Lecture 5 Notes – Mitochondria & Peroxisomes

Mitochondria: Definition, Roles & Significance

• "The human cell is a symbiosis of two life forms, the nucleus-cytosol and the mitochondrion" – Wallace (2007)
• NIH (2009) definition: organelles that convert energy from food into a usable cellular form (ATP)
• Key functional themes:
  • ATP production via oxidative phosphorylation (≈ >95\% of total cellular ATP)
  • Sequestration & release of $\text{Ca}^{2+}$ for signalling and metabolic regulation
  • Storage/release of pro-apoptotic factors (cytochrome c, AIF, SMAC/DIABLO)
  • Major endogenous source of reactive oxygen species (ROS); ROS act as signalling molecules but can also drive pathology
  • Generates heat through regulated proton leak (thermogenesis)

Evolutionary Origin (Endosymbiotic Theory)

• Pre-eukaryotic anaerobe engulfed an aerobic prokaryote (primitive $\alpha$-proteobacterium)
• Engulfed cell retained its own genome ⇒ double-membrane organelle
  • Outer membrane: derived from host plasma membrane
  • Inner membrane: original bacterial membrane, retains cardiolipin & impermeability
• Evidence:
  • Circular mtDNA, prokaryote-style ribosomes (55–60S), fission/fusion reminiscent of bacterial division
  • Maternal inheritance pattern (no mtDNA mixing at fertilisation)

Ultrastructure

• Outer mitochondrial membrane (OMM)
  • Contains VDAC (Voltage-Dependent Anion Channel) ⇒ permeable to ions/solutes up to ≈ $5\,\text{kDa}$
• Intermembrane space (IMS)
  • Reservoir for pro-apoptotic proteins; reflects cytosolic ionic composition
• Inner mitochondrial membrane (IMM)
  • Forms cristae; houses respiratory complexes I–V, transporters, ion channels
  • Impermeable; high protein/lipid ratio; cardiolipin-rich; key to membrane potential
• Matrix
  • Citric acid cycle enzymes, mtDNA, mitoribosomes, transcription/translation machinery

Mitochondrial Proteome & Import

• Genetic duality:
  • $\sim20{-}30$ polypeptides encoded by 16.6 kb human mtDNA (mostly core subunits of complexes I, III, IV & V, plus rRNAs & tRNAs)
  • $\sim2000$ proteins constitute the complete mitochondrial proteome ⇒ majority nuclear-encoded
• Import pathways
  • TOM complex (Translocase of Outer Membrane) = general entry gate
  • TIM23/TIM22, OXA, SAM, MIA, etc. route proteins to matrix, IMM, OMM, IMS respectively
  • Targeting sequences: N-terminal amphipathic $\alpha$-helices for matrix; multiple internal signals for carrier proteins; BUT many mito-proteins lack obvious motifs ⇒ reliance on chaperones/receptor recognition
• Clinical tie-in: mutations in import machinery (e.g., TIMM8A) cause neurodegenerative diseases (e.g., Mohr-Tranebjærg syndrome)

Morphology, Dynamics & Cellular Distribution

• Shape range: punctate spheres ⇄ elongated interconnected tubules (dynamic fusion/fission balance)
• Distribution tailored to energy demand:
  • Cardiomyocytes: tightly packed between myofibrils, near $\text{Ca}^{2+}$ release sites
  • Neurons: cluster at synapses & nodes for ATP/$\text{Ca}^{2+}$ buffering
  • Sperm tail: helically wrapped for flagellar motility
• Network plasticity regulates stress responses, metabolism, apoptosis; governed by mitofusins 1/2, OPA1 (fusion) and Drp1, Fis1 (fission)

Cellular Energy Metabolism (Fuel Pathways)

• Key incoming substrates:
  • Glucose ⇒ glycolysis ⇒ pyruvate
  • Fatty acids ⇒ CPT-1 (Carnitine Palmitoyltransferase-1) shuttle ⇒ $\beta$-oxidation
• Convergence:
  • Pyruvate Dehydrogenase (PDH) & $\beta$-oxidation generate acetyl-CoA + NADH
  • Acetyl-CoA enters tricarboxylic acid (citric acid) cycle ⇒ additional NADH/FADH$_2$
• Final common carriers:
  • NADH & FADH$_2$ deliver electrons to respiratory chain

Oxidative Phosphorylation & Chemiosmotic Coupling

• Steps (canonical sequence):
  1. NADH oxidised at Complex I; FADH$_2$ at Complex II
  2. Electrons traverse Complexes I → III → IV, ending with 1\/2\,\text{O}2 + 2e^- + 2H^+ \rightarrow \text{H}2\text{O}
  3. Proton pumping at I, III, IV establishes electrochemical gradient (proton-motive force, $\Delta p$):
     $\Delta p = \Delta \Psi + \frac{2.303RT}{F}\,\Delta pH$
  • Typical membrane potential $\Delta \Psi \approx -180\,\text{mV}$
  1. ATP Synthase (Complex V) harnesses gradient: $\text{ADP} + P<em>i + nH^+</em>{IMS} \rightarrow \text{ATP} + nH^+_{matrix}$
  2. Reaction is reversible; during anoxia or high cytosolic ATP, ATP hydrolysis can run “backwards” to maintain $\Delta \Psi$
• Peter Mitchell’s 1961 Nature paper formalised the chemiosmotic theory
• Membrane potential utilized for:
  • Nutrient/ion transport (e.g., Asp/Glu carrier)
  • Heat production via uncoupling proteins (UCP1-3)
  • Controlled proton leak regulating ROS levels & basal metabolic rate

Mitochondrial Ion Channels & Transporters

• Key entities (location ≈ IMM unless noted):
  • $\text{Ca}^{2+}$ Uniporter (MCU): matches ATP output to workload by sensing cytosolic $\text{Ca}^{2+}$ released near ER/sarcolemma
  • $\text{K}<em>{ATP}$, BK$</em>\text{Ca}$ channels: influence matrix volume, redox state, cardio-protection
  • Permeability Transition Pore (PTP): high-conductance, opens during $\text{Ca}^{2+}$ overload/oxidative stress ⇒ depolarisation & necrosis
  • NCX (Na$^+$/Ca$^{2+}$ exchanger): removes matrix $\text{Ca}^{2+}$ in excitable tissues
  • UCPs: facilitate regulated leak for thermogenesis (e.g., brown adipose) & ROS control
  • VDAC (OMM): gate for metabolites (ATP/ADP, pyruvate, small ions)
  • MAC (Mitochondrial Apoptosis Channel, OMM): oligomerised Bax/Bak forms conduit for IMS proteins during apoptosis

Calcium Handling & PTP Dynamics

• Under physiological stimulation, micro-domains of high $[\text{Ca}^{2+}]$ near ER/SR trigger MCU uptake ⇒ boosts dehydrogenase activity & ATP synthesis
• Pathological overload ⇒ PTP opens → loss of $\Delta \Psi$, matrix swelling, rupture of OMM, necrosis/apoptosis
• PTP modulation: inhibited by cyclosporin A (via cyclophilin D), favoured by inorganic phosphate, ROS, high $[\text{Ca}^{2+}]$

Mitochondria in Apoptosis

• IMS stores cytochrome c, AIF, SMAC/DIABLO
• Apoptotic stimuli activate Bax/Bak insertion ⇒ MAC formation ⇒ release of IMS factors
  • Cytochrome c binds Apaf-1 + dATP ⇒ apoptosome ⇒ caspase-9 activation
  • SMAC/DIABLO antagonises IAPs (inhibitor of apoptosis proteins)
  • AIF triggers caspase-independent chromatin condensation/fragmentation
• Cross-talk with PTP (necrosis) & ROS production intensifies cell death cascades

Experimental Assessment of Mitochondrial Function

1. Fluorescent Imaging
   • NADH autofluorescence (\u2191 reduced state ⇒ \u2191 signal)
   • Membrane potential dyes: TMRM/TMRE, Rh123; quenched/redistributed with $\Delta \Psi$ changes
   • Genetically encoded reporters: mito-cameleon (\text{Ca}^{2+}); mito-ATeam (ATP/ADP); roGFP (redox)
   • Example: Quercetin addition to neuronal cultures raises TMRM signal ⇒ hyperpolarisation
2. Respirometry (Seahorse XF 96, Clark-type electrodes)
   • Measures oxygen consumption rate (OCR) & extracellular acidification rate (ECAR)
   • Inhibitor protocol (typical concentrations):
     • Oligomycin ($2\,\mu\text{M}$) ⇒ blocks ATP synthase ⇒ isolates proton leak
     • FCCP ($0.5{-}2\,\mu\text{M}$) ⇒ uncoupler ⇒ reveals maximal respiratory capacity
     • Rotenone/Antimycin A ⇒ inhibit Complex I/III ⇒ non-mitochondrial OCR baseline
   • Calculated parameters: basal respiration, proton leak, ATP-linked respiration, maximal respiration, spare respiratory capacity (SRC)
   • Flavonoids (e.g., epicatechin + quercetin) ↑ SRC, confer neuro-protection against ischaemic stroke in vivo
3. Patch-clamp of mitoplasts (IMM only) to record ion channels (e.g., PTP flickers, $\text{K}_{ATP}$ conductance)

Pathological Implications of Mitochondrial Dysfunction

• Metabolic disorders: Type II diabetes (impaired fatty-acid oxidation, ROS)
• Cardiomyopathies: ischemia/reperfusion injury, chronic heart failure (PTP opening, $\Delta \Psi$ collapse)
• Neurodegeneration: Parkinson’s (complex I loss), Alzheimer’s (A$\beta$-induced ROS), Huntington’s (mutant huntingtin-PTP sensitisation)
• Cancer: Warburg effect & oncometabolite accumulation; altered fusion/fission balance promotes metastasis
• Ageing: accumulation of mtDNA mutations, decreased SRC, ↑ ROS damage (Mitochondrial Free Radical Theory of Ageing)

Peroxisomes: Comparative Organelle Biology

• Single-membrane oxidative organelles; collaborate with mitochondria
• Functions:
  • $\beta$-oxidation of very-long-chain fatty acids (VLCFA) => FADH$_2$ & NADH hand-off to mitochondria for ATP production
  • Detoxification: catalase decomposes $2\text{H}<em>2\text{O}</em>2 \rightarrow 2\text{H}<em>2\text{O} + \text{O}</em>2$
  • Biosynthesis: plasmalogens (myelin lipids), bile acids
• Genetic defects (e.g., Zellweger spectrum) highlight peroxisome-mitochondria metabolic crosstalk

Key Numerical / Statistical References

• >95\% of cellular ATP derived from oxidative phosphorylation
• $98\%$ of total body $\text{O}_2$ consumption occurs via mitochondria
• Membrane potential magnitude: $\Delta \Psi \approx -180\,\text{mV}$ (matrix negative)
• Human mtDNA encodes $\sim 20{-}30$ proteins; mitochondrial proteome ≈ $2000$ proteins

Ethical, Philosophical & Clinical Considerations

• Mitochondrial replacement therapy ("three-parent embryos") to prevent mtDNA disease raises bioethical debates on germ-line modification
• Maternal inheritance leads to gender-biased transmission of disorders; genetic counselling essential
• ROS: dual role as signalling messenger versus mutagenic agent necessitates balance; antioxidant supplementation trials yield mixed outcomes
• Targeting mitochondrial metabolism (e.g., metformin, dichloroacetate, IACS-010759) is an emerging anti-cancer strategy; requires vigilance for systemic toxicity

Integrated Summary

• Structural compartments (OMM, IMM, IMS, matrix, cristae) underpin highly orchestrated energy conversion and signalling functions.
• Dual genomic control dictates complex protein import logistics; any disruption can propagate multi-system diseases.
• Chemiosmotic coupling (proton-motive force) links electron transport to ATP synthesis, ion homeostasis and heat; reversible under stress.
• Ion channels (MCU, PTP, UCP, etc.) fine-tune metabolism and decide cell fate (survival, apoptosis, necrosis).
• State-of-the-art imaging and respirometry reveal dynamic bioenergetic parameters, informing therapeutic interventions (e.g., flavonoids, PTP inhibitors).
• Peroxisomes expand oxidative capacity and lipid metabolism, illustrating inter-organelle metabolic networking.