mitochondrial life cycle and endosymbiosis evidence

Question 1

what is mitochondrial fission and mitochondrial fusion?

Answer 1

Mitochondrial fusion: physical merging of outer (MOM) then inner (MIM) mitochondrial membranes

Mitochondrial fission: Breaking apart of a mitochondrial section into two

Question 2

why is it that mitochondrial morphology can change dramatically?

Answer 2

mitochondria are dynamic organelles, often existing as a large network

their length is determined by a balance between fission and fusion

a shift in this balance between fission and fusion is what can result in a dramatic change in morphology

Question 3

when and why do we see

more fusion?

more fission?

Answer 3

More fusion = (fewer but) elongated conjoined network of mitochondria - beneficial when not dividing:
If mutations are present, longer conjoined mitochondria are more likely to have some WT functional mt-genomes making enough proteins such that oxidative phosphorylation can go on unaffected

More fission = more of the smaller, less connected mitochondria - useful in cell division - mitochondria cannot be made organically but are inherited, so this is required before cell division to ensure both daughter cells get a good no. of mitochondria

Question 4

what proteins are involved in fusion

Answer 4

Mitofusins (outer mitochondrial membrane fusion)

OPA1 in mammals/Mgm1 in yeast(GTPases used in inner mitochondrial membrane fusion)

Question 5

explain how fusion occurs

also - how does one of the proteins involved have another function

Answer 5

Docking/tethering - On the outer membrane a mitofusin from each forms a dimer

GTP hydrolysis occurs to provide energy for the outer membranes to fuse

Inner membranes fuse in same way (GTP) but via Mgm or OPA1

Note - Mgm1/OPA1 are also found at cristae, but individually, to
maintain the structure (only when there are two does fusion occur)

Question 6

what genes are involved in fission?

what is seen in mutants of dnm1 and Fis1?

Answer 6

dnm1 (yeast) or Drp1 (mammals)
Fis1 gene

Mutations in dnm1 or Fis1 gene result in large nets of mitochondria due to failed mitochondrial division (Bleazard et al. 1999)

Question 6

fusion in mitochondria experiment?

Answer 6

Used two different mitochondrial stains on two yeast cells before fusion

After fusion you can see on the overlay there’s rapid joining/network forming between the two yeast’s mitochondria, so fusion happens
Conclusion: Mitochondrial networks from haploid yeast cells fuse together in the diploid zygote

Question 6

how does mitochondrial fission occur?

Answer 6

1) Drp1 recruitment
Fis1 recruits Drp1 to membrane

2) Oligomerization Multiple Drp1 molecules join together to form ‘scission machine’

3) Fission - GTP hydrolysis fuels membrane scission

Question 7

a gene for fusion was identified in drosophila experiments - what gene what experiment?

Answer 7

looked at fly sperm development - see huge reorganisation of mitochondria - they fuse and form networks, this fusion is essential for fertility - without it males are sterile

Identified fzo gene - encoding founder mitofusin GTPase
Temperature sensitive fzo1 mutants showed no fusion/networks, but punctate dots for mitochondria

Conclusions - Fzo1p is an integral mitochondrial membrane protein Mitochondrial fusion cannot occur without functional mitofusin

Question 8

give two things that demonstrate how mitochondrial fusion is essential

Answer 8

Mitochondrial fusion in mammals -
Knockout mice for Mfn1 and/or Mfn2 die due to placental defects; cells have fragmented mitochondria (Chen et al., 2003)

Human neurodegenerative disorder Charcot-MarieTooth disease type 2A results from mutations in human mitofusin Mfn2 (Zuchner et al. 2004)

Question 9

what determines the balance between fission and fusion?

Answer 9

The balance between fission and fusion is determined by levels of mitofusins, OPA1/Mgm1, and Drp1/Dnm1

Therefore… this balance is determined by the up and down regulation of these proteins OR/AND their activity

Question 10

give some ways in which fission proteins are often regulated

Answer 10

Often regulated by phosphorylation and ubiquitination

E.g. Drp1 activity controlled by
phosphorylation at different
sites

Also - regulated by cell cycle via PKC/Cyclin B/PKA

Question 11

give some ways in which fusion proteins are regulated

Answer 11

More commonly ubiquitinated and destroyed

E.g. ubiquitin-mediated degradation of Fzo1

Proteolytic cleavage can be used to go between active and inactive form

Question 12

what is mitophagy?

Answer 12

Autophagy of the mitochondria
Allows the cell to degrade sections of damaged mitochondria, in response to change in membrane potential - depolarisation

Question 13

explain a mitochondria’s cycle when healthy vs when damaged

Answer 13

Healthy - cycle between fusion and being in a network, with fission when dividing etc… they are polarised

Damaged/depolarised -
Fission sections off the damaged section, fusion is inhibited in the organelle

have the chance for RECOVERY - e.g. defective mitochondrial proteins in the membrane can be removed via ubiquitin-proteasome system. If successful they can re enter the fusion-fission cycle of healthy mitochondria
If they cannot recover, they are destroyed in mitophagy…

Question 14

how does mitophagy occur?

Answer 14

At a functional membrane potential, the outer membrane protein PINK (a serine-threonine kinase) is imported into the mitochondria and degraded

If depolarised, PINK remains on the surface and recruits PARKIN, an E3 ubiquitin ligase

PARKIN ubiquitinates the mitochondrial proteins and the section is destroyed by mitophagy

NOTE - so if the mitochondria could somehow get mutated mtDNA in a section being degraded it would be easy to destroy

Question 15

Parkinson’s disease - what is it and how are mitochondrial dynamics relevant?

Answer 15

Degeneration of the dopaminergic neurons in substantia nigra

Familial PD linked to mutations in PINK1, PARKIN and mtDNA (amongst others)

Has been shown that having problems with mitochondrial dynamics and mitophagy can increase risk of Parkinson’s

Question 16

autosomal dominant optic atrophy -
what mutations are seen?

what are the symtpoms?

Answer 16

Mutation in OPA1 (optic atrophy 1 gene), also associated with mtDNA depletion

Symptoms include eye issues, ataxia, deafness all as a result of decreased OxPhos

Question 17

Charcot Marie tooth disease type 2A - what is it, how are mitochondrial dynamics relevant?

Answer 17

The most common hereditary peripheral neuropathy, symptoms affect hands and feet

Problems with fusion - mutations in the Mfn2 gene.

Can also impact mitochondrial motility
PROBLEM = reduced OxPhos. Without the regular fusion and fission, you can’t get the damaged sections removed and degraded

Question 18

from a quick look at mitochondria and chloroplasts, what evidence is there for endosymbiosisis theory?

Answer 18

Double membrane system - similar to bacteria (cp and mt)

Just that they have their own DNA, with small genomes (kind of similar to bacteria but acc not, like cp and mt genomes are smaller. α-proteobacteria have genomes of 4-9 Mb; mtDNA is 15-350kb ,so not miles away but still shorter)

Ribosomes are found in mitochondria, and they are different to those found in the cytoplasm/more like prokaryotic ones

Question 19

how is cpDNA and mtDNA similar to prokaryotic (and so provides evidence for the endosymbiosis idea)?

Answer 19

Circular and double stranded

Free from associated proteins (mostly) such as histones, unlike nuclear DNA

CpDNA is organised into operons

Question 20

what differences in the way mt and cp DNA function are there that support the endosymbiosis theory?

Answer 20

The protein-coding sequences of organelle genes are more like those from bacteria than like the nuclear genes of eukaryotes

In the nuclear genetic code, UGA is a stop codon, whereas in the mitochondrial genetic code UGA codes for tryptophan; AUA codes for isoleucine in the nuclear genetic code, whereas AUA codes for methionine in the mitochondrial

Question 21

give an example of how chloroplasts are similar to prokaryotes

Answer 21

chloroplasts very similar to cyanobacterium, thylakoids and all

Question 22

briefly, what is the theory on how endosymbiosis occured?

Answer 22

There was likely one engulfment - that bacteria like thing became the mitochondria

Then a second engulfment producing a separate lineage of cells - plant cells - becoming a chloroplast

Question 23

what is gene transfer (in terms of endosymbiosis)?

Answer 23

Over time genes have moved from the mitochondria and chloroplasts to the nucleus (explains why the genomes are smaller than prokaryotic ones)

Question 24

comparing chloroplasts and cyanobacteria, why might the chlorplast geneome be smaller?

Answer 24

Chloroplasts contain 60-100 genes; cyanobacteria have over 1500

some of the cyanobacteria genes may have been lost due to no longer being needed - different environment

also could be due to redundancy (nuclear genome)

gene transfer to nucleus

Question 25

give some stats that back up gene transfer, and what this now means about the organelles’ independence

Answer 25

In land plants 11-14% of nuclear DNA originates from chloroplasts

So the mt and cp genomes are no longer independent - there are over 1000 nuclear-encoded gene products that are required for mitochondrial function
so…
Both mitochondria and chloroplast function relies heavily on effective nuclear-cytoplasmic interaction

Question 26

what is maternal effect?

Answer 26

Inheritance of gene products stored in the egg, transmitted to the offspring following fertilisation. These maternally-encoded proteins can exert a phenotypic effect on the offspring, regardless of offspring genotype

Can be essential for embryo development, or can cause a transient effect later overridden by offspring gene expression or loss of the gene product