2010 Lecture 7

From CellBiology

Mitochondria

Mitochondria fl.jpg 08lungtem.jpg

Welcome to the Matrix!

Introduction

This lecture introduces the cytoplasmic organelles that produce the energy required for cellular processes to occur. In recent years mitochondria have also been shown to have important roles in other cellular functions, in particular, cell death by apoptosis. This second role will be covered in detail in later lectures in this current series.

Mitochondrion is singular, Mitochondria is plural

Labeled Mitochondria Movie (1 frame/2 seconds)

Mitochondrial motility in cultured hippocampal neurons

Lecture Audio

The University has a system for automated recording of lectures called Lectopia. Lectopia requires login using your student number and unipass. I will be adding the link to each iLecture Audio following the Lecture. Due to the automated recording method, most lectures begin 4-5 minutes into MP3 recordings and occasionally stop before the lecture does. 2010 All Audio Files


Archive

(MH - note that content will not match exactly current lecture structure but has been selected as having similar content)

Objectives

  • Broad understanding of processes requiring energy within the cell
  • Brief understanding structure and function of plant chloroplast
  • Understand the structure and function of plant and animal mitochondria
  • Brief understanding of mitochondria evolution
  • Brief understanding of mitochondrial abnormalities

Double Membrane Organelles

Mitochondrion structure cartoon
  • Nucleus - all eukaryotes
  • Chloroplasts - plants
  • Mitochondria - plants and animals

History Mitochodria

1857 Kölliker discovers mitochondria in muscle

1929 Karl Lohmann discovered ATP

1940s and 1950s ATP is formed in cell respiration in mitochondria and photosynthesis in chloroplasts of plants

1960 Efraim Racker and co-workers isolated, from mitochondria, the enzyme "F o F 1 ATPase" now call ATP synthase

1963 There’s DNA in those organelles DNA is directly visualized in first chloroplasts and then mitochondria, from the JCB Archive.


1992 Wallace identified degenerative disease caused by mtDNA mutations

1997 Nobel Prize in Chemistry - The three laureates have performed pioneering work on enzymes that participate in the conversion of the "high-energy" compound adenosine triphosphate (ATP).

  • Paul D. Boyer and John E. Walker "for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)"
  • Jens C. Skou "for the first discovery of an ion-transporting enzyme, Na+, K+ -ATPase

Evolution Mitochondria

  • primitive Eubacterium
  • symbiotic relationship with eukaryotic cell
    • circular DNA
    • see antibiotic-induced deafness due to similarity of mitochondrial and bacterial ribosomes
  • genes transferred to nucleus
  • mitochondrial genome bp
    • 366,924 Arabidopsis
    • 16,569 Human
    • 5966 Plasmodium

Chloroplasts

  • Double membrane cytoplasmic organelle
  • present in photosynthetic Eubacteria, algae and plants
    • thought to originate as an endosymbiotic cyanobacteria (blue-green algae)

Function

  • photosynthesis
  • chlorophyll captures light energy
  • chloroplasts interact with peroxisomes

Structure

  • flat discs usually 2 to 10 micrometer in diameter and 1 micrometer thick. In
  • plants 5 μm in diameter and 2.3 μm thick
  • inner and an outer phospholipid membrane
  • intermembranous space
  • stroma
    • stacks of thylakoids (site of photosynthesis)
    • contains copies of small circular DNA
    • ribosomes
    • proteins transported to the chloroplast

(MH - will not cover this cell organelle in any depth in current course)

Mitochondria

  • Greek, mito = thread; chondrion = granule
  • Located throughout cytoplasmic compartment
    • has itself several membrane enclosed compartments
    • each compartment has different function
  • Ancient aerobic organisms in symbiosis (endosymbiosis)
  • present in all cells

Mitochondria Function

  • Energy production
    • Respiratory chain
  • Signaling
  • Apoptosis role
    • Programmed cell death

Mitochondria Structure

Mitochondrial membraneous compartments
Mitochondrion rat liver
  • Double membrane
  • outer membrane
  • intermembrane space
  • inner membrane
  • crista (plural, cristae)
    • originally considered specialized folds of the inner membrane
    • variable invaginations with narrow tubular connections to each other and by crista junctions to the peripheral region of inner membrane
  • matrix

Mitochondria Shape

  • Come in different shapes & sizes
  • Can rapidly change shape (minutes)

EM: Mitochondria EM: Mitochondria EM: Mitochondria Mitochondrial Morphology

Mitochodrial Movie


Mitochondria Location

  • cells with high energy requirements: Muscle, sperm tail, flagella
  • generally located where energy consumption is highest in the cell
  • Mitochondria (fibroblasts)
  • Mitochondria (sperm)
    • Packed around initial segment
    • Energy for sperm motility, microtubules (9+2)

Mitochondria Components

Mitochondrial membraneous compartments

Outer Membrane

  • porin - membrane channel, allows ions and metabolites into the mitochondria (<5000 daltons)

Intermembrane Space

  • similar to the cytosol with respect to the small molecules it contains
  • also enzymes that use ATP

Inner Membrane

Cardiolipin.jpg

  • cardiolipin - phospholipid, makes membrane impermeable to ions
  • transport proteins - permeable to molecules required in the matrix

Cristae

  • increase inner membrane surface area
    • tubular, vesicular or flat cristae
  • Adenosine triphosphate (ATP) synthase
  • respiratory electron transfer chain proteins
  • transport proteins

Matrix

  • metabolic enzymes of citric acid cycle (=Krebs) (100s of enzymes) (MH- do not need to know biochemical details of this cycle)
  • genetic material DNA, tRNA, ribosomes

Mitochondria DNA

Eukaryotic mitochondrial genomes
  • double stranded circular DNA (mitoDNA. mtDNA)
  • 1981 complete human sequence (16,569 nucleotides)
  • 37 genes
    • encodes 13 polypeptides involved in oxidative phosphorylation
    • remaining genes transfer RNA (tRNA) and ribosomal RNA (rRNA)
  • multiple copies within the matrix
  • maternally inherited
  • remainder encoded by nuclear DNA
  • proteins made in cytosol and imported into mitochondria



Links: Home Reference - Mitochondrial DNA

Mitochondria Protein Synthesis

  • yeast - Petite mutants
    • all mitochondrion-encoded gene products missing
    • forms small anaerobic colonies
    • organelle is constructed entirely from nucleus-encoded proteins

Proteintransport.gif

Many mitochondrial proteins are encoded by nuclear DNA

  • synthesis begins in the cell cytoplasm
  • imported into the mitochondria
    • targeting similar to signal sequence for RER
  • once in matrix signal sequence is cleaved (by Hsp70)
    • protein then folds (by Hsp60)
  • proteins for mitochondrial membrane or intermembranous space
  • additional signal following matrix localization

Mitochondrial targeting signal (MTS) - alternating amino acid pattern (amphipathic helix) with a few hydrophobic amino acids and a few plus-charged amino acids at the N terminus.


Links: Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA | Home Reference - Mitochondrial DNA

Mitochondrial Division and Fusion

Mitochondria Fission

Mitochondrial fission
Blocking mitochondrial fission causes autophagy
  • Mitochondrial Division
  • Divide independently of the whole cell cycle
  • Generated by existing mitochondria
  • inward furrowing like bacterial division
    • mitochondria lack FtsZ ring (seen in bacteria)
    • rely on dynamin on the cytosolic face for fission

Mitochondrial inheritance

Mitochondrial Fusion

Mitochondrial fusion
  • when two separate mitochondria join as one
  • fission and fusion considered to be balanced
  • disruption causes normal tubular network of mitochondria to fragment into short rods or spheres
  • Requires large GTPases
  • Mitofusins 1 and 2 (Mfn1, Mfn2) and OPA1
    • Mfn1 and Mfn2 for outer membrane fusion
    • OPA1 for inner membrane fusion

Links: Preventing Mitochondrial Fission Impairs Mitochondrial Function and Leads to Loss of Mitochondrial DNA | Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases]

Energy Production

Respiration

  • Raw Materials
    • Oxygen
    • Pyruvate & Fatty Acids
  • Products
    • Carbon Dioxide
    • Adenosine Triphosphate (ATP)

Apoptosis

Mitochondrial morphologies during apoptosis

Mitochondria in addition to energy production, have a second major function related to programmed cell death by apoptosis.

  • cytochrome C release activates caspases
  • other changes include
    • electron transport, loss of mitochondrial transmembrane potential
    • altered cellular oxidation-reduction
    • Bcl-2 family proteins (pro- and antiapoptotic)
  • Vesicular Mitochondria
    • begin to appear during the release of cytochrome C which initiates mitochondrial mediated apoptosis
    • transformation from normal morphology
    • with an inner boundary membrane connected to lamellar cristae via crista junctions
    • multiple vesicular matrix compartments
    • facilitates membrane fission or fragmentation as the matrix is fragmented at this stage
    • fragmentation of the mitochondrion requires only outer membrane fission

(MH- this topic will be covered again in the Cell Death Lecture)

Links: Lecture - Cell Death 1 | Lecture - Cell Death 2 | Movie - Vesicular Mitochondria | Role of mitochondria in apoptosis | Ibioseminar - Apoptosis Part 2: Factors Involved in the Intrinsic Pathway of Apoptosis (27:39 minutes)

Methods

Dynabead Isolation

  • Mitochondria isolation using magnetic beads conjugated to antibodies to mitochondrial surface markers.

MitoTracker Probes

  • MitoTracker probes are cell-permeant mitochondrion-selective dyes.

Links: Bovine pulmonary artery endothelial cells (BPAEC) - LysoTracker Red and MitoTracker Green | Bovine pulmonary artery endothelial cell - probes to mitochondria (red), peroxisomes (green), and the nucleus (blue) | Molecular Probes - Probes for Mitochondria | Spectral characteristics of the MitoTracker probes |

Apoptosis Assays

  • Cytochrome c Releasing Apoptosis Assays - detection of cytochrome c translocation from mitochondria into cytosol during apoptosis. Commercial Kit PDF
    • Cytochrome c normally located in the space between the inner and outer mitochondrial membranes
  • Mitochondrial transmembrane potential changes (JC-1, cationic dye) Commercial Kit 1 | Commercial Kit 2
    • Healthy cells - accumulates and aggregates in the mitocondria, bright red fluorescence.
    • Apoptotic cells - altered mitochondrial transmembrane potential causes dye to remain in the cytoplasm in monomer form, green fluorescence.

MitoChip

  • Microarray of both strands of the entire human mitochondrial coding sequence (15,451 bp).
  • oligonucleotide probes synthesized using standard photolithography and solid-phase synthesis, and is able to sequence >29 kb of double-stranded DNA in a single assay.

The Human MitoChip: a high-throughput sequencing microarray for mitochondrial mutation detection. Maitra A, Cohen Y, Gillespie SE, Mambo E, Fukushima N, Hoque MO, Shah N, Goggins M, Califano J, Sidransky D, Chakravarti A. Genome Res. 2004 May;14(5):812-9. PMID: 15123581 | http://genome.cshlp.org/content/14/5/812.abstract Genome Res. 2004]

Abnormalities

Human polymorphic numts PMID: 15361937

Nuclear transfer of mitochondrial DNA

  • mitochondria to the nucleus generates nuclear copies of mitochondrial DNA (numts)
  • Integration can appear as neutral polymorphism or associated with human diseases (insertion of mtDNA into genes,5 known cases), the mitochondrial genome remains intact in the individuals.
  1. insertion at the breakpoint junction of a reciprocal translocation between chromosome 9 and 11.
  2. insertion into splice site mutation in the human gene for plasma factor VII (causes severe plasma factor VII deficiency, bleeding disease)
  3. insertion into exon 14 of the GLI3 gene causes a premature stop codon (associated with Chernobyl)
  4. insertion into exon 2 of MCOLN1, eliminated proper splicing of the gene (mucolipidosis IV).
  5. insertion in exon 9 of the USH1C gene (Usher syndrome type IC)

Above examples from: PMID: 15361937

Mitochondrial Myopathies

Friedreich's ataxia (FRDA)

  • mutation in gene for frataxin (a mitochondrial iron (Fe) chaperone)
  • mechanism may be a mitochondrial iron (Fe) loading and reactive oxygen species
  • animal models show H2O2 is an important pathogenic substrate underlying the phenotypes arising from frataxin deficiency
  • Genes and Diseases - Friedreich's ataxia

Charcot–Marie–Tooth type 2A

  • inherited neuropathy
  • caused by mutations in mitofusin 2
    • proper regulation of mitochondrial dynamics required in neurons

Leber Hereditary Optic Neuropathy

Leber hereditary optic neuropathy
  • maternally inherited cause of blindness Genes and Diseases - LHOM
  • mutation of mitochondrial DNA (mtDNA)
    • three common mtDNA mutations: G11778A, T14484C, G3460A

Aminoglycoside-induced deafness

Other

Adenoid Cystic Carcinoma (ACC)

Mitochondrial mutations in adenoid cystic carcinoma of the salivary glands. Mithani SK, Shao C, Tan M, Smith IM, Califano JA, El-Naggar AK, Ha PK. PLoS One. 2009 Dec 30;4(12):e8493. PMID: 20041111

"Mitochondrial mutation is frequent in salivary ACCs. The high incidence of amino acid changing mutations implicates alterations in aerobic respiration in ACC carcinogenesis. D-loop mutations are of unclear significance, but may be associated with alterations in transcription or replication."

Cyclic Vomiting Syndrome?

Links: Genes and Diseases -Mitochondria and Disease | GeneReviews - Mitochondrial Disorders Overview | Genes and Diseases - Friedreich's ataxia | Muscular Dystrophy Association - Mitochondrial Myopathies | The Cleveland Clinic - Mitochondrial Myopathies | Genes and Diseases | OMIM - Online Mendelian Inheritance in Man

References

Textbooks

Essential Cell Biology

  • Chapter 13 Energy Generation in Mitochondria and Chloroplasts
  • Chapter 14 Intracellular Compartments and Transport

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

  • The Cell - A Molecular Approach - III. Cell Structure and Function 10. Bioenergetics and Metabolism - Mitochondria, Chloroplasts, and Peroxisomes
  • Mitochondria

Search Online Textbooks

Books

PubMed

  • PubMed is a service of the U.S. National Library of Medicine that includes over 18 million citations from MEDLINE and other life science journals for biomedical articles back to 1948. PubMed includes links to full text articles and other related resources. PubMed
  • PubMed Central (PMC) is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) in the National Library of Medicine (NLM) allowing all users free access to the material in PubMed Central. PMC
  • Online Mendelian Inheritance in Man (OMIM) is a comprehensive compendium of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 12,000 genes. OMIM
  • Entrez is the integrated, text-based search and retrieval system used at NCBI for the major databases, including PubMed, Nucleotide and Protein Sequences, Protein Structures, Complete Genomes, Taxonomy, and others Entrez

Search Pubmed

Reviews

  • Mitochondrial fragmentation in neurodegeneration. Knott AB, Perkins G, Schwarzenbacher R, Bossy-Wetzel E. Nat Rev Neurosci. 2008 Jul;9(7):505-18. Review. PMID: 18568013
  • Emerging functions of mammalian mitochondrial fusion and fission. Chen H, Chan DC. Hum Mol Genet. 2005 Oct 15;14 Spec No. 2:R283-9. Review. PMID: 16244327

Articles

  • Bcl-x L increases mitochondrial fission, fusion, and biomass in neurons. Berman SB, Chen YB, Qi B, McCaffery JM, Rucker EB 3rd, Goebbels S, Nave KA, Arnold BA, Jonas EA, Pineda FJ, Hardwick JM. J Cell Biol. 2009 Mar 9;184(5):707-19. Epub 2009 Mar 2. PMID: 19255249


Mitochondrion Images

For a full selection of see Cell Biology Images - Mitochondria Images


Movies

  • Apoptotic cells show a transient loss in mitochondrial membrane potential Waterhouse et al. suggest that the transient loss of mitochondrial membrane potential that can be seen in individual apoptotic cells may be masked by cell-to-cell asynchrony when looking at a population of cells.
  • Mitochondria slow down for calcium Mitochondria move along microtubules, but Yi et al. find that they slow down when calcium levels rise, probably so that they can help buffer the ion back to normal levels.


Honours Projects - Dr Mark Hill

Project 1: Development of stable neural lines expressing GFP-tagged mitochondria.

Project 2: Analysis of mitochondrial motility under conditions of cellular stress.


2010 Course Content

Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cell Mitochondria | Cell Junctions | Cytoskeleton Introduction | Cytoskeleton 1 Intermediate Filaments | Cytoskeleton 2 Microtubules | Cytoskeleton 3 Microfilaments | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | Revision

Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Immunochemistry | Cell Knockout Methods | Cytoskeleton Exercise | Confocal Microscopy | Microarray Visit | Tissue Culture 1 | Tissue Culture 2 | Stem Cells Lab | Stem Cells Analysis

Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G