Mitochondria

What is Ubiquinone?

A lipid soluble molecule that delivers its electrons to the next complex in the ETC once it has been reduced to QH2. These can be derived from Complex I or II.

How can fluorescence microscopy be used to measure mitochondrial function?

Allows assessment of mitochondrial function to assess mitochondrial states within living cells (in vivo) and at the level of the single cell in response to physiological or pathophysiological conditions. NADH (blue) and FAD (green) are both fluorescent so you can measure their redox states without adding dye. NB: NAD and FADH2 aren't fluorescent. Therefore, more respiration, less blue, more green. In cyanide poisoning (Complex IV inhibition): more blue less green. W/ uncoupler, less blue, more green. This technique can be used to report ion the oxygenation state of intact tissues.

Why is calcium overload an issue? Give an example.

Associated with cell death in many disease models. i.e. ischaemic reperfusion of the heart. Damage is mediated by the mitochondrial permeability transition pore (PTP), which is thought to open between the two dimers of ATP synthase when it undergoes a conformational change with CYCLOPHILIN D. This pore releases damaging molecules which the mitochondria has within it i.e. CC. mPTP is closed by ATP and the drug cyclosporin A. This binds to cyclophilin D and delays opening of PTP. Excessive calcium causes depolarisation, mitochondrial membrane potential is lost and the mitochondria swells. This is measured by a swelling assay - absorbance of transmitted light changes. High peroxide/free radical concentrations lower the calcium threshold and so encourage ca overload. The PTP is a druggable target

How is this achieved?

Autophagy/Mitophagy. Mtiochondria depolarize and lose their membrane potential. This signals for mitophagy (this could be how IF1 causes cancer! - high expression of IF1 plays a prominent role in tumor progression and was correlated with the regulation of tumor energetic metabolism by controlling the synthase activity of the H+-ATP synthase. encourages metastasis). LC3 is part of the autophagosome membrane that can be used as a marker. See autophagy for role in parkisonsons

How and why does calcium accumulate in the mitochondria?

Ca accumulates in the mitochondria for the same reason as TMRM. It does this through the mitochondrial calcium uniporter (MCU). This is regulated by MICU1 proteins, which interact with MCU and prevents mitochondrial calcium overload (negative regulator). EMRE, the scaffold protein, is also involved. Ca is removed via the sodium/ca exchanger (NCLX). If there is a calcium signal, mitochondria take up calcium and modulate the signal.

What is complex III?

Coenzyme Q-cytochrome c reductase. Pumps protons through the membrane and passes its electrons to cytochrome c for transport to the fourth complex of proteins and enyzmes. i.e. Q passes to III passes to CC. Cytochrome C is the acceptor of electrons from Q. CC can only accept one electron at a time (rather than 2 like Q).

How is MCU regulated?

MICU1 sits in the intermembrane soace and regulates opening of MCU (only allowed when threshold reached). MICU1 KO mice have very poor exercise tolerance. This is because calcium is taken up at resting concentration causing excessive uptake. This is expensive and unnecessary (futile ca cycling). Wastes proton gradient in maintaining the calcium gradient.

What role does mitochondria play in physiology?

Major providers of energy dependent processes in cells (this is why we need O2!) They house proteins that intiate cell death They accumulate calcium and help shape spatial and temporal patterning of calcium signals, which are key to many cell processes They have their own maternally inherited DNA. NB: mitochondrial biogenesis and replication involves regulation of synthetic pathways from both nuclear and mitochondrial genes. They house a number of synthetic enzymes involved in key processes - manufacture of haem, biosynthesis of steroids and carbonic anhydrase (for acid production in the stomach).

Explain mitochondrial fission.

Mediated by DRP1 (a GTPase) DRP1 wraps around the outer membrane and restricts it so it splits. Often associated with mitochondrial damage. Cerebellar ataxia.

How can mitochondrial potential be measured?

Tetramethylrhodamine methylester (TMRM) is a member of the lipophilic cation fluorescence family. It can cross membranes and has a net positive charge. This means it will accumulate in a compartment with a negative membrane potenial. Therefore, when mitochondria respire, they take up lots of TMRM and become brighter. The brighter the fluorescence, the faster the rate of respiration. NB: if an uncoupler is added, the dye will leave the mitochondria becasue the potential is lost.

What is complex II?

Succinate dehydrogenases. Doesn't pump across the membrane. NB: this means there are fewer ATP derived from fadh2 than nadh.

What is their role in respiration?

Take up pyruvate and other substrates by active transport. These are fed into the citric acid cycle which keeps NADH and FADH2 in their reduced form. They are oxidised in the ETC: Electrons are transferred through complexes I or II (II = flavo), then II and IV (mitochondrial core complexes). The electrons are transferred to oxygen by cytochrome c to generate water. Each step pumps protons into the intermembrane space to generate the mitochondrial membrane potential. This gradient drives ATP synthase. Protons move back in through the ATP synthase motor, driving synthesis of ATP. ANT transporter (adenine nucleotide translocator) exports ATP from the mitochondrial matrix, and imports ADP to replenish supplies so the cycle can continue.

Why do cells die after the opening of the mPTP?

Collapse of mitochondrial membrane means no oxidative ATP generation. Mitochondria may even consume ATP to maintain gradient. This leads to energetic collapse, run down of ionic gradients and the cells will die. Bax translocates to the outer membrane to conduct cytochrome C to the cytosol without disrupting the mitochondrial structure. Release of pro-apoptotic factors. Cyctochrome C and AIF (apoptosis inducing factor) that lead to activation of caspases and cell death. CC activates caspase 9 etc...

What is complex IV?

Cytochrome C Oxidase. The cytochrome holds an O2 molecule very tightly between iron and copper ions until the O2 is completely reduced. Reduced O2 picks up 2 hydrogen to produce H20. Removal of H ions from the system contributes to the ion gradient used in the process of chemiosmosis.

What relevance does this have to pathology?

Frameshift mutations of MICU1 in children cause muscle weakness, mild learning difficulties and progressive extrapyramidal motor disturbances.

Describe the mitochondrial genome.

37 genes 13 protein encoding, for proteins essential to ETC and ATP synthesis. Most mitochondrial proteins are encoded in the nucleus and then imported. mtDNA is packaged as nucleoids. These are packaged with proteins but no histones. These may make mtDNA more vulnerable to damage/mutation. Inheritance is maternal.

Why are ca oscillations important?

Key trigger to early development in the oocyte. Mitochondria will stay close to ER which will give out Ca which the mitochondria take up. Mitochondrial calcium signals outlast cytosolic. Mitochondria take longer to recover! Each calcium promotes more reduced state of NADH or FADH2 as the CAC is stimulated. Upregulates rate-limiting enzymes of the CAC. More energy needed. Uptake is sudden and rapid after 2mM. Threshold!

What is their structure?

Double membraned organelle Outer membrane and inner are distinct from one another (different lipid compositions and protein content) and have an intermembrane space Inner membrane forms cristae, made from cardiolipin and expands SA for bioenergetic transformation.

What is IF1 and what effect does it have in O2 deprivation?

IF1 is a natural inhibitor of ATP synthase. It reduces the mitochondrial membrane potential. In O2 deprivation, the ATP synthase tries to work backward as an ATPase in order to maintain the membrane potential, consuming ATP. IF1 stops this from happening without affecting the ATP synthesis during oxidative phosphorylation. Depletion of ATP can cause cell death! IF1 is upregulated in a number of cancers and is thought to be tumorigenic (upreg of aerobic glycolysis and inhib of oxidative phosphorylation w/ concurrent mitochondrial hyperpolarization). Therefore, in siRNA IF1 KO, there is higher fluorescence.

When do mitochondria need to be removed?

If dysfunctional or in excess.

How can this knowledge be used to improve the prognosis of these patients?

In ischaemia there is: progressive fall in ATP, a rise in calcium, a rise in Pi. The mitochondrial potential collapses and mitochondria can't take up calcium. At reperfusion, mitochondria repolarise, calcium is high, Pi is high and ATP is low. Mitochondria can accumulate calcium under conditions which are ideal for mPTP opening. This leads to cell death. This is a paradox - can't recover from infarct w/o O2, but reperfusion increases risk of further injury. Cyclosporin A administered with O2 with a fibrinolytic agent can reduce damage and salvage tissues.

What is complex I?

NADH dehydrogenase. Can pump 4H+ across the membrane.

How can respirometry be used to measure mitochondrial function? What does the addition of ADP, uncouplers, Oligomycin or Antimycin A do?

The Clarke electrode (oxygen electrode in a sealed container) measures oxygen consumption. If ADP is added, respiratory rate increases. NB: adp won't cross membrane of intact cell. Uncouplers (CCCP) break the coupling of ATP production with the ATC. They are weak acids or proton channels (either allow H+ re-entry or increase matric H+), which get rid of the proton gradient. ETC therefore runs at maximum in an attempt to make up for deficit of ATP. This therefore demonstartes maximum rate at which ETC can run. Oligomycin is a drug that inhibits the proton channel in ATP synthase. This is a way of measuring how much oxygen consumption is attributed to ATP synthesis. Antimycin A binds to cytochrome C reductase and therefore inhibits the ETC.

What is mitochondrial biogenesis? How is it mediated?

The process by which a cell increases their individual mitochondrial mass (in response to increased energy expenditure or caffeine - induces ca signalling mediates the increase). PGC1 is the master regulator. It is a transcriptional co-activator involved in energy metabolism (defects cause neurological disease). PGC-1 regulates the ratio of nuclear to mitochondrial proteins (this must remain constant). AMPK (AMP-activated protein kinase) is of major importance. AMPK acts as an energy sensor of the cell and works as a key regulator of mitochondrial biogenesis. AMPK activity has been shown to decrease with age, which may contribute to decreased mitochondrial biogenesis and function with aging. Activates PGC-1. When activated, PGC1 is activated, it initiates expression of many TFs like NRF1 and NRF2 (nuclear respiratory factor - increase expression of nuclear mitochondrial proteins). Also they initiate TFAM (mitochondrial transcription factor A), which translocates from the nucleus to the mitochondria and initiates transcription of mitochondrial DNA.

Explain mitochondrial trafficking.

They are always moving around, dividing and fusing. Their velocity is increased when the nerve is stimulated. V important in peripherl nervous system - dysfunction leads to peripheral neuropathy. Charcot-Marie Tooth (CMT) syndrome can be caused by defects in proteins that are responsible for trafficking.

What are some common features of mitochondrial disease?

Variable combinations of myopathy (most common), CNS disorders, diabetes, deafness, blindness and seizures. Many characterized by sudden, severe episodes of lactic acidosis. Diseases caused by mtDNA are usually heteroplasmic (some DNA is mutated and some not so there is a mixture of more than one type of mitochondrial genome). Therefore there is varied expression of disease. Severity seems to depend on the mutant load. They do NOT show mendelian inheritance.

Explain mitochondrial fusion.

When mitochondria fuse, they share proteins and mtDNA 'kiss and run'. This keeps the population happy as it replenishes proteins in ageing organelles. Requires both membranes to fuse. OPA-1 mediated fusion of inner membranes - mutations causes optic atrophy. Mfn1/2 mediates outer membrane fusion - mutations in MFN2 are associated with peripheral neuropathies (CMT).