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

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MTC April 7 2011

Contents

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

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.

Pranav:

Comments: [1]

David

      1. I agree with Shing that there are concentrated bright spots in most of the microtubes some do not have them.
      2. The ones that do not have the bright spots seem to be brighter the the microtubes that were polymerized in H2O this could be a real result or just how the intensity were adjusted
         to generate the color data. 
      3. The size of the microtubes does not seem to matter in terms of the brighter spots that were present, some it seems are just bright spots.

Boleszek

      1. As Sheng notes, there are indeed quite a few small segments that do not move much. There
         may be small patches where kinesin is not properly oriented which is responsible for the
         lack of movement in some of these smaller segments.
      2. The overlapping of the H20 microtubules seems to produce produce bright regions that
         look much like the permanent bright spots seen in the D2O data. If the permanent bright 
         spots seen in the D2O data are the result of two overlapping microtubules with uniform 
         fluorophore distributions, then we would expect the overlapping and permanent bright 
         spots to look the same. However, overlapping in the D2O sample seems to produce bright 
         regions that are not as bright as the permanent bright spots. It is possible then that 
         the D2O is somehow encouraging fluorophores to bunch up on the microtubules. Perhaps it 
         is altering the way taxol binds to the microtubules.
      3. I notice an interesting event at approximately 0:24. A microtubule with a bright head 
         approaches the bottom left corner from above and seems to drop its bright head off.
         Importantly, the bright head remains bright after falling off. It could be that the 
         microtubule was pushing a shorter segment along and so they were never attached. It is 
         also possible that the bright head is a bundle of taxol and fluorophores.

Alex:

      1. These microtubules have distinct bright spots that appear to be part of the microtubule
         structure due to their stationary position on each tube.
      2. The average size of these microtubules is shorter, and there are numerous small segments 
         that remain stationary and are moved by the kinesin.
      3. I thought about the possible ribbon structure theory associated with polymerization in D2),
         but after comparison with the normal H2O sample, and my current theory about intensity being
         correlated with the layering of polymerized tubuline, I believe that the ribbon structures 
         would appear with less intensity than the normal H2O sample.

Melissa:

      1. As has been mentioned multiple times, the microtubules have very distinct bright spots, most located at their midpoints.  This could be due to the microtubules layering in that area when forming, or as Boleszek notes, the D2O affects the way the fluorophores attach to the microtubules. Its higher density possibly creates irregularity in the concentration of fluorophores throughout the sample, although the tendency for the bright spots to consistently be near the midpoints of the microtubules could discredit this, since if this were the case, one might expect bright spots in various locations of the microtubules - not just the midpoints.
      2. The sizes of the microtubules vary widely from one to the next.  However, their sizes are ultimately much shorter than the H2O samples.  Formation in the D2O could hamper the attachment of tubulin to each other, causing this.
      3. Sheng and Boleszek mention that the shortest of the microtubules tend to move very little.  Their small size might make it difficult for the kinesin to effectively move those microtubules, as compared to the longer microtubules.

Microtubules polymerized in H2O

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

Pranav:

Comments[2]

David

     1. The microtubes that were polymerized in H2O seem to have an overall higher intensity than those polymerized in D2O but do not have the intense regions that were seen in the D2O microtubes.
     2. These microtubes seem to have an average length that is longer than those that were polymerized in D2O.
     3. When two of the microtubes cross over one and other they seem to get more intense this does happen in the sample that were polymerized in D2O but the intensity seems to be diminished 
        when compared to those polymerized in H2O.  I think that the reason for this is that the microtubes that were polymerized in D2O might have a higher concentration of the labeled tubulin
        that seems to be concentrated more in one or two spots as compared to those microtubes that were polymerized in H2O.

Alex:

      1. As noted above these microtubules polymerized in H20 have consistent luminance through the bodies    
      2. The average size of this sample appears to be larger overall and consists of fewer small segments
      3. The intensity of the microtubules increases where two intersect or pass over each other. Assuming 
         that the gain in our microscope was not changed for each sample, the intensity of these overlaps 
         seems to correspond to the intesity of the bright segments on the microtubules polymerized in D2O. 
         This could lend itself to the idea that the "bright spots" in the D2O sample could be overlapping 
         microtubules. 
      4. We seem to have a higher concentration of microtubule formations in this sample, but Andy did comment 
         in the lab that the D2O sample was much older than this H2O sample which could or could not have some 
         effect on this.

Melissa:

      1. The microtubules created in the H2O do not have the bright spots located near their centers, as the microtubules in the D2O sample do.  This could possibly be due to the difference in density between H2O and D2O, changing the ease and regularity with which the microtubules could form.
      2. At around 32 sec in this movie, a very long microtubule travels across the upper right corner.  The H2O microtubules, all in general, seem to be much longer than those of the D2O sample, possibly again due to the less dense characteristics of the H2O, which could facilitate microtubule formation moreso.
      3. The range in size between all microtubules in this sample seems to be much smaller than the D2O sample - most microtubules are similar in size and fairly long.
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