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LT17-18 Mitochondria
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Mitochondria structure, function, experiments used to elucidate function, mitochondrial disease
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MitochondriaFunctions
Mitochondria central to life, necrotic and apoptotic cell death
Mitochondrial dysfunction has been implicated in a large array of disease
Major providers for energy for energy dependent processes in cells (most of our
physiology is adapted to efficiently transport oxygen to cells for mitochondrial
respiration)
House proteins that initiate apoptosis
Accumulate calcium – help shape spatial and temporal patterning of calcium
signals key to many cellular processes
Contain own DNA – inherited maternally: Mitochondrial biogenesis and
replication involves regulation of synthetic pathways from both nuclear and
mitochondrial genomes
House synthetic enzymes involved in key processes = manufacture of haem,
biosynthesis of steroids
Respiration
Peter Mitchel Nobel Prize (1978) – Chemiosmotic
scheme for coupling respiration and
phosphorylation
Linkage of electron transport, proton pumping and
ATP synthesis
First evolved in bacteria – aerobic eukaryotic cells
than adopted the mechanisms through engulfing
aerobic bacteria to form mitochondria and later in
lineages leading to algae and plants, engulfed
cyanobacteria to form chloroplasts
Process
1. Mitochondria use pyruvate (glucose + sugars) and fatty acids (fats) as fuel
Fuel molecules are transported across IMM and converted to crucial metabolic
intermediate acetyl coA by enzymes in the matrix
2. Acetyl groups of acetyl coA then oxidised in the matrix via citric acid cycle –
cycle converts the carbon atoms in acetyl coA to CO 2 (waste product)
Cycle generates high-energy electrons, carried by the activated carrier
molecules NADH and FADH2
3. NADH and FADH2 donate high energy electrons to electron-transport chain in
the mitochondrial membrane , thus being oxidised back to NAD+ and FAD
Electrons passed along chain to molecular oxygen to form water
, Passage of high-energy electrons releases energy which is used to pump protons
across IMM, resulting proton gradient drives synthesis of ATP (oxidative
phosphorylation)
Experimental Techniques – How do we know and study mitochondria?
Oxygraph chamber
Can measure absolute concentration of oxygen as it
declines OR oxygen consumption rate
- Isolated mitochondria/cells can be used
- Oligomycin inhibits ATP synthase – by adding
oligomycin, can deduce how much oxygen is needed to
produce ATP
- Then add uncoupler to deduce the mitchondria’s
maximum capacity to respire (uncouplers dissipate the
proton gradient)
Fluorescence Microscopy
Probe aspects of mitochondrial function to assess
mitochondrial states within living cells at the level of
the single cell in response to physiological or
pathophysiological conditions
Cardiac mitochondria – make up 40% of cell volume
Measuring mitochondrial potential
Mitochondria isolated from rat heart, bathed in TMRM (Tetramethylrhodamine
methyl ester) (expts. with Helen Maddock)
TMRM are fluorescent dyes which are cell permeant, cationic , red-orange, readily
sequestered by active mitochondria
Delocalised positive charge -Attracted and accumulate in compartments with
negative membrane potential (so in mitochondria)
The larger the membrane potential, larger the concentration of dye, larger the
fluorescence
Give substrate to mitochondria and observe fluorescence
Another method is to add uncoupler – leads to the fluorescence diffusing then
returning after uncoupler washes away
Origin of mitochondria – endosymbiotic theory
, Mitochondrial DNA similar to bacterial DNA – encodes 13
essential proteins of respiratory chain and mRNAs
Majority of mtproteins are encoded in nucleus and imported
Some organism (eg. fungi) have a more complex
mitochondrial genome
Genomes vary in size from 80-100kb in fungi and plants
(containing introns), 20kb in mammals (compact, no introns
and can overlap)
Human mtDNA: 16.6kb, 37 genes
Mitochondrial biogenesis/replication requires coordinate synthesis of new and
protein from both mitochondrial and nuclear DNA and DNA replication
Mitochondrial DNA
Each mitochondria contains hundreds of copies of mtDNA packaged as nucleoids –
each cell has thousands of copies
How does mtDNA partition or segregate during mitochondrial biogenesis? Is biogenesis
coordinated with the cell cycle?
Many diseases associated with specific mutations of mtDNA – most show a maternal
inheritance and mitochondrial heteroplasmy
Causes a variety of problems – myopathy, CNS disorders, diabetes, lactic acidosis
etc. (No mendelian inheritance – severity of symptoms depend in part on mutant
load)
Mitochondrial biogenesis – controlled by(?) research
Mitochondria membrane potential is increased in myotubes exposed to caffeine for
5 hours a day for 5 days: longer term treatment with caffeine at low doses causes
regular calcium treatments
Mitochondria proteins, DNA copy number also increased – driven by calcium
(intermediary which couples signals that increase in energy supply with increase
mitochondrial biogenesis)
Mitophagy
Dysfunctional or surplus mitochondria are
removed by autophagy
Nutrient deprivation-induced type 1
mitophagy in GFP-LC3 (Kim et al., 2011)
Damage mitochondria by shining a laser on
them – autophagosome membrane encloses
mitochondria, this fuses with lysosome and
is then degraded
, Mitochondrial populations are constantly mobile
Mitochondrial trafficking – long axonal
tracts in vivo via transgenic mouse
expressing CFP in neuronal mitochondria
Mitochondrial proteins may move between
mitochondria through fusion and fission
events
Calcium Signalling
Contrary to popular belief – mitochondria does not rely of ATP:ADP ratio – this
ratio is too critical for cell function, it could be detrimental if it changed: not
enough conclusive evidence for this
Calcium in the matrix upregulates rate limiting enzymes of the citric acid cycle
Intra-mitochondrial calcium concentration within matrix is low – calcium moves along
an electrochemical gradient (negative potential inside, low calcium concentration
maintained via the sodium-calcium exchanger)
Mitochondrial calcium uniporter identified by Rizzuto and Mootha groups (2011)
- Consists of complex of proteins MCU (previously cdc109a) – channel forming
transmembrane protein probably forming tetrameric channels
- MICU1 probably confers calcium ion sensitivity on the channel, cdc109b appears
to act as a dominant negative – overexpression reduces and knockout increases
calcium flux
Increase in intramitochondrial calcium concentration increases the rate of ATP
synthesis, matching energy supply demand - using luciferin to identify (Jouaville et
al., 1996)
Changed in work that require ATP usually associated with rise in [Ca 2+] –
transmission of calcium into the mitochondria matches energy supply with demand
Short-term response = increase ATP made, long-term response = increase
biogenesis
Still do not know if biogenesis in the brain is regulated this way
The purpose of Programmed Cell Death
Tissue development/reorganisation – shaping organs (eg. removal of interdigital web
to form a mature mouse paw or hand during embryonic development)
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