2021 Fiscal Year Research-status Report
Mechanical stability of microtubules investigated under unidirectional strain using combined fluorescence microscopy and high-speed atomic force microscopy
| Project/Area Number |
20K03889
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| Research Institution | Center for Novel Science Initatives, National Institutes of Natural Sciences |
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Principal Investigator |
Ganser Christian 大学共同利用機関法人自然科学研究機構(新分野創成センター、アストロバイオロジーセンター、生命創成探究, 生命創成探究センター, 特任助教 (50846095)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | Microtubules / Mechanical Properties |
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| Outline of Annual Research Achievements |
Microtubules are an important part of the cytoskeleton and are under contant mechanical stresses. In order to study microtubules under such conditions it is necessary to have tools available to reliably apply stress in a non-destructive way during observation. A stretching mechanism was developed - including a protocol to reliably immobilize microtubules - to apply stress during high-speed atomic force microscopy observations.
Microtubules could be fixed to the strechable silicone and PDMS substrates and imaged with high-speed atomic force microscopy. Further, streching of the substrates could be used to apply stress to the microtubules and stressed microtubules could be imaged by high-speed atomic force microscopy.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Currently, microtubules can be fixed to the substrates and stretched. Fixing microtubules is achieved by coating the surface with kinesin, which attaches to the tubulin dimers. This procedure holds the microtubules in place securely while still allowing deformations in the microtubule lattice without causing stress spikes which would cause irreversible damage - such as rupture events. This process is being optimized with respect to the concentration of kinesin coating and surface passivation. It is necessary to keep the microtubules fixed securely especially with respect of future motor protein studies.
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| Strategy for Future Research Activity |
In the next steps it is planned to study motor proteins kinesin and dynein moving along stressed microtubules. It is expected to observe for the first time motor protein movement with high-speed atomic force mircoscopy under deliberately stressed conditions. There will be a focus on fixing the kinesin-tubulin bond for surface fixation. If walking kinesin or dynein should be observed, the surface kinesin needs to be fixated in order to inhibit the gliding movement of microtubules. One possibility is to utilize glutaraldehyde, but for this to be properly utilized it is necessary to make sure that such glutaraldehyde fixation does not change the tubulin structure significantly. If tubulin structur is changed, it is possible that this will affect motor protein movmement.
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