Normal mitochondrial function -lecture 2

Question 1

What was originally thought about the complexes involved in the ETC ?

Answer 1

originally thought they were all distinct entities in the membrane but actually they form supercomplexes

Question 2

What are the different combinations of the super complexes and why is it thought that they occur?

Answer 2

3 + 4
1 + 3
1 + 3 + 4
not fully understood but appears to relate to oxygen and substrate levels and likely serves to reduce ROS released form complexes 1 and 3

Question 3

What is different about ATP synthase ?

Answer 3

it is scarcely part of the super complex unit but it can be found co-migrating as a dimer with other super complexes

Question 4

What is the function of complex 4 in supercomplexes?

Answer 4

it is able to increase the catalytic activity by modifying the conformation of the super complex

Question 5

What is the advantage of having super complexes?

Answer 5

• easier to mop up radical agents so there are less ROS

- also means they can immediately pass on electrons

Question 6

What is an important property about mitochondria?

Answer 6

their extremely hyper polarised membrane potential- it can reach -150 to -180mV in the presence of ADP

Question 7

Why do mitochondria have such a negative membrane potential?

Answer 7

because of the generation of the proton gradient by OXPHOS

- It is a good measure of how healthy the mitochondria are

Question 8

What is JC1 staining used for ?

Answer 8

it is used for staining mitochondrial membrane potentials because it accumulates at very negative potentials forming JC1 complexes

Question 9

What was shown about the JC1 staining in wt and mutant mouse embryonic fibroblasts?

Answer 9

in normal mouse embyronic fibroblasts there is lots of jc1 complex staining but in the defective cells the membrane potential is depolarised due to OXPHOS not functioning properly as there are defects in the fission process so there is little staining

Question 10

What does the mitochondrial membrane potential form and what does this mean for calcium ions?

Answer 10

It forms an intracellular gradient between intracellular millieu and the mitochondria which has a big heavy gradient for positive charged ions such as calcium ions
- when calcium uniporter is opened you immediately get calcium flowing into mitochondria down its concentration gradient therefore the mitochondria act as a calcium buffer when cytosolic calcium rises- this may smooth calcium dynamics

Question 11

What does chronic hypoxia do ?

Answer 11

it loads the mitochondria with calcium and therefore individuals that have suffered periods of ischemia, are more likely to suffer from dementia

Question 12

Which complexes produce ROS and what is a common one thats produced?

Answer 12

complex 1 and 3
- generate ROS specifically superoxide radicals that are highly damaging to mitochondrial DNA, membrane lipids and proteins and if they leak into the cell they can cause cellular damage

Question 13

What is the main defence against superoxide radicals ?

Answer 13

superoxide dismutases
1) MnSOD in mitochondria
2 + 3) Cu-Zn-SOD in the cytosol and extracellular space

Question 14

What do dismutases do to superoxide radicals ?

Answer 14

they are very catalytically active enzymes and they dismutate superoxide radicals to hydrogen peroxide
- although hydrogen peroxide is a ROS, at low levels it isn’t too bad and it is broken down by catalase to water and oxygen

Question 15

What is interesting about ROS?

Answer 15

although they are damaging to cells they can also act as signalling molecules and are therefore important at a certain level

Question 16

What are some other substances that remove ROS ?

Answer 16

Peridoxin and glutathione

Question 17

What proteins are involved in fission process?

Answer 17

FIS1 and dynamic related protein 1 (DRP-1)

• Drp1 is a member of the large GTPase family ad is important in the final parts of the process
• FIS1 is present throughout the outer mitochondrial membrane and is thought to recruit DRP1 from the cytosol so it can localise at punctate foci on the outer mm

Question 18

What does DRP1 do in fission ?

Answer 18

thought to form a ring around the mitochondrion and mediate constriction process at the plane of organelle fission

• maybe to act as a mechanoenzyme that changes angle of spiral on GTP hydrolysis similar to dynamin
• the 20nm internal diameter of dynamin helix is too small to wrap around typical 500-700nm wide mitochondrion so DRP1 helices could wrap around the mitochondrial constriction site like a twisted rope

Question 19

What proteins are involved in fusion ?

Answer 19

dynamin family members - OPA1, mitofusin 1 and mitofusin 2

• OPA1 is a large GTPase located in inner and outer membrane
• mitofusin 1 and 2 form homo and hetero complexes with each other

Question 20

Why is the n-terminal of mitofusin 1 needed for fusion?

Answer 20

because it is orientated towards the cytosol while the c-terminal coiled coil domain also faces the cytosol where it coordinates docking of mitochondria to one another through antiparallel binding to the c-terminal coiled coil domain of mitofusin 1 or 2 molecules on adjacent mitochondria

Question 21

What was seen in mice with defects in fission ?

Answer 21

in WT mice the mitochondria are nicely spread out with long parts while in the mutant mice no fission is occurring so you get masses of mitochondria altogether because they can fuse but they cant break apart
these mutants die early of a massive cardiac related myopathy after about 6 months

Question 22

What was seen in mice with defects in the fusion process?

Answer 22

unable to sit up and they suffer many neurological defects

• point mutations in dynamin proteins cause embryological lethality in homozygotes
• defects in mitofusin genes lead to disruption of the fusion process causing splaying of legs, animals lose any locomotor control, even just small defects cause substantial phenotypic defects therefore a large defect is likley to be catastrophic

Question 23

What are MAMs?

Answer 23

mitochondrial associated membrane - tethering of mitochondria and endoplasmic reticulum
- enable communication between the ER and mitochondria
- made up of an area of the ER rich in several lipid biosynthetic enzyme activities causing it to become reversely attached to a mitochondria
MAMs are associated with many disease states such as AD

Question 24

Why are these MAM contact sites important?

Answer 24

appear to be needed for key cellular events such as transport of calcium from the ER to mitochondria, import of phosphatidylserine into mitochondria from ER for decarboxylation, formation of autophagosomes, regulation of morphology, dynamics and functions of mitochondria and cell survival

Question 25

Define autophagy:

Answer 25

basic catabolic mechanism involving degradation of unnecessary or dysfunctional cellular components though actions of lysosomes - ensures survival during starvation by maintaining cellular energy levels

• functioning properly it ensures synthesis, degradation and recycling of cellular components
• cytoplasmic constituents are isolated from the rest of the cell in autophagosome and then fuse with lysosome to be degraded

Question 26

Define mitophagy:

Answer 26

specialised form of autophagy that can either promote cell survival by removing damaging mitochondria or initiate cell death - however if this process goes wrong it is likely to cause a disease

Question 27

What were the proteins involved in mitophagy first known to be involved in ?

Answer 27

involved in mitochondria of parkinson’s disease patients but unsure of function

Question 28

What happens to mitochondria when they are stressed?

Answer 28

it causes them to undergo fission event so they break apart

Question 29

When the mitochondria get stressed and undergo fission what happens?

Answer 29

usually one part of the mitochondria is a good part and it will continue to go onto fusion event and return to normal
but
the other part is still depolarised so it is bas and therefore the best thing to do is get rid of it
- bad part associates with protein called PINK present in the cytosol
- PINK associates with outer mito membrane but it is internalised into mitochondria under normal conditions in healthy mitochondria
- healthy mitochondria internalse it and break it apart but defective ones cant
PINK starts to accumulate on the outer membrane and it attracts parkin causing it to be ubiquitinated and targeted for proteosomal degradation

Question 30

define necrosis:

Answer 30

traumatic cell death with debris, scarring and necrotic tissue

Question 31

define apoptosis:

Answer 31

programmed cell death, recycling of components, no scarring. it is vital in development but may also have a role in degenerative disorders
- intrinisic signalling pathway involves non-receptor mediated intracellular signals, inducing activites in the mitochondria that initiate apoptosis

Question 32

What are examples of stimuli for the intrinisic pathway to initiate programmed cell death?

Answer 32

viral infections or damage to cell by toxins
free radicals or radiation
they cause changes in the inner mitochondrial membrane resulting in the loss of transmembrane potential causing the release of pro-apoptotic proteins such as cytochrome c into the cytosol

Question 33

What are some examples of mitochondrial disease?

Answer 33

pearson syndrome
leigh syndrome
leber hereditary optic neuropathy

Question 34

What are mitochondrial dysfunction usually due to ?

Answer 34

due to OXPHOS problems

- can manifest themselves in different ways often due to excess ROS production or failure of antioxidant defences