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Cell Biology III (Chapter 16: The Cytoskeleton (Microtubules (A protein…
Cell Biology III
Chapter 14: Energy Conversion: Mitochondria and Chloroplasts
Two types of electron transport systems
Mitochondrion
There is an . . .
Inner membrane
cristae accommodate electron transport proton and ATP synthesis
Cristae folds provide an increase in surface are giving the mitochondrion more locations for ATP production to occur.
In fact, without them, the mitochondrion would not be able to keep up with the cell's ATP needs.
It also allows for more compaction of the cell
The matrix hosts the citric acid cycle to produce NADH
Outermembrane
freely permeable to ions and small molecules once it is less than 5 kD
Interact with other membrane systems such as ER to induce mitochondrial fission and mediates lipid exchange between the two
Chloroplasts & Photosynthesis
Resemble mitochondria but have separate thylakoid compartment
electron transport operates in the thylakoid membrane
Stacked thylakoids are interconnected by unstacked thylakoids
The lumen is called thylakoid space
Chloroplasts capture energy from sunlight and use it to fix carbon
When Light reacts with the chlorophylls, a photon knocks an e- out of the chlorophyll in the first reaction center and this e- then moves along a transport chain to the second reaction center
During this process, protons are pumped into the thylakoids and the electrochemical potential is used to synthesize ATP in stroma
The 1st reaction center regains its electrons from water to produce oxygen. Finally, electrons are loaded together with proton to NADP- to produce reducing power NADPH in the second reaction center
The dark reaction takes place in stroma to use ATP and NADPH to convert CO2 to 3 - phosphoglycerate
1 more item...
A photosystem consists of an antenna complex and a reaction center
The reaction center contains
a pair of chlorophyll molecules
A light-harvesting antenna complex contains
many chlorophylls
and a few carotenoids and is engaged in light absorption and resonance energy transfer
Carotenoids also protect the photosystem from damage
The Thylakoid Membrane contains 2 different photosystems working in series
Plastoquinone is similar to ubiquinone, and cytochrome b6-f complex is homologous to cytochrome c reductase (mobile electron carrier)
An electron is knocked out by light photon but is replenished from the splitting of water by photosystem II
Plastocyanin takes the place of cytochrome c in mitochondria
Plastocyanin and ferredoxin are soluble electron carriers
Photosystem II leads to plastoquinone, cytochrombe b6-f complex and plastocyanin then photosystem I which goes to ferredoxin
The chloroplast ATP synthase uses the proton gradient generated by the photosynthetic light reactions to produce ATP
A steeper proton gradient across the thylakoid membrane than the mitochondria:
Protons are generated by water oxidatoin
pumped by cytochrome b6 - f complex from stroma to thylakoid space
and the consumption of protons to produce ATP allows the electrons to synthesize NADPH with ferredoxin
Carbon fixation uses ATP and NADPH to convert CO2 into sugars
the initial step in carbon fixation is catalyzed by ribulose biphosphate carboxylase (Rubisco)
ends with 2 molecules of 3 - phosphoglycerate
Physical Arrangement of the Photosystems in the Thylakoid membrane explains how the light reactions proceed
Why are thylakoids stacked?
Increases surface area
save space/ less energy
Increases stability
once there is too much light they shut off
provide protection
This stacking only occurs in PSII and LCHII due to their flat surfaces
Why do you have 2 photosystems in separate locations?
As the electrons travel through cytochrome b6f complex to Photosystem I via an electron transport chain set in the thylakoid membrane, the energy fall is harnessed to transport hydrogen through the membrane, into the thylakoid lumen and provide a potential energy difference between the lumen space and chloroplast.
This is used to generate ATP
How does this physical arrangement of the photosystems retain their electron transport integrity?
Why are ATP synthases in the peripheral grana thylakoids or stroma thylakoids?
The electron transport chain
found in the . . .
in the
thylakoid membranes of chloroplast
in photosynthetic eukaryotes
inner mitochondrial membrane
where it serves as the site of oxidative phosphorylation through the action of ATP synthase
The Proton Pumps of the Electron - Transport Chain
Transition metals and quinones accept and release electrons readily
6 cytochromes have
a Fe atom in mitochondrial cells
held by 4 nitrogen atoms of a
porphyrin ring
(heme, blood color iron in hemoglobin and green color with
Mg in chlorophyll
)
8 iron-sulfur clusters contain 2 to 4 iron atoms bound by an equal number of sulfur atoms and are linked to
cysteine side chains via covalent sulfur bridges
Quinone is hydrophobic and can
freely move in lipid bilayer
NADH transfers its electrons to oxygen through 3 large enzyme complexes embedded in the
inner membrane
3) Cytochrome c Oxidase
contains hemes and cooper atoms
cytochrome c serves as a mobile carrier
involves condensation of H+ and O2 into water
2) Cytochrome c Reductase
cytochrome c serves as a mobile carrier
1) NADH dehydrogenase
Succinate dehydrogenase
involves . . .
ubiquinone
serves as a mobile carrier
citric acid cycle
NADH -> NAD+
Has a flavin mononucleotide and 8 iron-sulfur clusters
moving protons from matrix to crista space
The Genetic systems of mitochondria and chloroplasts
Biogenesis of respiratory proteins in human mitochondria
Most protein components in the respiratory chain are encoded by nuclear genome
There are only 13 mitochondria - encoded proteins and all are involved in oxidative phosphorylation
22 mt transfer RNAs and 2 mt ribosomal RNAs are required for translation of these mRNAs on the mitochondrial ribosomes
Animal mitochondria contain the simplest genetic systems known
The organization of the human mitochondrial genome: It is less than 0.001% of the nuclear genome and contains only 2 rRNA genes, 22 tRNA genes and 13 protein-coding genes
Chloroplasts and Bacteria share many striking similarities
The organization of the liverwort chloroplast genome: the genome evolves a bit slower compared to mitochondria since it is similar in all higher plants in 3 major processes: transcription, translation, and photosynthesis. It includes 80-90 proteins and 45 RNAs with 37 or more tRNA
Organelle Genes are Maternally Inherited in Animals and Plants
In animals, a sperm cell only contains a few mitochondria. As sperms mature, the DNA is degraded in their mitochondria
sperm mitochondria is also eliminated from the fertilized egg by autophagy
In 2/3 rd of plants, the chloroplasts from the pollens fail to enter the zygote and chloroplast genes are maternally inherited
In other plants, chloroplast inheritance is bi-parental and defective chloroplasts cause variegation
Chapter 16: The Cytoskeleton
Microtubules
Microtubules are hollow tubes made of 13 protofilaments
The subunit is a tubulin heterodimer
GTP with alpha tubulin is embedded deep or tightly
Either GTP or GDP can be associated with beta - tubulin. GTP tubulin tends to polymerize
beta is at the plus end and alpha is at the minus end
the minus ends grow and shrink more rapidly (beta)
The interface between dimer is much like the one between alpha - beta interface plus lateral alpha - alpha and beta - beta contacts
A protein complex containing gamma - tubulin nucleates microtubules
2 copies of gamma tubulins with a pair of accessory proteins form the gamma - tublin small complex (gamma - TuSC)
Seven copies of the gamma- TuSC associate to form a spiral structure with the last gamma - tubulin beneath the first, resulting in 13 exposed gamma - tubulin subunits
In many cells, the gamma - TuSC spiral associates with additional proteins to form the gamma - tubulin ring complex (gamma - TuRC), which nucleates at the minus end of a microtubule
Microtubules Emanate from the Centrosome in Animal Cells
The centrosome in the microtubule organization center (MTOC) of animal cells. Located near the nucleus, centrosome is organized by a pair of cylindrical centrioles (made of modified microtubles) arranged at right angles and consists of a matrix of fibrous proteins
A centrosome with attached mircrotubules which are nucleated at their minus ends by the gamma - tubulin ring complexes and the plus end points outward
Microtubule - binding proteins modulate filament dynamics and organization
The microtubule dynamics inside a cell are governed by a variety of proteins that bind tubulin dimers or microtubules
They are called microtubule associate proteins or MAPs
3 types
gamma - TuRC
nucleates assembly and remains associated with minus end
kinesin -13
induces catastrophe and disassembly
XMAP215
stabilizes plus ends and accelerates assembly
Microtubule plus - end binding proteins modulate microtubule dynamics and attachments
A microtubule switches from a growing to a shrinking state (the frequency of catastrophes) or from a shrinking to a growing state (the frequency of rescuces)
Kinesin -13 is a member of the kinesin motor protein superfamily and binds to plus ends prying them apart
Destabilization
XMAP215 binds tubulin dimers and delivers them to the microtubule plus end
Stabilization
2 Types of Motor proteins move along microtubules
Kinesins carry organelles or vesicles on their long coiled - coil tails, and walk toward the plus ends with their 2 nucleotide binding motor heads
The lagging head leaves it tubulin binding site, passes the leading head, and rebinds to the next tubulin binding site
ATP hydrolysis in the lagging head and binding of ATP by the leading head pull the rear head forward
Cytoplasmic dynein
Dyneins have minus end directed movement and are used for organelle and vesicle trafficking and positioning the centrosome and nucleus during cell migration
Microtubules and motors move organelles and vesicles
Dynein walks toward the minus end
Dynactin mediates the attachment of dynein to a membrane - enclosed vesicle or organelle
Myosin and actin
Actin-based motor proteins are members of the myosin superfamily
it's globular head contains the walking or moving machinery, hydrolyzes ATP, and walks toward the plus end of actin filamanets
In contrast, the actin filament moves toward its minus end
myosin II has 2 heavy chains and 4 light chains
The 4 light chains bind close to the head of the heavy chain
The coiled - coil tails of the heavy chain bundle with the tails of other myosin II to form thick myosin filaments
Sliding of myosin II along actin filaments causes muscles to contract
The Z - discs are the attachment sites for the plus end of actin filaments
The M - line (midline) is the location of proteins that link adjacent myosin II filaments, each myosin filament has 300 heads
When sarcomere contracts, the myosin filaments slide past one another toward the plus end of actin filaments
Accessory proteins
Tropomodulin
- caps and stabilizes the minus end of actin filaments
Nebulin
- binds actin filaments to influence their length
Titin
: extend from Z disc and associate with myosin thick element as molecular spring and ruler
Myosin Structure:
A dimer with 2 identical motor heads that act independently
each has a catalytic core and an attached lever arm
a coiled-coil rod ties the two heads together and tethers them to the thick filament
at beginning heads contain bound ADP and phosphate and have a weak affinity for actin
once a head docks properly onto an actin subunit phosphate is released
this strengths the binding of the myosin head to actin &triggers the force generating the movement of the actin filament
ADP dissociates and ATP binds to the empty nucleotide binding site and causing detachment of myosin
Then hydrolyzed and returned to pre-stroke state
Actin filament does not slide back because of other myosin molecule attachment
Intermediate filaments and septins
Cytoskeletal filaments adapt to form dynamic or stable structures
Intermediate filaments extend across the cytoplasm to provide cells mechanical strength, twist into strong cables to form keratin filaments that span at sites of cell-cell contact called desmosomes to hold epithelial cell sheets together or cell- matrix contact called hemidesmosomes, and line the inner face of nucelar envelope (nuclear lamins form a meshwork to provide anchorage sites for chromosomes and nuclear pores)
Intermediate filament structure depends on the lateral bundling and twisting of coiled-coils
The monomer pairs with another monomer to form a dimer of wound coiled-coil
Two dimers then line up to form a tetramer
Lateral association of 8 tetramers and tetramers are packed together to form rope-like filaments
Antiparallel, no ATP binding, no polarity
Actin and actin-binding proteins
Actin subunits assemble head-to-tail & contain a +/- end
Influence filament dynamics and organization
Thymosin
: binds to subunits, prevents assembly
Formin
: nucleates assembly and remains associated with growing plus end
Arp2/3 complex
: nucleates assembly to form a web and remains associated with the minus end
Tropomodulin
: prevents assembly and disassembly at minus end
Profilin
: binds subunits, speeds elongation
Monomer availability controls actin filament assembly
actin monomers bound by
thymosin
are locked from binding to and elongating the plus end, preventing their association with neither plus nor minus end of the actin filament
Profilin
binds actin monomers to elongate the filament, blocking its association with minus end of the actin filament
Actin - Nucleating Factors Accelerate Polymerization and Generate Branched or Straight Filaments
Dimeric formins associate with the plus end, and facilitate the addition of a new actin monomer. Each monomer has a binding site for monomeric actin
Profilin and formins
Fromins
facilitate the addition of actin to the growing plus end
when profilin is bound. Formins actually have binding sites to
recruit profilin
2 actin related proteins, Arp2 and Arp3 have their plus end but NOT the minus end similar to actin, preventing them from forming filaments
How?
Arp 2 and Arp3 form an inactive complex and an activating factor then binds the complex nucleating actin filament growth at the plus end
Acts more efficiently when bound to a pre-existing actin filament
Serves as a nucleation site for new actin filaments
Function & origin of the cytoskeleton
The Cytoskeleton determines cellular organization & polarity
In intestinal epithelial cells, bundled actin filaments support microvilli at the apical surface
Below a circumferetial band of actin filaments is connected to cell-cell adherens junction (anchor cell to each other and support plasma membrane)
microtubules run vertically from top to bottom and provide the global coordination of organelles and vesicle transport
Intermediate filaments are anchored to desmosomes (cell-cell connection) and hemidesmosomes (attached to the extracellular matrix)
Cytoskeletal filaments adapt to form dynamic or stable structures
Actin filaments
determine the shape of cell surface
(strength to its thin lipid bilayer and cell projections); are necessary for
whole cell locomotion
(muscle); and
drive the pinching of one cell into two
Muscle contraction
A neuron stimulates a muscle cell and an action potential sweeps over the plasma membrane of the muscle cells, the action potential releases internal stores of calcium which trigger a contraction
Muscle cells distribute calcium ions quickly throughout cytosol
T tubules criss-cross and promote the release of calcium cells during stimulation
Chapter 19: Cell Junctions and the Extracellular Matrix
Cell-cell junctions
Cadherins mediate homophilic adhesion
The N-terminal domain of a cadherin in one cell binds to the N-terminal domain of another cadherin from another cell. Ath the dege, it is filled with Ca 2+ to limit flexing
Catenins link classical cadherins to the actin filaments
Adherens junctions respond to forces generated by the actin filaments
Mechanotransduction at cell is through proteins in the cadherin complexes that alter their shape when stretched by tension
3 adaptor proteins link cadherins to the actin filaments
Undertension from an adjacent cell, a domain in a alpha-catenin is unfolded to expose a binding site for vinculin. Vinculin is able to recruit more actin filaments
Actin filaments are pulled by non-muscle myosin II downward
Huluwa (Hwa) is essential for the organizer and body axis formation
It binds to and promotes the degradation of Axin in a way independent of Wnt ligand/ receptor binding, resulting in stabilization and nuclear translocation of beta-catenin for activating organizer-specific target gene expression
Desmosomes give Epithelia mechanical strength
The epithelial cells are indirectly connected to one another through desmosomes and to the basal lamina through hemidesmosomes
Tight junctions form a seal between cells and a fence between plasma membrane domains
Tight junctions seal the adjacent cells to prevent flow of lumen fluid into extracellular fluid and the backflow of glucose from teh basal side into the gut lumen
Help confine the various transport proteins to different regions or domains of the plasma membrane by acting as diffusion barrier
Tight junctions contain strands of transmembrane adhesion proteins
2 families of proteins with 4 transmembrane domains
Their 2 termini are both on the cytoplasmic side where they interact with large scaffold adaptor proteins that organize and link the sealing strands to actin filaments
A
Gap Junction
Connexon is made of 6 transmembrane connexin
Gap junctions only allow the passage of small molecules (<1000 Daltons) but not macromolecules
Connexon are found only in vertebrates
Cell-matrix junctions
Selection mediate transient cell-cell adhesions in the bloodstream
Selectins on endothelial cells lining blood vessels bind specific oligosaccharides (low affinity) on glycoproteins or glycolipids of WBCs
Integrins on WBCs bind to specific Ig- family proteins on the surface of endothelial cells strongly that enables white blood cells to leave the blood-stream
Membranes of the immunoglobulin superfamily mediate Ca2+ 0 independent cell-cell adhesion
Intracellular cell adhesion is expressed on endothelial cells and other cell types and binds heterophilically to an integrin on WBCs
Neural cell adhesion molecule is expressed in neurons and many other cell types and mediates homophilic binding (non-major but fine-tuning compared to cadherins)
Integrins are transmembrane heterodimers that link the extracellular matrix to the cytoskeleton
Integrin is a transmembrane protein
Its N-terminal extracellular domain binds Arp-Gly-Asp (RGD) sequences in fibronectin or other matrix proteins
Beta integrin C - terminal intracellular domain binds to adaptor proteins such as talin that interacts with actin filaments
In hemidesmosomes, integrin anchors the cell to outside basal lamina
It attaches karetin intermediate filaments inside via adaptor proteins BP230 and plectin
Integrins can switch between an active and an inactive conformation
They fold into a compact inactive structure, but are more extended when becoming active
Activation of integrins by intracellular signaling
In platelets, thrombin binds to its receptor to initiate a signaling cascade that leads to activation of Rap1
Rap1 interacts with RIAM to recruit other proteins such as talin and kindlin that interacts with beta-integrins to trigger its activation
Cell - Matrix adhesions respond to mechanical forces
Talin is a tension sensor at the cell-matrix junction
Tension across cell-matrix junctions stimulates the recruitment of vinculin and other actin-regulatory proteins, strengthening the attachment of the junction to cytokelton
Viculin binding sites are hidden and inaccesible
Tension stretches the 12 alpha - helixes and expose the vinculin binding site
Vinculins are fluorescently labeled after the talin protein was stretched and excess vinculin solution was washed away, determine if vinculin was able to bind to talin protein
The extracellular matrix of animals
2 MAJOR ways in which animal cells are bound together
In epithelial tissue, cytoskeletons link from cell to cell through adhesion junctions
Adhesion sites or junctions include all four types of junctions involving actin and intermediate filaments
Cell-matrix attaches epithelial tissue to the connective tissue
4 Types of junctions
Tight Junction
seals gap between epithelial cells
Cell-cell anchoring junctions
contains
adherens junction
connects actin filament bundle in one cell with that in the next cell
desmosome
connects intermediate filaments in one cell to those in the next cell
channel-forming junction
Gap junction
Allows the passage of small water-soluble molecules from cell to cell
cell - matrix anchoring junctions
contains
Actin-linked cell-matrix junction
anchors actin filaments in cell to extracellular matrix
hemidesmosome
anchors intermediate filaments in a cell to extracellular matrix
A general organization for the 4 cell-cell and cell-matrix connections:
Transmembrane adhesion proteins link the cytoskeleton from cell to cell or to extracellular matrix with the help or adaptor proteins