BMB170a_2011_LECTURE7

BMB170a_2011_LECTURE7 - – – – Actins Cytoskeleton...

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Unformatted text preview: – – – Actins Cytoskeleton Myosins Actin-binding proteins – MreB, Microtubules – Tubulins Dyneins Kinesins MAPs – FtsZ IFAPs TypeIII IF proteins Crescentin MinD Eukaryotic cytoskeleton Microtubules - green Microfilaments - red Intermediate filaments - yellow Fibroblast cell Cellular movement driven by microfilaments Amoeba pseudopod driven by actin polymerization Microfilaments Provide mechanical support for the cell – Shape – Movement – Junctions 7nm in diameter Polarized +/- ends G-actin One of the most abundant proteins in eukaryotic cells (~100 M - 5% of protein) ~43 - 47 kD Highly conserved - 5% divergence across higher eukaryotes First isolated in 1942 in Hungary by Brúnó Straub (Salt dependent polymerization) Uncomplexed actin monomer bound to ADP (1j6z) - 4th structure F-actin Nucleotide stimulates polymerization Slow hydrolysis of nucleotide Apo form depolymerizes 13 molecules per 6 lt-handed turns per repeat “Holmes model” Stable interface is a trimer Model constrained by disulfide crosslinking F-actin bound to myosin Actomyosin – Rabbit F-actin & Dictyostelium myosinS1 – Force generation Cryo-EM reconstruction In rigor ATP & ADP structures Actin network Arps have actin folds Binds nucleotide with low affinity (none in structure) p40 binds to actin (WD40 fold) p20/p34 form core Arp2/3 complex Arp3 p34 p20 p16 Arp2 (partial) p40 Robinson et al Science p21 in vitro Listeria and actin Listeria monocytogenes – causative agent of listeriosis – systemic infection – can cross blood barriers meningitis & encephalitis bad for babies (NO soft cheeses and no hotdogs!) Listeria in Xenopus extract ActA stimulates Actin polymerization Actin MreB mre operon B. subtilis MreB structure T. maritima et al Microtubules Tubulin-GFP cells – Kinesins – Dyneins / tubulin (1tub) 55kD proteins Both bind GTP - only can hydrolyze GTP form polymerizes, GDP falls apart Dimer solved by electron xtallography of Zn stabilized sheets Taxol – anti-cancer drug – 1963 from Pacific Yew – $1.6 bln in 2000 (+) and (-) ends Microtubule structure cryo structure induces curve Motor Proteins – – – In vitro – FtsZ structure M. jannaschii Tubulin family Intermediate filaments Eukaryotic cells contain about 70 genes Stable, tough, durable structures ~10 nm in diameter, 80-100Å long Multiple types - all cell specific – I & II Acidic/Basic Keratins – III: Vimentin; Desmin; Glial fibrillary acidic protein (GFAP); peripherin – IV: Neurofilament (L,M,H) – V: Nuclear lamins A, B, & C – VI: Nestin Form higher order structure Form homo- & hetero-dimers (align ends) Dimers form tetrads aligning ends Protofilament - joins tetramers end to end Further lateral association No +/- ends – – cytoskeleton Crescentin Caulobacter crescentus Clathrin coated vesicles Endocytosis of receptors Binding of adaptor proteins to cellular receptors causes clathrin binding Series of EM stained images Clathrin lattice Trimers form icosahedral lattice Clathrin structure Complete lattice from cryoEM (8Å) Smallest lattice from 28, 36, 60 triskelions Light and heavy chains – – – – – – – – – – – – – – – – – – – – – – – – – – – into 3 A.U.’s – – – – T=3 Johnson & Speir JMB (1997) 269:665-75 – – – – – – – – A B C A, B C – – – – – – e.g., TBSV e.g., STNV JMB – – et al – – – – TBSV Movie B Figures A red blue C green Polio TBSV blue green blue red red Rice et al PNAS et al PNAS – – – et al Science – – – – – Curr Opin Micro ...
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This note was uploaded on 01/03/2012 for the course BI 170a taught by Professor List during the Fall '09 term at Caltech.

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