User:Steven J. Koch/MTC/April 7 2011
MTC April 7 2011
Molecular Motors
- Viral packaging motor
- Flagellar motor(s)
- Archaea, Prokaryotes, Eukaryotes
- Wikipedia flagellum article: http://en.wikipedia.org/wiki/Flagellum
- E. Coli swimming movie http://www.rowland.harvard.edu/labs/bacteria/showmovie.php?mov=swimming_ecoli
- Electron density: http://www.youtube.com/watch?v=vA0hw29Q26g
- D2O results (Berg et al. 2010)
- Archaea, Prokaryotes, Eukaryotes
- Dynein, Myosin, Kinesin,
- Famous kinesin animation: http://www.youtube.com/watch?v=4AnPVuzF7CA&feature=related
- ATP Synthase
- Microscopy: http://www.youtube.com/watch?v=oFgMTdVRi6I
- ATP Synthase animation: http://www.youtube.com/watch?v=J8lhPt6V-yM&feature=related
- From above: http://www.youtube.com/watch?v=H0RchC0FbYU&NR=1
- What happens if we drive the motor with force? Mendeley: Itoh, H., Takahashi, A., Adachi, K., Noji, H., Yasuda, R., Yoshida, M., et al. (2004). Mechanically driven ATP synthesis by F1-ATPase. Nature, 427(6973), 465-468. Retrieved from http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14749837.
- Polymerases / exonucleases / helicases / topoisomerases / ...
- RNA, DNA Polymerase reminder
- Ribosome wikipedia: http://en.wikipedia.org/wiki/Ribosome
- DNA Repair claymation: http://www.youtube.com/watch?v=9PEdqBuDMHM
- Helicase
- http://www.youtube.com/watch?v=UWNhwceMjfk
- Dan Johnson single-molecule helicase
- Topoisomerase
- Animation: http://www.youtube.com/watch?v=EYGrElVyHnU
- Keir Neuman experiment
- RNA, DNA Polymerase reminder
Microtubules field trip / HW--With Andy Maloney
- Read up on Tubulin / microtubules
- Andy's tubulin description: http://openwetware.org/wiki/User:Andy_Maloney/Kinesin_%26_Microtubule_Page#Tubulin
- 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.
- "Tweak" the tubulin polymerization solution--Andy will prepare it. Don't do same as someone else.
- Perform Experiment
- Thursday, April 14 9:30 AM At the CHTM, Don't Be Late!
- 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
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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:[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.
Microtubules polymerized in H2O
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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:[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.