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Lecture 5 Notes – Mitochondria & Peroxisomes

Page 1

Lecture & Contact
• Lecture 5: Mitochondria and Peroxisomes
• Instructor: Yalda Moayedi — ym2994@nyu.edu

Contextual framing
This lecture integrates cell biology, bio-energetics, signalling and pathology to explain how mitochondria and peroxisomes influence overall cellular physiology and disease.

Page 2 — Objectives

  1. Mitochondrial morphology & evolutionary origin

  2. Central role in cell-energy metabolism

  3. Oxidation–phosphorylation coupling (chemi-osmotic model)

  4. Experimental imaging & respirometry approaches

  5. Mitochondria in pathology (metabolic, neuro-degenerative, apoptotic)

  6. Peroxisomes — catabolism & lipid metabolism
    Take-away: By the end of the lecture you should be able to mechanistically link structure ↔ function ↔ disease.

Page 3 — What are Mitochondria?

“The human cell is a symbiosis of two life forms, the nucleus-cytosol and the mitochondrion” — Wallace, 2007.

Definitions & Core functions
• NIH (2009): “Mitochondria are structures within cells that convert the energy from food into a form that cells can use.”
• ATP production via oxidative phosphorylation (OXPHOS)
• Ca²⁺ sequestration & release — fast intracellular signalling, regulation of enzymes (e.g., PDH, TCA dehydrogenases).
• Reservoir of pro-apoptotic factors (Cytochrome c, AIF, SMAC/DIABLO).
• Major source of reactive oxygen species (ROS); ROS act both as signalling molecules and deleterious agents when uncontrolled.
Concept link: Mitochondria are not just “powerhouses”; they are hubs that couple metabolism, signalling and death pathways.

Page 4 — Evolutionary Origin

• Endosymbiotic theory: anaerobic pre-eukaryote engulfed an aerobic proteobacterium.
• Result: double-membrane organelle with remnants of its own genome.
• Stepwise events:

  1. Internal membrane development in host.

  2. Engulfment of aerobic bacterium.

  3. Gene transfer to nucleus; loss of bacterial cell wall; emergence of modern mitochondrion.
    Illustrative implication: Dual genetic control (nuclear + mtDNA) accounts for distinctive inheritance patterns and disease presentations.

Page 5 — Ultrastructure

• Outer membrane (OMM) — contains porins (VDAC) → permeable to <~5 kDa solutes.
• Inner membrane (IMM) — highly selective; houses ETC complexes and ATP synthase.
• Cristae — IMM folds ↑ surface area → ↑ ETC capacity.
• Intermembrane space (IMS) — accumulation site for pro-apoptotic proteins.
• Matrix — TCA cycle, β-oxidation, mtDNA, ribosomes.
Structure–function motif: Spatial segregation underlies proton gradient formation and metabolite channeling.

Page 6 — Genetics & Protein Import

• Maternal inheritance of mtDNA (≈16.5 kb, circular).
• Encodes ~20–30 polypeptides (core ETC subunits), 2 rRNAs, 22 tRNAs.
• Full mitochondrial proteome ≈ 2000 proteins — majority (95 %) nuclear-encoded.
• Cytosolic translation → post-translational import across OMM & IMM.
• Mitochondria possess complete but minimal translation machinery (55S ribosome, fMet-tRNA, etc.) for their encoded subset.
Clinical note: Mutations in either nuclear or mtDNA compromise OXPHOS → heterogeneous mitochondrial diseases.

Page 7 — Import Machinery

• TOM (Translocase of Outer Membrane) — general entry gate.
• TIM23/TIM22 (Inner-membrane complexes) — sort matrix vs. membrane proteins.
• OXA — inserts proteins translated inside matrix back into IMM.
• Targeting sequences:
– N-terminal amphipathic helices for matrix import (cleavable).
– Internal signals for IMM, IMS or OMM residency.
• Paradox: Many proteins lack obvious signals → import governed by chaperone recognition & structural motifs.

Page 8 — Disease Spectrum

Mitochondrial dysfunction contributes to:
• Metabolic — Type II diabetes, cardiomyopathy.
• Neurodegeneration — Parkinson’s, Alzheimer’s, Huntington’s.
• Cancer (metabolic reprogramming, Warburg effect).
• Aging (ROS, mtDNA mutations).
Mechanistic threads: Energetic failure, ROS imbalance, mitophagy defects, release of death factors.

Page 9 — ATP Production Dominance & Distribution

• >95\% of cellular ATP derives from OXPHOS.
• Mitochondrial distribution is cell-type specific:
– Cardiomyocytes: packed between myofibrils for immediate ATP to contractile apparatus.
– Neurons: enriched at synapses & nodes for local signalling.
– HeLa cells, astrocytes, lymphocytes show varied morphologies reflecting metabolic demand.
Physiological insight: Positioning ensures minimal diffusion distance for ATP, Ca²⁺ and ROS signals.

Page 10 — Networking & Dynamics

• Mitochondria form elongated tubular networks via fusion (MFN1/2, OPA1) and fission (DRP1).
• Continuous reticulum allows content mixing, complementation of mtDNA mutations and rapid redistribution.
Clinical angle: Excessive fission → fragmented mitochondria in neurodegeneration; fusion defects → optic atrophy.

Page 11 — Density Governed by Demand

Examples:
• Cardiac muscle — dense, orderly rows parallel to myofibrils.
• Sperm tail — helical sheath around flagellum supplies propulsion ATP.
Organizational logic: spatial optimisation for highest local ATP turnover.

Page 12 — Cellular Energy Metabolism Overview

Key molecules/pathways:
• Glucose → Glycolysis → Pyruvate.
• Pyruvate + PDH → Acetyl-CoA + NADH.
• Fatty acids transported by FAT/CD36; imported into mitochondria via CPT-1 & carnitine shuttle → β-oxidation → Acetyl-CoA + NADH + FADH₂.
• Acetyl-CoA enters TCA cycle → more NADH/FADH₂.
Integration: Carbohydrate & lipid catabolism converge on reducing equivalents (NADH, FADH₂) that power ETC.

Page 13 — Electron Transport Chain (ETC) Steps

  1. NADH from TCA donates 2 e⁻ to Complex I (NADH dehydrogenase).

  2. FADH₂ (succinate dehydrogenase) feeds e⁻ into Complex II.

  3. Electrons transit via CoQ, Complex III, cytochrome c, Complex IV (cytochrome c oxidase) where O2 is terminal acceptor → H2O.

  4. Coupled proton pumping (++ out of matrix) across Complex I, III, IV creates membrane potential (Δψ ≈ −180 mV).

  5. ATP synthase (Complex V) uses proton re-entry to phosphorylate ADP → ATP.
    Equation: \text{ADP} + Pi + 4H^+{\text{IMS}} \rightarrow \text{ATP} + H2O + 4H^+{\text{matrix}}

Page 14 — ATP Synthase Reversibility

• Under ischemia or ETC inhibition, F₁F₀-ATPase can hydrolyze ATP to pump protons out, sustaining Δψ.
• Protective in short term (keeps Ca²⁺ uniporter silent), but catastrophic if cytosolic ATP drops.

Page 15 — Proton-Motive Force & Chemi-Osmosis

Total driving force: \Delta p = \Delta \Psi - (2.303RT/F)\;\Delta pH
• Δψ (electric) dominates; ΔpH adds minor component.
• Conceptualized by Peter Mitchell (1961 Nature) → Nobel 1978.
Physiological roles: ATP synthesis, metabolite transport (e.g., ADP/ATP translocase), protein import, heat production (via UCP).

Page 16 — Oxidative Phosphorylation Coupling

• Oxidation: e⁻ flow from NADH → O₂ (exergonic).
• Phosphorylation: Endergonic ADP + Pi → ATP, driven by Δψ.
Additional outcomes of Δψ:

  1. Ion transport (Ca²⁺ uptake, K⁺ flux).

  2. Thermogenesis via uncoupling proteins (UCP1,2,3).

  3. Proton leak — basal uncoupling moderates ROS but wastes energy.
    • Dysregulated leak can be protective (mitigate ROS bursts) or damaging (energy deficit).

Page 17 — Mitochondrial Ion Channels & Carriers

Diagram highlights:
• VDAC (OMM) — metabolite gateway.
• MAC — apoptosis channel (BAX/BAK dependent).
• Ca²⁺ Uniporter — low μM affinity; activity ∝ Δψ.
• KATP — proposed volume regulator.
• BKCa — Ca²⁺-activated K⁺ channel.
• Permeability Transition Pore (PTP) — large, non-selective; opens during stress → depolarization & death.
• NCX — Na⁺/Ca²⁺ exchanger balances matrix Ca²⁺.
• UCP — controlled proton leak for heat (brown fat).
Clinical correlation: Pharmacological modulators (e.g., cyclosporin A inhibits PTP) are cardioprotective.

Page 18 — Calcium Handling & Stress

• High Δψ drives Ca²⁺ uptake → matches energy supply with workload (activates TCA dehydrogenases).
• Overload → PTP opening → collapse of Δψ, swelling, release of death factors.
Equation for driving force: J{Ca} \propto \Delta \Psi \times [Ca^{2+}]{cyto}
Implication: Fine tuning is vital; disruption during ischemia/reperfusion leads to cell loss.

Page 19 — Apoptosis Control

• Cytochrome c, AIF, SMAC/DIABLO stored in IMS.
• MAC opening releases them →
– Cytochrome c + Apaf-1 + dATP → apoptosome → caspase-9 activation.
– AIF translocates to nucleus → DNA fragmentation.
• Mitochondria therefore dictate intrinsic apoptotic threshold.

Page 20 — Functional Assays Overview

Intact mitochondria measurements:

  1. NADH autofluorescence — redox state.

  2. Δψ imaging (e.g., TMRM, Rh123).

  3. ATP sensors (luciferase, FRET probes).

  4. O₂ consumption — Clark electrode, Seahorse.

  5. Patch-clamp — channel currents on mitoplasts.
    Experimental caveat: Membrane integrity is essential; disrupted prep alters Δψ and transporters.

Page 21 — Example: Flavonoid Modulation

• TMRM assay shows Quercetin acutely increases Δψ in cultured neurons.
Mechanistic possibilities:
– Stimulates ETC (e⁻ donation?).
– Slight uncoupling → ↑ respiratory flux → over-compensatory hyper-polarization.
Research relevance: Natural compounds can fine-tune mitochondrial bioenergetics; therapeutic angles in neuroprotection.

Page 22 — Respirometry Platforms

• Seahorse XF Analyzer measures OCR (O₂ consumption rate) & ECAR (extracellular acidification rate) in live cells.
• Clark-type electrode: Pt cathode, O₂ diffuses through membrane; current ∝ O₂ reduced to H_2O.
• Schematic links OCR to ETC steps (Complex I → IV) and Δψ (−180 mV).
Quantitative output enables drug titration, metabolic phenotyping.

Page 23 — OCR Tracing Protocol

Sequential additions:

  1. Oligomycin (ATP synthase inhibitor) → reveals Proton leak OCR.

  2. FCCP (uncoupler) → drives Maximal respiration (collapses Δψ).

  3. Rotenone + Antimycin A (Complex I & III block) → Non-mitochondrial respiration baseline.
    Parameters extracted:
    • Basal OCR, ATP-linked OCR, Spare respiratory capacity (max – basal), Coupling efficiency.
    Interpretation: Higher spare capacity = better ability to handle stress.

Page 24 — Flavonoids Enhance Spare Capacity

• Combined Epicatechin + Quercetin (E+Q) raise spare respiratory capacity → improved resilience of neuronal mitochondria.
• In vivo: E+Q reduce infarct size in stroke models.
Mechanism: probable mild uncoupling & antioxidant action reduce ROS-induced ETC inhibition, preserving function.

Page 25 — Pathological Context: Ischemia

From Duchen 2004:
• ATP depletion within minutes in cardiomyocytes during ischemia.
• Massive mitochondrial depolarization → no contractile ATP; rigor contraction → cell death.
Therapeutic strategies: maintain Δψ, inhibit PTP, supply alternative fuels.

Page 26 — Peroxisomes

• Single-membrane organelles distinct from mitochondria.
Functions:

  1. β-oxidation of very-long-chain fatty acids, producing FADH₂ & NADH (later fed to mitochondria).

  2. Detoxification of alcohols & ROS (catalase converts H2O2 \rightarrow 2H2O + O2).

  3. Lipid biosynthesis (plasmalogens essential for myelin).
    Division of labour: Peroxisomes initiate oxidation, mitochondria finish oxidative energy harvest.

Page 27 — Summary Points

• Compartmental anatomy: OMM, IMM, cristae, IMS, matrix.
• Majority of proteins are nuclear-encoded & imported.
• ETC uses NADH energy to erect Δψ (oxidation).
• Δψ drives ATP synthesis, ion transport, heat, and can be regenerated in reverse.
• Mitochondria are gatekeepers of apoptosis via regulated release of IMS factors.
• Functional assays (imaging, respirometry) dissect bioenergetic states.
• Pathologies stem from energy failure, Ca²⁺ overload, ROS overproduction, defective dynamics.
• Peroxisomes complement mitochondria in lipid catabolism and detoxification, producing reducing equivalents that feed cellular metabolism.