?

A

by feeding galactose to cancer cells grown on glucose that usually depend on glycolysis for ATP (gal can only be burned by mito so it drives respiration). This generates ROS (potential damage), and cause an increase in MDVs.

83
Q

why does galactose drive respiration?

A

because it can only be burned by the mitochondria which generates more ROS and potential damage

84
Q

what type of cell did they use to study MDVs?

A

cos7 cells

85
Q

How many TOM20/PDH MDVs are found in cos7 cells normally?

A

about 100 TOM20 MDVS, and about 10 PDH+ MDVs

86
Q

What did they give to cos7 cells to increase ROS (en plus de galactose)? what did it do?

A

XO: Xanthine oxidase/Xanthine generates ROS in the cytosol
Anti A: AntimycinA blocks complex III of the electron transport chain, leading to high ROS production within mitochondria
-> both increase MDVs

87
Q

immunogold EM revealed that TOM20+ MDVs target what organelle? what did they conclude from that

A

to multivesicular bodies to degrade mitochondrial content

88
Q

what did the study about MDV analysis in animals brain (in neurons) found?

A

it found an abundance of protrusions and MDVs in the dendrites of neurons, and almost none in axons.
found multiple MDVs per mitochondria in about 20% of mitos

89
Q

what are PINK1 and Parkin?

A

mitochondrial proteins that when dysfunctional cause the loss of mitochondria electrochemical potential and Parkinson’s

90
Q

what is PINK1’s normal function?

A

its a kinase that gets imported and rapidly turned over/degraded

91
Q

what is parkin function?

A

its a Ub E3 ligase that ubiquitinates a host of OMM proteins to tag the mito for mitophagy

92
Q

how can PINK1 and Parkin recognize dysfunctional mitochondria?

A

when mito is dysfunctional, you loose the electrochemical potential needed for protein import.
PINK1 kinase import fails; PINK1 is stuck at the translocon; PINK1 phosphorylates Parkin and Ub; Parkin (a Ub E3 ligase) ubiquitinates OMM proteins, tagging mito for mitophagy

93
Q

what kind of vesicles is Parkin recruited to?

A

matrix positive MDVs that don’t express TOM20 (the TOM20 vesicles machinery is different than the matrix vesicles)

94
Q

“Additional experiments showed PINK1 and Parkin were required to generate ______ ______ “

A

matrix-positive MDVs

95
Q

what are the different methods of mitochondrial quality control?

A
  1. mitochondrial proteases in matrix and IMS
  2. selective ub-mediated degradation by proteasome
  3. MDVs carry larger damaged cargo to lysosomes
  4. Parkin recruitment for mitophagy
96
Q

proteases in mito matrix and IMS degrade what kind of proteins?

A

oxidized, mainly soluble proteins

97
Q

what proteins can be degraded by proteasomes?

A

outer membrane proteins that get ubiquitinated by cytosolic E3 ligases and retranslocated by chaperones

98
Q

in what cases are proteins degraded via MDVs transport to lysosome?

A

MDVs can carry larger complexes and sculpt the proteome. They can lead to the real loss of an individual protein when needed

99
Q

what is the main pathway of MDVs?

A

to multivesicular bodies and then to the lysosome for degradation

100
Q

what are some secondary pathways of MDVs?

A

Bacterial defense: MDVs can carry enzymes that help degrade bacteria to phagosomes and then to PM.
Can also go to MVBs which then fuses with PM to release mito content for signaling

101
Q

how can MVBs drive signaling?

A

by fusing with PM and releasing mitochondrial content

102
Q

what is MAPL?

A

mitochondrial anchored protein ligase

103
Q

what were MDVs first described as?

A

small, 70-200nm vesicular profiles that carried selected cargo

104
Q

what 2 fates of MDVs were described so far?

A
  1. peroxisomes
  2. late endosomes/MVBs
105
Q

how did prof McBRide first discover MDVs transport to peroxisome?

A

seeing MAPL in confocal and EM imaging doing unusual budding and going to a small fraction of peroxisomes, not lysosome

106
Q

what was required for MDV transport to peroxisomes? (on slide with MAPL discovery)

A

retromer complex

107
Q

what are peroxisomes?

A

suborganelle derived from primitive mitochondria involved in metabolic tasks, ROS scavenging, innate immunity response

108
Q

what metabolic tasks are peroxisomes involved in?

A

bile acid synthesis, fatty acid oxidation, plasmalogen synthesis for myelin

109
Q

where do the enzymes for fatty acid oxidation in mito and peroxisomes come from?

A

from alphaproteobacterial lineage

110
Q

what are similarities between mito and peroxisome? (4)

A

shared metabolic tasks, same fission machinery, ER required for biogenesis, same transcriptional pathways for biogenesis

111
Q

what is PPAR? what is it required for?

A

peroxisome proliferator activated receptor: required for mito and peroxisome biogenesis

112
Q

why are MDVs delivered to peroxisomes?

A
  • steady state: to carry cargoes that can’t be taken up by peroxisomal import machinery
  • during biogenesis: to bring some essential proteins and fuse with ER to build peroxisome (import machinery from mito and ER are sent in vesicles and fuse)
113
Q

what are peroxisomes a hybrid of?

A

hybrid organelle of ER and mitochondria

114
Q

essential peroxisome proteins are found where, before being brought by MDVs to build peroxisome?

A

in mito and in ER

115
Q

what are iron sulfur clusters?

A

something mitochondria do for respiration

116
Q

what is the evolutionary hypothesis for MDVs?

A

early mito (a-proteobacterium) shed vesicles with hydrolytic enzymes to attack archea and would enter it and not be degraded because it offered a major metabolic benefit

117
Q

what are peroxisomes a derivative from? they evolved to do what?

A

mitochondria. they evolved to carry the most oxidative damaging reactions

118
Q

what is the hypothesis of how endosomes evolved?

A

the new hydrolytic machinery brought by a-proteobacterium vesicles in a host needed iron from outside the cell

119
Q

what is the primary function for delivery of MDVs to the peroxisome?

A

protein biogenesis

120
Q

what is special about cos7 cells?

A

TOM20+ MDVs are abundant in this cell type

121
Q

about how many TOM20+ MDVs are present in steady state in cos7 cells

A

about 100

122
Q

remember: at steady state, MDVs ….

A

deliver cargo to peroxisomes (protein biogenesis) or MVBs (degradation)

123
Q

what was this modules paper about?

A

what are MDVs and what is the machinery that forms it? MIROs and DRP1

124
Q

what type of MDVs did the paper study? why?

A

TOMM20+; because they are present at steady state

125
Q

opposed to tomm20+ MDVs, PDH+ MDVs are more dependent on?

A

dependent on oxidative triggers

126
Q

MDV formation is independent of what?

A

independent of the autophagy and mitochondrial division machinery

127
Q

are MVDs single or double membraned?

A

can be either!

128
Q

there are emerging links between MDV pathway and what diseases?

A

amyotrophic lateral sclerosis, Parkinson’s disease and Alzheimer’s disease

129
Q

they characterize TOMM20+ MDVs with what 2 characteristics in the abstract?

A

cargo specificity and their punctate structures of approximately 160 nm

130
Q

what OMM protein is excluded from TOM20+ MDVs? what was it used for in the paper?

A

MAPL (and PDH). can be tagged to remove other parts of mito membrane that aren’t MVDs (artifactual small fragments of mito)

131
Q

specifically what epitope tags were used for rapid isolation of MVDs and mito membrane with antibodies?

A

HA tag for TOM20 (TOM20-HA) and Halo tag for MAPL (MAPL-Halo)

132
Q

why was the Halo tag used?

A

to generate a chimera between MAPL (protein of interest) and the tag (permeant fluorophores, magnetic resins, etc)

133
Q

MAPL-Halo tag allows to remove what from the mitochondrial membrane?

A

allows to remove the parts that aren’t MVDs who are double-labelled with Halo AND Ha

134
Q

what makes the HALO tag so nice to use?

A

it has a well-defined binding pocket for which many ligands have been engineered. very versatile

135
Q

what is the HA tag on TOM20?

A

peptide from hemagluttinen for which there are many commercial antibody tools

136
Q

how did they determine the proteins enriched in the MDV fraction vs the proteins in the total mito membrane?

A
  • They cultured cos7 cells with TOM20-Ha and MAPL-Halo -> homogenization to break the cell -> isolated the mitochondria content by centrifugation -> used the Halo link resin to isolate the MDVs from OMM contaminants -> immuno-isolation of MDVs with HA tag -> did lipidomics and proteomics.
  • They cultured cos7 cells with TOM20-Ha and MAPL-Halo -> homogenization to break the cell -> isolated the OMM proteins by slower centrifugation -> immuno-isolation of MDVs with HA tag -> did lipidomics and proteomics.
137
Q

why do they use homogenization to break the cell instead of chemical technique?

A

because they don’t want to change the biochemistry of the organelle by using acid

138
Q

why is the homogenization step even there?

A

to isolate mitochondria from other organelles and cellular debris

139
Q

how did they isolate the MDVs from the rest of the OMM?

A

by using a Halo link resin to which all the mito with MAPL binded. what was left in the supernatant is the MAPL-negative MDVs

140
Q

how did they determine OMM protein content?

A
  1. culture cos7-TOMM20HA-MAPLHalo cells
  2. homogenization (break the cell)
  3. isolate mitochondria with swelling and mechanical force
  4. swelling + mechanical force to pull out intact pieces of the OMM
  5. 10 000g centrifugation
  6. immuno-isolation of OMM fragments with HA tag
  7. MS/MS lipidomics + proteomics
141
Q

how did they determine MDVs protein content?

A
  1. culture cos7-TOMM20HA-MAPLHalo cells
  2. homogenization (break the cell)
  3. 15 000g centrifugation spins down heavy (nucleus, mito) stuff
  4. pre-clearing of supernatant with halo link resin (removing OMM contaminants)
  5. immuno-isolation of TOM20+ MDVs with HA tag
  6. MS/MS lipidomics + proteomics
142
Q

why did they do this whole immuno-isolation thing with MDVs and OMM?

A

to see if MDVs selectively enrich specific cargos or just take random cargos in the OMM

143
Q

how many proteins were found to be at least 2x more abundant in the MDVs vs OMM?

A

107 proteins (vs 1500 total mito proteins)

144
Q

how did they validate the 107 MDVs proteins?

A

repeated proteomics and normalization calculations / comparisons between MDVs and OMM proteomic results (3 biological replicates)

145
Q

in the graph with the circles showing the 107 MDVs abundant proteins, what is the Y vs X axis?

A

Y = statistical significance (p-value) of the peptide count over 3 biological replicates
X = fold change in peptide counts within the MDV fraction relative to the mitochondrial membranes

146
Q

figure 2

A

xxxxxx

147
Q

what did they surprisingly not found in the MDVs proteome?

A

no SNARE proteins for vesicle fusion, no coat proteins like clathrin or AP complexes, retromer, and no obvious fission type dynamins to pinch them off

148
Q

can we be sure that the machinery that was not found in proteomics is not in MDVs?

A

no because they could have fallen off during purification

149
Q

what did they identify in MDVs following proteomics?

A

OMM beta-barrel type proteins (highly embedded TM proteins) whose degradation pathway was unknown

149
Q
A
150
Q

why does it make sense to find OMM beta-barrel type proteins (highly embedded TM proteins) whose degradation pathway was unknown in MDVs?

A

they are highly embedded in the membrane therefore are prime candidate for why you would have vesicles just to remove them

151
Q

what mitochondrial proteins were used that is not in TOM20 MDVs to confirm cargo selectivity by confirming that the cargo doesn’t colocalize with those proteins?

A

MRPL12 and PDH (MDV negative mitochondrial markers

152
Q

how did they verify the 16 MDVs endogenous cargoes with fluorescence?

A

immunofluorescence: colocalized with TOM20 + not colocalization with PDH/MRLP12 (mito tags)

153
Q

what % of MDVs contain IMM cargoes and what can we conclude from it?

A

at least 50%; meaning MDVs are double membraned

154
Q

after identifying the cargos, what did they want to find out?

A

are the cargos present in MDVs are monomeric or are as full complexes?

155
Q

what is cool about BN (blue native) -PAGE? what did this feature allow them to find?

A

no SDS used = no denaturation of protein in the gel, can analyze stable multiprotein complexes; found that full protein complexes exit in MDVs

156
Q

how did the BN-PAGE result show them that full protein complexes were in MDVs?

A

found that the cargos were part of complexes of about the same weight (350kDa) as mito TOM complexes

157
Q

what did they fin when loosing USP30 and therefore dysregulating TOM complex?

A

an increase in TOM+ MDVs: increased turnover of TOM complexes via MDVs

158
Q

“TOM+ MDVs facilitate the lysosomal delivery and degradation of difficult-to extract β-barrel proteins and multi-subunit complexes in response to ________ ________,
thus building a mitochondrial stress response”

A

functional impairments

159
Q

what kind of protein complexes did they find in MDVs appart from TOM complexes?

A

ETC complexes (COX1, vDAC3, NDUFA9, ATP5A proteins part of ETC complex)

160
Q

The fact that complexes are intact opens the possibility that what?

A

that MDVs could carry functional enzyme complexes, for degradation or of active transport

161
Q

how do MDVs bud off of mitochondria?

A

mitochondria elongates until MDVs is pinched off

162
Q

why did they use bafilomycin A1 to inhibit lysosomal acidification?

A

to see if it affects MDVs in lysosomes; and yes there are more MDVs in the lysosome because they are not being degraded

163
Q

give an example of how MDVs could do active transport of functional components

A

is could fuse with PM (like MVBs do) to deliver complexes to other cells

164
Q

what are MIRO proteins?

A

GTPases found in MDVs that are anchored in mito and peroxisome membranes with calcium regulated domains ELM-1/2.
They bind motor proteins and are essential for fission/fusion.

165
Q

what are ELM-1/2? what do they do?

A

calcium regulated domains in miro GTPAses that bind SAM and MICOS strong parts of mitochondria

166
Q

what are SAM and MICOS?

A

strong complexes of mito membrane.
MICOS anchors both IMM and OMM

167
Q

what do miro gtpases do?

A

Binds to adaptor proteins that link to kinesin and dynein motor proteins to drive mitochondrial and MDV movement

168
Q

what happens when MIRO1 and MIRO2 were silenced?

A

number of MDVs dropped by half when 1 or both were silenced, and mitochondria hyperfuses

169
Q

what are Armcx1, 3, and 10?

A

MDV proteins that bind Miro/Trak and regulates mito distribution and motility in neurons

170
Q

what happened when ARMCS proteins were silenced?

A

silancing only 1 had no effect, silencing 2 reduced number of MDVs by half

171
Q

what does the decrease in MDVs caused by MIRO and ARMCX silencing tell us?

A

MDVs formation required the mitochondrial movement machinery (MIRO and ARMCX)

172
Q

now that they know that MIRO proteins are needed for MDVs formation, they wanted to know if __________ were needed

A

microtubules

173
Q

what is nocodazole?

A

drug that depolymerizes microtubules (is another way to look at requirement for cytoskeleton in MDV formation)

174
Q

what happened to MDVs when treated with nocodazole?

A

blocks MDVs formation, mitochondria hyperfuses: number of MDVs reduced from like 95 to like 22

175
Q

did MIRO silencing and nocodazole+ affect only TOM+MDVs?

A

no it also affected PDH+ MDVs!

176
Q

what experiement did nocodazole blocking MDVs formation allow for?

A

allowed to follow how long it took for the pre-existing MDVs to reach late endosomes because it blocked the formation of new MDVs

177
Q

what result did they get by following pre-existing MDVs after nocodazole?

A

The clearance of pre-existing vesicles to the late endosome shows a half-life of ~30 minutes

178
Q

what did MDV lipidomics reveal? what does this tell us?

A

2x enrichment in phosphatidic acid (PA): central lipid in membrane budding events that allows negative curvature

179
Q

how did they test for PA necessity in MDVs?

A

they expressed mLipin1B, enzyme that hydrolyzes (modifies) phosphatidic acid, and also expressed a mLipin1B mutant with no hydrolytic activity

180
Q

what were the results of using mLipin1B on MDVs? what about the mutant mLipin1B?

A

causes loss of MDVs.
mLipin1B mutant without hydrolytic activity had no effect.

181
Q

what is AGPAT5? why does it make sense that it is found in MDVs?

A

enzyme that generate phosphatidic acid. there is high PA in MDVS.

182
Q

what happened when they silenced AGPAT5?

A

it reduced the number of MDVs by half, showing that PA is critical in MDVs formation

183
Q

what did they conclude from the reduction in nb of MDVs from mLipin1B and AGPAT5 silencing?

A

phosphatidic acid is CRITICAL in MDVs formation

184
Q

what protein did they look at to try to understand the scission machinery of MDVs? why?

A

DRP1 because it mediates mitochondrial fission

185
Q

how did they study DRP1 in MDVs?

A

they generated CRISPR knockout cell lines, one lacking DRP1 and a control lacking dynamin DNM2.

186
Q

what is dynamin DNM2 KO?

A

knockout of microtubule machinery used as a control, showing that KO of microtubule machinery has no effect on the WT

187
Q

what did they find out about MDVs in DRP1 KO cells?

A

found completely fused mitochondria and NO MDVs, to be expected since DRP1 is essential for mitochondrial fission

188
Q

what was the control in the DRP1 KO experiment?

A

a control dynamin DNM2 (i dont know why they used it tho)

189
Q

what happened when they rescued DRP1 in DRP1 KO cells by re-expressing it?

A

MDVs came back

190
Q

what is different in DRP1 expression during MDV scission vs mito division?

A

DRP1 only expressed for 2s during MDV scission and only in small foci compared to 5s long big foci for mito division

191
Q

what did they find out about DRP1 adaptor’s role in MDV formation?

A

KO of 2/3 DRP1 adaptors (MIDs) only had a moderate effect on DRP1 formation.
KO of all 3 stopped MDV formation.

192
Q

what is CCCP? what happens to MDVs when it is given to cos7 cells?

A

something that triggers mitos fragmentation by increasing the function of DRP1

193
Q

what happens to MDVs when CCCP is given to cos7 cells?

A

it makes no difference in MDVs

194
Q

what is cycloheximide CHX? what happens to MDVs when we give it to cos7 cells?

A

something that blocks protein translation which triggers DRP1 inhibition and causes mitos hyperfusion. But, it stimulates MDVs

195
Q

what is forskolin? what happens to MDVs when we give it to cos7 cells?

A

something that inhibits adenylate cyclase which blocks DRP1 and mito fission causing mito hyperfusion. but, it stimulates MDVs (like cycloheximide)

196
Q

inhibiting mito fission (with CHX and forskolin) does what to MVDs?

A

stimulates MDVs formation

197
Q

what do the opposite effects of CCCP, cycloheximide, and forskolin on mito vs DRP1 tell us?

A

that the regulation of DRP1 recruitment of MDV scission sites is distinct from mito divison.

198
Q

what did a mutation in DRP1 GTP binding domain of DRP1KO cells do to MDVs? what can we conclude?

A

it did not rescue the MDVs (that were lost from DRP1 KO).
signaling and recruitment of DRP1 to MDV scission sites is distinct

199
Q

mutations of DRP1 phosphorylation sites that usually stimulate (S616) or inhibits (S637) mito fission did what to MDVs?

A

they both rescued MDV formation (??) in DRP1 KO cells,, doesn’t make sense, therefore tells us that the signaling and recruitment of DRP1 to MDV scission sites is distinct

200
Q

so DRP1 plays a role in MDVs formation, BUT ….

A

not the same way it does in mito fission (different active and binding sites)

201
Q

what did they find is thought to promote MDV formation as reflection of import failure?

A

ubiquitination

ANAT365 (10 decks)

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