Lecture15 - Kymographs: time Change in direction depending...

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MCB110L Lecture 15 03/09/11 Andreas Martin
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Outline: new data on directional movement of Cin8 along MT Protein purification, separation criteria (remaining slides from lecture 14 )
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anaphase + + + + = Cin8p or Kip1p = Kar3p kinesin-1 kinesin-5 (mitotic) Eg5 (vertebr.) Cin8 (yeast) Known so far: Kinesins usually move to the + end of MT (based on MT sliding exp.)
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Single Cin8 tetramers on individual microtubules move to the minus end new paper on Cin8 movement: “Directional Switching of the Kinesin Cin8 Through Motor Coupling” Roostalu J. et al ., Science 2011 (Scienc express , DOI: 10.1126/science.1199945) http://www.sciencemag.org/content/early/2011/02/23/science.1199945.full.pdf Cin8- mGFP Alexa568 - labeled MT (polarity marked) Kymographs of Cin8-mGFP movement along MT time Single molecule imaging by TIRF
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when crosslinking two microtubules Cin8 moves to the plus end -> sliding of antiparallel MT
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Unformatted text preview: Kymographs: time Change in direction depending on number of MT-interacting motors surface gliding assay - end directed movement + end directed movement mechanically coupled Cin8 ensembles switch to + end directionality switch does not depend on tetramer state of Cin8, immobilized dimer (C-term. trunc. Cin8 871 ) also switches = potential response to mechanical constraints Evanescent wave: = nearfield standing wave, formed at boundary between media with different refractive indices n intensity shows exponential decay with distance from interface; = f( θ , λ , n) penetration depth ( d ) usually 50 – 300 nm image from: MicroscopyU, FSU 1. Sample 2. Evanescent wave 3. Cover slip 4. Immersion oil 5. Objective 6. Excitation beam 7. Emission beam (signal) 1 2 3 4 5 6 7 TIRF: Total Internal Reflection Fluorescence modified from Wikipedia d...
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This note was uploaded on 09/12/2011 for the course MCB 120L taught by Professor Fairclough during the Spring '08 term at UC Davis.

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Lecture15 - Kymographs: time Change in direction depending...

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