Lecture 25 - Extranuclear inheritance IV

Question 1

What do mitochondria have?

Answer 1

Mitochondria are organelles & have different behaviour in different cellular contexts. Link to implications on mitochondrial genome.

Mitophagy - removal non-functional parts of mitochondrial networks.

Question 2

Describe mitochondrial morphology

Answer 2

Size & shape of mitochondrial varies between cell types with cell cycle stages.

Mitochondria exist as constantly-evolving networks within the cell cytoplasm rather than as totally separate organelles.

Question 3

What is mitochondrial dynamics?

Answer 3

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

Mitochondrial fission: breaking apart of a mitochondrial section into 2

Mitochondrial length is determined by balance between fusion & fission

Morphologies can change dramatically by a shift in this balance - mitochondria are ‘dynamic’ organelles.

Question 4

Why is mitochondrial dynamics essential?

Answer 4

• mitochondria can’t be made; they have to be inherited
• accommodating cell growth
• ATP generation in oxygen-poor regions of cells
• redistribution of mitochondria with cell division
• genetic complementation
• important for cell survival

Mitochondrial dynamics includes fusion & fission. Due to double membrane - out membrane fusion occurs first, then fission.

This is useful as mitochondria has to be inherited. Breaking down is useful for distributing mitochondria into different cells.

Mitochondria are mainly needed for energy production, which is more efficient where there are networks of mitochondria. This helps with ATP generation in oxygen-poor regions of the cell. Compensation can occur if there is an elongated network, whereas it is unlikely to occur in mitochondria-poor areas of cell.

Question 5

What are the 3 central players in mitochondrial dynamics?

Answer 5

• Mitofusins (outer mitochondrial membrane fusion)
• OPA1/MGM1 (inner mitochondrial membrane fusion)
• DRP1/DNM1 (division of outer & inner mitochondrial membranes)

Question 6

What are the GTP-hydrolyzing proteins (GTPases) that belong to the dynamin superfaily?

Answer 6

OPA1 - mammalian
Mgm1 - yeast

Drp1 - mammalian
Dnm1 - yeast

Question 7

How was mitochondrial fusion investigated?

Answer 7

MitoTracker Red - selectively localises to mitochondria; covalenty attaches to membrane proteins

Mito-GFP - expressed in only one parental strain; under control of Gal1/10 promoter

Mitochondrial networks from haploid yeast cells fuse together in the diploid zygote.

YEAST STUDY - looking at what happens to mitochondrial network after fusion 2 yeast cells. Shows mitochondrial populations merge - biparental. 2 populations come together, fuse & genomes mix across zygote.

Question 8

Describe mitochondrial fusion investigation into fly?

Answer 8

Looking at mutations that made flies sterile. What happens to mitochondrial network during sperm cell development. Problems with mitochondrial fusion resulted in sterility. Onion stage sperm, elongating sperm stage.

• Drosophila melongaster sperm development: mitochondria undergo dramatic reorganization
• sterile male flies observed due to failure in mitochondrial fusion
• fzo gene identified: encodes the founding member of the conserved mtiofusin GTPase family

Mutations in fzo prevent mitochondrial fusion & result in sterility

Question 9

What was discovered regarding genes involved in mitochondrial fusion?

Answer 9

Temperature-sensitive fzo1 mutants.

Fzo1p is an integral mitochondrial membrane protein

Mitochondrial fusion cannot occur without mitofusin (fzo1)

25 - permissive
37 - restricted temperature

Fused network of mitochondria, but looks different at restricted temperature.

Not fusing at 38 degrees with each-other, but there is also breakdown.

Fzo1p - integral mitochondrial membrane protein.

Question 10

Describe mitochondrial fusion in mammals

Answer 10

KO mice lacking Mfn1 and/or Mfn2 die due to placental defects; cells have fragmented mitochondria

Human neurodegenerative disorder Charcot-Marie-Tooth disease type 2A results from mutations in human mitofusin Mfn2

Question 11

What is involved in inner mitochondrial membrane fusion?

Answer 11

Inner membrane protein Mgm1 (yeast) or OPA1 (mammals): Large GTPase localised to inner mitochondrial membrane

Temperature-sensitive mutants tested for fusion in vitro assay

Yeast grown at different temperatures. Different mutant were introduced into mgm1 gene - Expressed as a % of wild type.

Mutations have a significant impact on amount of fusion taking place, with a reduction in fusion taking place in restricted vs permissive temperatures.

Deformed mitochondria

Conclusion - Mutating mgm1 results in a decline in fusion events & an increase in deformed mitochondria.

Defects in mgm1 mutants specifically relate to problems with inner mitochondrial membrane fusion.

Question 12

Summarize mitochondrial fusion

Answer 12

1) Docking/tethering - mitofusin dimers form
2) GTP hydrolysis - outer membranes fuse
3) Tethering & fusion - inner membranes fuse via Mgm1 (yeast)/ opa1 (mammals)

Question 13

What occurs during Docking/tethering of mitofusins?

Answer 13

from 2 pieces of mitochondria - outer membranes come together

Question 14

What occurs during GTP hydrolysis?

Answer 14

fusion of outer mitochondrial membranes

Question 15

What occurs during Tethering & fusion?

Answer 15

Inner mitochondrial membranes docking together via OPA1. GTP hydrolysis causes membranes to fuse.

Individual copies of OPA1 protein at junction site of cristae. OPA1 is essential for maintaining the structure of cristae, if it is just 1 copy, alongside its function in fusion (where 2 or more copies are found).

Question 16

What occurs during mitochondrial fission?

Answer 16

Constant remodelling & rearrangement of mitochondria

• mitochondria are not always fusing but have an equal, balanced activity of division (fission) within most cells.
• important for the remodelling & rearrangement of mitochondrial networks, as well as for enabling mitochondrial segregation during cell division.
• mutations in dmn1 gene results in large nets of mitochondria, due to failed mitochondrial division
• Dnm1 (yeast)/Drp1 (mammals) protein physically associates with other copies of itself in curved structures on outer surface of mitochondria.
• Curved Drp1/Dnm1 structures constrict & pinch off mitochondria using the energy from GTP hydrolysis

Question 17

What are the effects of different mutations of genes involved in mitochondrial fission & fusion?

Answer 17

Fzo1-1 mutation - problems with mitochondrial fusion

dnm1 mutation - when fission can’t occur, get increasingly large sections of mitochondria joined together in nets

fis1 mutation - problems with fission (need dnm1 & fis1)

Question 18

What is a mitochondrial network doing at any given time?

Answer 18

Fission & fusion - balanced by the proteins driving each interaction.

Question 19

Summarize of mitochondrial fission

Answer 19

1) Drp1 recruitment - Fis1 recruits Drp1 to membrane

2) Oligomerization - multiple Drp1 molecules join together to form scission machine

3) GTP hydrolysis fuels membrane scission

Question 20

How does regulation of mitochondrial fusion & fission occur?

Answer 20

Balance between mitochondrial fusion & division is determined by levels of mitofusins, OPA1/Mgm1, and Drp1/Dnm1

Up-and-down regulation occurs in different cell contexts

Regulation occurs in different ways in various cell contexts & at many levels:
- transcriptional & post-transcriptional regulation
- protein stability/cleavage
- protein conformation
- modifications such as phosphorylation
- protein localization via association with binding partners

Push towards fission = increased fission proteins & decrease fusion proteins & visa versa

All features interact together to dictate what is occurring (fusion/fission)

Question 21

What are the mitochondrial fission proteins?

Answer 21

• Phosphorylation
• Ubiquitination
• e.g. Drp1 activity controlled by phosphorylation at different sites.
• Through action of PKC/Cyclin B/PKA

Range of mechanisms to regulate the balance of proteins.

Question 22

What are the mitochondrial fusion proteins?

Answer 22

• Proteolysis
• Ubiquitination
• E.g. ubiquitin-mediated degradation of Fzo1
• E.g. proteolytic cleavage of inner membrane dynamins (OPA1/MGM1)

Range of mechanisms to regulate the balance of proteins.

Question 23

What is mitophagy?

Answer 23

Damaged or defective mitochondria

Tagged with specific kinases & ubiquitin ligases

Mitochondrial fusion disabled

Destruction by mitophagy

Question 24

What is the role of mitophagy?

Answer 24

Mitophagy = mitochondrial autophagy

Important for maintenance of healthy mitochondrial population

Response to changes in mitochondrial membrane potential

Defective mitochondrial proteins can be removed via ubiquitin-proteasome system

Question 25

What is autophagy?

Answer 25

mechanism of destruction of unwanted cellular components

Question 26

What is mitophagy?

Answer 26

mitochondrial autophagy Ensures removal of damaged mitochondria - maintaining healthy mitochondria Section of mitochondria can become damaged through changes to the membranes - e.g. oxidative phosphorylation to occur, movement of protons across membrane is needed & membrane potential is needed at a certain level. A loss of this membrane potential (e.g. depolarization) leads to section of mitochondria is tagged for destruction & the cell will prevent fusion of that section of mitochondria, leading to destruction via mitophagy.

Question 27

Describe the mitochondrial life cycle

Answer 27

Mitochondrial networks under fission: - either depolarized - or polarized Depolarized then either undergo recovery or lower levels of fusion & undergo mitophagy. Polarized cells the undergo fusion & the cycle continues. Important mechanism to get rid of mutated genomes. Mechanisms are not fully discovered, but if section of mitochondria have mutated genomes, and decrease in oxidative phosphorylation, it makes sense that there is a mechanism to remove the mutated genomes.

Question 28

What difference is seen between healthy mitochondria & defective mitochondria?

Answer 28

Mitophagy Healthy mitochondria: - functional membrane potential - PINK1 (present on mitochondrial membrane) is imported & degraded. Defective mitochondria: - lack of membrane potential; PINK remains on surface (can't be internalized) & recruits PARKIN; Mitochondrial proteins ubiquitinated - destruction by mitophagy (flagging system) PARKIN problems lead to Parkinson's disease

Question 29

What does Mis-regulation of Mitochondrial Biogenesis lead to?

Answer 29

Neurodegenerative disease - Parkinson's disease (PD) is marked by degeneration of the dopaminergic neurons in substantia niagra - PD patients have reduced complex I activity in substantia niagra - Familial PD has been linked to mutations in Pink1, Parkin & mtDNA - Roles for mitochondrial dynamics, mitophagy & oxidative stress in the PD pathophysiology - Mitochondrial dysfunction observed during PD could be the result of defective mitophagy Defective mitophagy can have implications, such as the development of PARKINSON'S DISEASE.

Question 30

What is Autosomal Dominant Optic Atrophy?

Answer 30

- affects mitochondrial encoded complex 1 subunits - mutation in Opa1 (optic atrophy 1) - associated with mtDNA depletion - symptoms include opthalmonplegia (paralysis/weakness of eye muscles), ataxia, deafness - resulting from decreased OXPHOS. Not just eyes, but ataxia (movement problems) & hearing loss. Attributed to loss of oxidative phosphorylation. Mostly impacts the mitochondrial encoded complex 1 subunits & associated with mtDNA depletion.

Question 31

What is Charcot Marie tooth type 2A (CMT2A)?

Answer 31

It is the most common hereditary peripheral neuropathy - most of the CMT2A cases involve Mfn2 mutations, mainly in the GTPase domain. Can be gain of function or loss of function - defects in mitochondrial motility have also been linked to CMT2A - CMT2A patients have reduced OXPHOS which has been linked with increase in mtDNA deletions (mutations), as well as mitochondria depletion (copy number). Problems with shape of hands & feet, affecting sensory & motor neurons. Result of Mitofusin2 mutations. Also associated with problems with mitochondrial motility.

Question 32

Summarize mitochondrial networks

Answer 32

- Mitochondrial networks continually change within cells; balance of fusion & fission. - distribution of mtDNA during fission is important - defective mitochondrial with mutated mtDNA are eliminated from the network - mutations in vast numbers of genes can affect mitochondrial networks & hence impact on cell survival