User:Steven J. Koch/MTC/April 7 2011

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MTC April 7 2011

Molecular Motors

Microtubules field trip / HW--With Andy Maloney

  1. Read up on Tubulin / microtubules
  2. Design Experiment
    • "Tweak" the tubulin polymerization solution--Andy will prepare it. Don't do same as someone else.
      • A boring example: dilute the tubulin, expecting fewer or longer MTs.
      • Try not to do something "crazy."
      • We can't change temperature (at least not easily)...polymerization time is an option
    • Due Tuesday April 12 -- you can email it to me.
  3. Perform Experiment
    • Thursday, April 14 9:30 AM At the CHTM, Don't Be Late!
  4. Analyze data
    • Observations, make figure(s)
    • Upload at least one to figshare.com (requires linking to your Science 3.0 account)

Microtubule data

Below you will find the data that we took in the lab tour class. Both movies have the following characteristics.

  • We took data at 5 frames/s for a total of 10 minutes.
  • The movies have been sped up by a factor of 6 so you are watching it at 30 frames/s.
  • The movies are false colored using ImageJ's Green Fire Blue LUT.
  • Fluorescent images were obtained with rhodamine tagged tubulin. The tubulin was sourced from Cytoskeleton and was polymerized into microtubules using a 29:71 ratio of labeled tubulin:unlabeled tubulin. They have been fixed with 10 μM Taxol.
  • The EMCCD gain of the camera was 150.
  • We used a 100 W Hg lamp attenuated by 94%.
  • The objective used was a 1.42 NA PlanApo 60x objective held at a constant temperature of 33°C.
  • The pixel length is 166.7 nm/pixel which gives the field of view dimensions of 82x110 μm.
  • The glass was passivated with 1.0 mg/mL bovine alpha casein.
  • The concentration of kinesin used was 27.5 μg/mL.

For a complete description of how the assay was prepared and run, please see this page that describes a basic procedure on how to conduct a gliding motility assay.

Note: To complete this assignment, you must make comments about the movies. Be sure to add your signature to the comment by including '''~~~~:''' before you comment. Write down any and all observations you can make about the microtubules. Try not to overlap comments with your fellow students but, if you do, be sure to make them such that a discussion is started. Feel free to leave questions for Dr. Koch in the discussions below.

Microtubules polymerized in D2O

<html> <object width="480" height="390"><param name="movie" value="http://www.youtube.com/v/wOQT9X0mirA?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/wOQT9X0mirA?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="390"></embed></object> </html>

Add comments below

Sheng 

      1. there is a brighter spot on almost every microtube, but in different position and have different size. some has two bright spot.
      2. the size of microtube are very different.
      3. they move in different direction, and some move in a circle.
      4. some very small microtube stay in a same spot for a while.
      5. some move in and out of focus.

Microtubules polymerized in H2O

<html><object width="480" height="390"><param name="movie" value="http://www.youtube.com/v/uEc3HmGloNo?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/uEc3HmGloNo?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="390"></embed></object> </html>

Add comments below

sheng 

     1. there is no bright spot on the microtube.
     2. like the one in D2O, the microtubes have different size, and move in different direction, and some move in a circle.(and there is one microtube form and move in a 
        circle through the entire movie, start at 30s, on the left edge)
     3. sometime there is a small microtube swim along a longer one, and form a bright spot on the long microtube, but they separate in a second.
     4. some also move in and out of focus.
     5. the microtube seems more dense than the one in D2O.
     6. there are not many very small microtube and they don't stay in a same spot as long as the one in D2O
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