Jones - What is mt dynamics and why is it essential for good health? Flashcards
(93 cards)
1
Q
What are enlarged mt often indicative of?
A
- disease state
2
Q
When/where does mt size and shape vary?
A
- between cell types and w/ cell cycle stage
3
Q
Do mts exist as separate organelles?
A
- no, exist as constantly evolving networks w/in cell cyto → can fuse to form these networks
4
Q
What is mt length determined by?
A
- balance between fusion and fission
5
Q
Why are mts considered ‘dynamic’?
A
- morphologies can change dramatically by a shift in balance of fusion and fission
6
Q
Why does mt growth need to be dynamic?
A
- mt can’t be made, have to be inherited
- accom cell growth
- ATP gen in oxygen poor regions of cell
- redistribution of mt w/ cell division (beneficial to break up so this can happen)
- genetic complementation (some parts might have defect in eg. complex I, so can fuse diff mts to compensate for presence of some mtDNA mutations)
- important for cell survival
7
Q
What are the 3 central players in mt dynamics?
A
- mitofusins
- OPA1(/Mgm1)
- Drp1(/Dnm1)
8
Q
What do mitofusins, OPA1 and Drp1 have in common?
A
- all GTPases (can hydrolyse GTP) and belong to dynamin superfam
9
Q
What are each of the 3 central players of mt dynamics responsible for (mitofusins/OPA1/Drp1)?
A
Mitofusins = responsible for outer mt membrane fusion OPA1(/Mgm1) = responsible for fusion of inner mt membrane Drp1(/Dnm1) = responsible for division of outer and inner mt membrane
10
Q
What were lots of the the 1st experiments into mt fusion done in?
A
- yeast
11
Q
How did an experiment in yeast show mt fusion?
A
- stained w/ 1 of 2 stains
- 1 selectively localises to mt, by covalently attaching to mem prots
- other exp in only 1 parental strain, under control of Gal promoter (so only expressed when grown on galactose)
- can see after fusion of cells, there is almost complete fusion of 2 diff mt networks
12
Q
How did further experiments in to mt fusion in Drosophila identify a new gene?
A
- stained images of dev sperm cells
- as dev get elongation
- during these processes mt bodies fuse and form big network that is reshaped as sperm reshaped (= dramatic reorg of mt)
- sterile male flies observed due to failure in mt fusion
- observed some fertility defects in these sperm
- fzo gene identified
13
Q
What does fzo gene encode?
A
- encodes founding member of conserved mitofusin GTPase family
14
Q
What did looking at fzo equivalent yeast gene show?
A
- at restrictive temp 2 pops don’t fuse (ie. no mixing of mt networks)
- due to Ts fzo1 mutants
- conclusion: need functional mitofusin for mt fusion to occur
- in mutant cells have unopposed fission, mt able to divide but cant fuse, there is no conjoined network of mt
15
Q
What is the result of mitofusin KOs in mammals?
A
- KO mice lacking Mfn1 and/or Mfn2 die due to placental defects → cells have fragmented mt
16
Q
What condition results in humans from Mfn2 mutations?
A
- Charcot Marie Tooth disease type 2a
17
Q
What is Mgm1/OPA1, and where is it found?
A
- large GTPase localised to IMM
- Mgm1 in yeast
- OPA1 in mammals
18
Q
How was it discovered experimentally that Mgm1 had a role in IMM fusion, and why is this data not as convincing as it could be?
A
- Ts mutants tested for fusion in in vitro assay
- looking at no. of fusion events –> decreased at restrictive temp
- therefore mgm1 essential for inner mt membrane fusion
- BUT also decline at permissive temp → not addressed by authors (so not particularly clear data)
- overall prob is a reduction, but data not as convincing as could be
- from timelapse images can see fusion of OMM, but IMM doesn’t fuse
- conclusion: defects in mgm1 mutants specifically relate to problems w/ IMM fusion (and not OMM)
19
Q
What is the role of mitofusins in docking/tethering?
A
- mitofusin from 2 membranes come/dock together and tether 2 sections to each other
20
Q
Why is GTP hydrolysis important in mt fusion?
A
- allows OMMs of 2 mt bodies fuse
21
Q
Apart from IMM fusion, what is OPA1/Mgm1 important in?
A
- important role in maintenance of cristae structure
22
Q
What is mt fission important for?
A
- important for remodelling and rearrangement of mt networks, as well as enabling mt seg during cell div
23
Q
What do mutations to Dnm1 gene result in?
A
- large nets of mt due to failed mt division
24
Q
What is the mammalian equivalent of Dnm1 (yeast)?
A
- Drp1
25
How does Dnm1/Drp1 perform its role?
- oligomerises and physically assoc w/ other copies of itself in curved structures on outer surface of mt
- forming a scission machine
- curved Drp1/Dnm1 structures constrict and pinch off mt using energy from GTP hydrolysis
26
How was it determined experimentally that fis1 is important in fission?
- ts mutants grown at restrictive temp
- mt fluorescently labelled
- looked at WT, fzo1 mutants, fis1 del mutant and dnm1 del mutant
- if KO dnm1 get huge networks of mt forming through unopposed fusion
- if KO fzo1 get unopposed fission
- conclusion: role of fis1 important in fission, as look like other fission mutants
27
How is Drp1 recruited?
- fis1 bound to OMM and recruits it
28
How is balance to mt fusion and fission determined?
- levels of mitofusins, OPA1/Mgm1 and Drp1/Dnm1
29
In what diff ways can reg of balance between fusion and fission be determined, in diff cell contexts?
- protein stability
- protein cleavage
- protein conformation
- changes, eg. phosphorylation, ubiquitination, PTMs
- protein localisation via assoc w/ binding partners
30
What is mitophagy?
- autophagy of mt
31
Why is mitophagy necessary?
- allows cell to get rid of defective sections of mt, so important for maintaining healthy mt pop
32
What does mitophagy occur in response to?
- changes in mt membrane pot
33
How can defective mt prots be removed?
- via ubiquitin proteasome system
34
How are damaged or defective mt destroyed?
- tagged w/ specific kinases and ubiquitin ligases
- mt fusion disabled
- destruction by mitophagy
35
What is the mt life cycle?
*DIAG*
36
Do mt divide just for cell division?
- no, constantly happening even in normal cell growth
37
What happens to mt if defective depolarised section?
- fusion not allowed and degrad by mitophagy
| - or poss for mem pot to be recovered, if this happens then allowed to rejoin network
38
Why does there have to be mechanisms so always at least 1 copy of mtDNA
- so after division each section of mt network must contain mtDNA
39
What happens if fission is decreased?
- nucleoids cluster and lose even distribution
40
In what kind of cells has of close prox between nucleoids and mt division sites been observed?
- budding yeast and mammalian cells
41
How is mtDNA organised?
- into nucleoids, nucleoids distrib t/o mt network
42
What did time lapse fluorescence microscopy images show about what happens to mtDNA during mt fusion/fission, and what questions did this raise?
- nucleoids marked at mt division sites
- by 36s clear division of 2 parts of mt membrane
- dye showed nucleoids present at these division site
- present at 80% fission sites (63% at both tips, 37% at 1 tip)
- do nucleoids move to fission site?
- or does location of nucleoid determine site of fission?
- (lots of research ongoing, but don’t know)
43
What is ERMES?
- ER mt encounter structure
44
What was found experimentally about the ERMES complex and mt fission?
- ERMES present at fission sites and partly responsible for fission occurring
- could be that nucleoid attracts ERMES to site and fission occurs, or ERMES binds and attracts nucleoids to site
- conclusion: ERMES complex assoc w/ mt fission sites, present at 60% division sites
45
What is the importance of mt fusion/fission for mtDNA integrity?
- mtDNA undergoes freq mutations due to close prox w/ ROS gen site
- mt dynamics plays important role in compensating for these mutation
- mammalian cells w/ defects in mt fusion/fission machinery have reduced mtDNA content and an increased rate of mtDNA mutation --> not allowed to fuse back into network, v important to maintain integrity
46
What evidence is there for other protective mechanisms (apart from mt fusion/fission) for mtDNA integrity?
- complementation --> restoration of oxphos function t/ complementation in cybrids, saw signif increase in levels, but not fully returned to normal (suggests some complementation)
- deletion/repair of mutant mtDNAs --> when mixed mtDNA, either WT pref rep (and mutant diluted out), or WT used as template to repair DNA
- segregation of mutated mtDNAs --> mechanism to selectively attract all mutated mtDNAs to 1 area o fmt network to be effective, then just eliminate this area through mitophagy
47
What is a cybrid, and how can they be made in the lab?
- cytoplasmic hybrid
- might make in lab by treating bunch of somatic cells w/ eg. polyethylene glycol, disrupting mem, spin to remove nucleus and left w/ cytoplast, then fuse w/ somatic cells, so have somatic cell nucleus from 1 type and heteroplasmic mtDNA pop
48
What is a cytoplast?
- cyto w/ mt and other components of cyto in it
49
How does fusion/fission balance change at G1/S (inter)phase?
- needs increase in ATP levels to gen biomolecules, to increase efficiency of ATP synthesis mt fuse and elongate
- so more fusion/less fission
50
How does fusion/fission balance change at mitosis?
- mt fragmentation needed to ensure distrib of mt to daughter cells
- so more fission
51
What was found about fission of mt networks in mitosis, through confocal microscopy images?
- mt network stained red t/o interphase and diff stages of mitosis
- in interphase conjoined network
- rapid fission of network occurs early in mitosis
- come together at end before cytokinesis
- conclusion: mt networks mostly tubular during interphase
52
What conclusion was drawn from an experiment using RNAi knockdown of Drp1 in HeLa cells?
- Drp1 essential for mitosis related mt fission
53
How was reg of Drp1 in cell cycle specific manner determined experimentally, and what was found?
- showed Drp1 phos to activate it, by cdk1/cyclinB MPF complex
- aimed to identify which residue --> identified 4 poss ser residues that could be phos
- gel shift assay
- in WT just 1 band, phos causes v small shift in prot, so see v small increase in prot size --> 2 bands of gel for first 3 mutants (so not this site responsible, as mutating this Ser site and still get shift
- in last one only get 1 band, so mutating this ser site does stop phos --> therefore this is the residue getting phos
54
What did studies in yeast show about what happens to mt when cells divide?
- distrib of mt during asymmetric cell division --> role of cytoskeleton
- looking at cycle during G1 phase, get actin filaments lining up from potential bud sites, and on these filaments get mt aligning
- as bud starts to dev, mt start to move to mother cell tip (retrograde movement) or bud cell tip (anterograde movement)
- before cell splits mt anchored at poles so can make clear break for cell division
55
How is decision between retrograde and anterograde movement made?
- proposed that ‘fitter’ mt more motile and more likely to move to bud --> as takes more energy to move anterograde (could be mechanism to ensure ‘fitter’ mt as req more here than my mother)
- site specific anchorage retains mt to certain locations
- mt anchored in bud tip have fewer ROS
56
What do the quality control mechanisms for damaged mt involve?
- mt fusion repairs low functioning mt by intra-organellar complementation
- firstly try to mend --> molecular chaperones bind and stabilise unfolded prots
- if can't be then proteases w/in and outside organelle degrade mt prots
- mitophagy and mt fusion and fission eliminate mt that are beyond repair
57
What are the 2 important prots involved in mitophagy?
- PINK1 (PTEN induced putative kinase protein 1) = a ST kinase
- PARKIN (E3 ubiquitin ligase)
58
How does PINK1 and PARKIN differ in healthy and defective mt?
- healthy mt = functional mem pot, PINK1 imported and degraded
- defective mt = lack of mem pot, PINK1 remains on surface and recruits PARKIN, mt prots ubiquitinated, destruction by mitophagy
59
What disease has PARKIN been assoc w/?
- Parkinson's Disease
60
What is the hallmark of PD, and what does this result in ?
- degen of dopaminergic neurons in substantia nigra
- patients have reduced complex I activity in substantia nigra
- defective mitophagy so mt network can't stay healthy, reduced OXPHOS capacity, get problems w/ neurons
61
What mt related mutations has familial PD been linked w/?
- PINK1, Parkin and mtDNA
62
How does PD pathophysiology relate to mt?
- roles for mt dynamics, mitophagy and oxidative stress
63
What is autosomal dominant optic atrophy assoc w/, what are the symptoms and what causes it?
- assoc w/ mtDNA depletion
- degen of optic nerve --> symptoms inc ophthalmoplegia, ataxia, deafness
- results from reduced OXPHOS capacity
- largely effects mt encoded complex I subunits
64
What mutation causes autosomal dominant optic atrophy?
- mutation in OPA1 (mammalian inner mt mem fusion prot)
65
What is the most common hereditary peripheral neuropathy?
- Charcot Marie Tooth Type 2A
66
What is CMT2A caused by, and what does it result in?
- most cases involve Mfn2 (OMM fusion prot) mutations, mainly in GTPase dom, can be gain or loss of function
- defects in mt motility also linked
- patients have reduced OXPHOS, linked w/ increase in mtDNA deletion
67
What roles do mt play w/in the cell?
- apoptosis via caspase system
- KC
- oxphos
- Ca signalling
68
What was the main finding of a paper investigating effects of Dnm1 del on ageing, in yeast?
(Scheckhuber)
- reducing fission (Dnm1 del) results in increased life span and fitness in 2 fungal ageing models (P. anserina and S. cerevisiae)
69
What did a paper investigating effects of Dnm1 del on ageing, discover about P. anserina yeast, inc diffs in gene exp and morphology between WT and mutant strains?
(Scheckhuber)
- older cells had higher levels of Dnm1 than juvenile
- juvenile and mature WT cells had filamentous mt
- mutants juvenile and mature cells had elongated mt
- senescent cells (both mutant and WT) had fragmented mt
- therefore Dnm1p exp more w/ age and fragmentation increases w/ age
- mean lifespan in WT lower
- as less fragmentation leads to longer lifespan
70
What did a Dnm1 del prevent in a paper investigating effects of Dnm1 del on ageing?
(Scheckhuber)
- mtDNA reorg in old yeast
- delayed H2O2 release
etc. ..
71
What did a paper investigating effects of Dnm1 del on ageing, discover when reprod Dnm1 mutants in S. cerevisiae yeast?
(Scheckhuber et al)
- similar results to P. anserina
- deletion of Dnm1 counteracted age related fragmentation of mt, by forming nets of densely packed mitochondrial tubules
- similar mem pot in mutant to WT, so not reg by ETC
- both Dnm1 and fis1 del mutants increased lifespan and fitness of cells, comp to WT
72
What did a study into OPA1s roles find, by exp a prot in bacteria and yeast?
(Ban et al)
- OPA1 essential for fusion of IMM
- interaction of OPA1 w/ mems can stim higher order assembly, enhance GTP hydrolysis and lead to mem deformation in tubules
- mutant OPA1 results in fragmented mt morphology, preventing fusion
73
In a study into the role of OPA1, what was found out about the effects of lipid on OPA1 behaviour?
(Ban et al)
- assocs w/ -vely charged phospholipids (not w/ neutral)
- OPA1 + neutral phospholipids = basal level of GTP hydrolysis
- OPA1 + negative phospholipids = enhanced GTP hydrolysis
- w/ cardiolipin, higher order oligomers of OPA can form
- cardiolipin is in high conc in IMM, so prob binds OPA1, enhancing GTP hydrolysis and releasing more energy for IMM fusion
74
What can liposomes be used as a model for?
- mt mem
75
What were the findings of a study into the roles of OPA1, on mem tubulation and fusion?
(Ban et al)
- OPA1 alone responsible for tubulation
- GTP hydrolysis not req, just reg fusion activity by controlling interactions of OPA w/ IMM
- OPA1 facilitates fusion of mt mems
76
How is OPA1 disruption implicated in disease alleles?
- OPA1 disrupted in DOA (dominant optic atrophy)
- mutation causes loss of tubular mt, so loss of fusion activity
- DOA mutations affect liposome binding and GTPase activity --> and OPA1 is involved in lipid binding, reg of GTP hydrolysis and mem tubulation
77
What phenotype did a study of zebrafish find loss of MFN2 function could lead to?
(Chapman et al)
- CMT2A like phenotype
78
Why were zebrafish (and not mice) used for a study of MFN2 KOs?
(Chapman et al)
- MFN2 KO leads to placental defects --> lethal to mice
| - but zebrafish dont req placenta
79
What is the effect of MFN2 mutation on mt morphology, and how was this visualised?
(Chapman et al)
- mutations cause fusion failure
- visualised by staining mt
- applied model to humans, supporting theory that MFN2 mutations lead to abnormal mt morphology, leading to eg. CMT
80
How was it investigated whether MFN2 loss of function lead to loss of motor function?
(Chapman et al)
- investigated swimming behaviour (indicator of motor function)
- comp WT to heterozygotes and homozygous mutants
- loss of MFN2 function shown to be assoc w/ progressive motor defects in adult zebrafish
81
Is loss of MFN2 function assoc w/ defects in distal axon and abnormal NMJ pathology?
(Chapman et al)
- area of pre and postsynaptic compartments signif reduced
| - showed alt swimming in MFN2 del zebrafish is assoc w/ defects at NMJ
82
What was found when looked at MFN2 mutations and mt distrib along the axon?, and how did this relate to CMT2A
(Chapman et al)
- looked at muscle biopsies
- MFN2 mutations lead to alt mt distrib along the axons
- CMT2A patients have irregular distrib of mt in axons of muscle cells
- signif reduction in mt density and increase in inter mt distance in the proximal (ie. mt are further apart), but not the distal axon
- so MFN2 mutation can affect retrograde transport and leads to alt distrib along axon
83
What is the role of Pink1, according to a study in Drosophila?
(Yang et al)
- interacts w/ mt fission/fusion machinery and modulates mt dynamics, linked to familial PD
84
How were the genetic interactions of Pink1 w/ mt fusion/fission pathways investigated?
(Yang et al)
- Drp1 overexp and Opa1 like heterozygosity restored dopamine levels
- so Pink1 may play role in reg dopaminergic physiology
- then overexp Pink1 in dopamine neurons
- -> induced mt clustering and changed mt morphology, may be caused by excessive fission
- -> supports Pink1 promoting mt fission
85
What is the relationship between Pink1 and Drp1 and how was this investigated?
(Yang et al)
- single and double knockdowns
- caused perinuclear aggregation of mt
- some cells from double knockdown had long continuous threads of mt (extreme fusion)
- disruption of mt function alone does not lead to extreme mt fusion
- interaction between Pink1 and Drp1 is specific for reg mt morphology
86
What prots are conserved as +ve regulators of mt fission?
| Yang et al
- Pink1
- Fis1
- Drp1
87
What is the relationship between Pink1, Fis1 and Drp1 in mammals?
- Drp1 may act ds of Pink1
| - Drp1 and Fis1 epistatic to Pink1
88
How do levels of fusion and fission prots (and eg.s of each) differ in HD patients?
(Reddy et al)
- high levels of fission prots (Drp1, Fis1) and low levels of fusion prots (MFN1, MFN2, OPA1) in HD patients
89
What is mutant Htt assoc w/ in affected brain regions of HD patients?
(Reddy et al)
- mt imbalance and mt dynamics impairs axonal transport of mt
- decreases mt function
- damages neurons
90
What is the effect of Htt on mt encoded genes?
| Reddy et al
- increased exp of mt encoded genes in complexes I, III, IV, V in patients
- may be compensation for loss of mt function caused by mutant Htt
- related to this loss of neurons mt function
91
What is the result of the fact that HD patients have lower no. neurons?
(Reddy et al)
- mt function lower
92
What was the result of determining oxidative DNA damage was present in HD patients?
(Reddy et al)
- may be due to increased assoc of mutant Htt w/ mt in neurons
- mutant Htt overexp involved in oxidative damage during disease progression
93
What did a study about the balancing of mt dynamics find about its effects on mt fitness, in S. cerevisiae?
(Bernhardt et al)
- made double Mgm1/Dnm1 mutant, had same morphotype as WT due to complementation
- assessed replicative lifespan of mutants --> mean lifespan of double mutant and Mgm1 mutant much shorter than Dnm1 mutant, as more sensitive to stress conditions
- reduced lifespan when Mgm1 del, so has role in responding to exogenous stressors
- energy metabolism analysis: single Dnm1 mutant not signif diff to WT in terms of resp capacity, but double mutant had signif lower ETC capacity and basic resp capacity comp to WT
- analysed mt mass and quality:
- -> Mgm1 del has all small colonies
- -> also Dnm1 del had more small colonies, double mutant had even more
- impairment in mt fission and fusion leads to reduction in mitophagy
- overall reduction of mitophagy key to giving rise to specific characteristics of double mutant
