User:Andy Maloney/Kinesin & Microtubule Page/Osmotic stress/The Force Exerted by a Single Kinesin Molecule Against a Viscous Load
These are my notes. Please read the article before you read my notes.
- Shows that by increasing the viscosity of the buffer used for the motility assay, they were able to get longer microtubules to move slower than short ones. This is due to the larger drag force of longer microtubules.
- The speed of the microtubule depended linearly on the drag force loading the kinesin.
- They state that they were able to extrapolate a maximum time-averaged drag force for kinesin, 4.2 ± 0.5 pN.
- They note that when a microtubule is moving in a gliding assay, the minus end of the microtubule is the end that is always leading.
- Buffer solution:
- 80 mM PIPES
- 1 mM EGTA
- 2 mM MgCl2
- pH 6.9 with KOH
- They used 2.5 mg/mL casein to coat the glass.
- Bovine brain kinesin stored in their buffer with .25 mg/mL casein.
- Bovine brain tubulin.
- They were able to make 1 - 5 µm microtubules by passing them through a 30 gauge needle.
- Ha! They heated the microscope objective by wrapping a copper pipe around it and used a heated closed water system.
- They note that their 100W Hg lamp only raised the temp in the observation field by less than 1˚C. I should see if I can figure out a way to check mine.
- They note that methyl cellulose and high molecular weight dextran are not good chemicals to change viscosity because they are long filaments that give different viscosities in different directions.
- BSA causes microtubule bundling. 20% (w/v).
- Their "visc-mix" (chemicals used to increase the viscosity) consisted of:
- 14.5% (w/v) trypsin inhibitor
- 15% dextran
- 7.5% Ficoll-400
- In their buffer.
- I don't know why, but they dialyzed the trypsin inhibitor because it would depolymerize microtubules.
- High viscosity and high amounts of kinesin did nothing to the velocity of microtubules.
- Low kinesin density and high viscosity caused the microtubules to slow.
- They state that speed is not affected my osmolality.
- They state that changing the ionic strength of the buffer doesn't affect speed.
- Wow! They were able to get the force produced from kinesin by looking at the speed of microtubules in viscous environments. And on VHS to boot!
- Man this thing reads like a dissertation.
We need a viscometer to measure the viscosity of our solutions so that we are no where near what these guys did. Wow, that was a tough read. But, extremely well written!