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Lecture 9

Lecture 9.docx

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Department
Biological Sciences
Course Code
BIOB10H3
Professor
Stephen Reid

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Lecture 9: Cytosolic Proteins and the Cytoskeleton Cytosol What is the cytoskeleton? •Fluid matrix surrounding organelles •Extensive protein network –Polar –Made on free ribosomes in the –Ions, sugar, ATP cytoplasm, no targeting sequence –Free ribosomes, amino acids •Three main components: –Many proteins 1. Microtubules 2. Actin Cytosolic Proteins 3. Intermediate Filaments •How are proteins targeted to cytosol? –They aren’t: targeting sequence = no Cytoskeleton -Functions targeting sequence •Scaffold –structural support and cell shape -proteins translated in cytosol and •Internal framework –organize organelles stay there by “default” within a cell •Movement –directs cellular locomotion and •Some examples: movement of materials within the cell –Clathrin and COP proteins : proteins •Machinery for cell division outside of the vesicles 1) Structure and Support –Signalling proteins: bring proteins 2) Intracellular Transport through the cell 3) Contractility and Motility –Glycolysis enzymes 4) Spatial Organization –Organelle proteins during “transit” -Peroxisomes associated with microtubules. –Chaperone proteins -Red-tubulin incorporated in micro tubles -Cytoskeleton Cytoskeleton is not static Cytoskeleton components 1) Microtubules Structure • Hollow, cylindrical • Set of globular proteins arranged in rows : profilaments • Contain 13 proto filaments – a- and b- tubulin subunits • Subunits polymerize to make microtubules • B- tubulin binds GTP to allow polymerization • Polymerize into proto filament -13 proto filaments arrange around a hollow core and forms the microtubule = entire rod like structure Interactions are mainly non-covalent 4nm= heterodimer = 2 balls . Protofilament = strings of those alpha –beta –alpha beta. 13 of those protofilaments=microtubules. •MT have polarity – A plus (+, alpha end) and minus (-, beta end) end – Structurally different – (-) end embedded in centrosome – (+) end extends towards – “Growth” from the positive end, heterodimers add on to the positive end and also fall off the positive end = “shrinkage” – MTs polymerize/depolymerize at the (+) end therefore it has direction. 1) Microtubules –Assembly •Microtubules assemble in two stages –Nucleation–small portion of the microtubule is initially formed –Followed by elongation •In vitro nucleation is a slow phase, in a cell (in vivo) nucleation occurs rapidly in special structures: Microtubule organizing centres (MTCOs) : provide an area in the cell specialized to perform nucleation •Best studied MTOC in animal cells is the centrosome - Two barrel shaped centrioles surrounded by pericentriolar material (PCM) –Pair of centrioles at right angles to each other Centrosomes Experiment 1)Cells treated with colcemid–disassembly of microtubules 2)Remove drug, fix and image cells (fluorescent tubulin) 3) Within 30 minutes labeled filaments radiating out of centrosome-microtubules don’t make contact with centrioles but terminate in PCM: pericentriolar matrix Other MTOCs •Basal body–structure where outer microtubules in a cilia and flagella are generated. •Plant cells lack MTOCs and their microtubules are organized around the surface of the nucleus. 1) Microtubules •Fraction of microtubules that remain associated with centrosome is highly variable by cell type •MTs rapidly turnover –Polymerize/depolymerize from (+) end –Results into “Dynamic instability”: idea that the proteins are constantly assembled and dissembled allows the structure to quickly respond to change. –Half life averages 10 minutes •MT stability can be increased –Binding of structural MAPs (MT-associated proteins) -prevent MTs from disassembly/ depolymerizing MAPs •Microtubule-associated proteins •Collection of proteins •Classical MAPs have one domain that attaches to the side of a microtubule and another that projects outward •Some MAPs can be seen on EM as cross-bridges connecting microtubules to each other •MAPs MT-binding activity controlled mainly by phosphorylation •Abnormally high level phosphorylation of a particular MAP (tau) is implicated in neurodegeneration (ex, Alzheimer’s, FTDP-17) -Alzheimer’s: tau is all tangled up on itself •MAPs can be used to stabilize microtubule structures •Stable MT are often used to transport organelles: transport “highways” Microtubules –Intracellular motility •Facilitate movement of vesicles and organelles through the cell •Movement away from the cell body (anterograde) and toward the cell body (retrograde) •Provide “tracks” for a variety of motor proteins: Microtubule motors. Axonal transport: Cytoskeleton help the axon project outwards structurally and has to transfer signals. Mitochondria is transported to the axon to provide the nerve with ATP. Organelles and vesicles don’t make in contact with the MT it requires motor protiens Microtubule motors •Multiprotein complexes which move vesicle/organelle “cargo” along MTs •2 domains –Microtubule binding domain –Cargo binding domain Motor moves according to polarity of MTs –Dynein–moves to the (-) end of MTs (retrograde) –Kinesin–moves to the (+) end of MTs (anterograde) They are both ATPases- use ATP to drive movement. Microtubule Motors -Dynein •Minus end-directed microtubular motor •Contains 2 heavy chains (polypeptides) –Large, globular, force generating head –Stalk with MT binding site - Site of ATP hydrolysis : use of energy for force –Stem •Contains several intermediate and light chains (polypeptides) that -bind to dynactin: protein which mediates binding to cargo - Stalk is where ATP is synthesized - Intermediate light reactions has dynactin that binds to cargo 0heavy chain is made up of a stem and a head. Microtubule Motors -Kinesin •Member of a protein super family called KLPs (kinesin-like proteins) •Plus end directed microtubular motor •2 heavy chains –Globular head, ATPase–produces force –MT binding domain •2 light chains: binds to kinesin receptor on ves
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