Flashcards in Normal mitochondrial function -lecture 2 Deck (34):
What was originally thought about the complexes involved in the ETC ?
originally thought they were all distinct entities in the membrane but actually they form supercomplexes
What are the different combinations of the super complexes and why is it thought that they occur?
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
What is different about ATP synthase ?
it is scarcely part of the super complex unit but it can be found co-migrating as a dimer with other super complexes
What is the function of complex 4 in supercomplexes?
it is able to increase the catalytic activity by modifying the conformation of the super complex
What is the advantage of having super complexes?
- easier to mop up radical agents so there are less ROS
- also means they can immediately pass on electrons
What is an important property about mitochondria?
their extremely hyper polarised membrane potential- it can reach -150 to -180mV in the presence of ADP
Why do mitochondria have such a negative membrane potential?
because of the generation of the proton gradient by OXPHOS
- It is a good measure of how healthy the mitochondria are
What is JC1 staining used for ?
it is used for staining mitochondrial membrane potentials because it accumulates at very negative potentials forming JC1 complexes
What was shown about the JC1 staining in wt and mutant mouse embryonic fibroblasts?
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
What does the mitochondrial membrane potential form and what does this mean for calcium ions?
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
What does chronic hypoxia do ?
it loads the mitochondria with calcium and therefore individuals that have suffered periods of ischemia, are more likely to suffer from dementia
Which complexes produce ROS and what is a common one thats produced?
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
What is the main defence against superoxide radicals ?
1) MnSOD in mitochondria
2 + 3) Cu-Zn-SOD in the cytosol and extracellular space
What do dismutases do to superoxide radicals ?
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
What is interesting about ROS?
although they are damaging to cells they can also act as signalling molecules and are therefore important at a certain level
What are some other substances that remove ROS ?
Peridoxin and glutathione
What proteins are involved in fission process?
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
What does DRP1 do in fission ?
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
What proteins are involved in fusion ?
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
Why is the n-terminal of mitofusin 1 needed for fusion?
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
What was seen in mice with defects in fission ?
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
What was seen in mice with defects in the fusion process?
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
What are MAMs?
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
Why are these MAM contact sites important?
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
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
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
What were the proteins involved in mitophagy first known to be involved in ?
involved in mitochondria of parkinson's disease patients but unsure of function
What happens to mitochondria when they are stressed?
it causes them to undergo fission event so they break apart
When the mitochondria get stressed and undergo fission what happens?
usually one part of the mitochondria is a good part and it will continue to go onto fusion event and return to normal
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
traumatic cell death with debris, scarring and necrotic tissue
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
What are examples of stimuli for the intrinisic pathway to initiate programmed cell death?
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
What are some examples of mitochondrial disease?
leber hereditary optic neuropathy