2020 Fiscal Year Annual Research Report
Imaging myosin-driven stress fiber contraction with molecular resolution by high-speed AFM
| Project/Area Number |
20H03218
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| Research Institution | Kanazawa University |
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Principal Investigator |
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | High-speed AFM / Bioimaging / SPM / actin / cytoskeleton / myosin |
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| Outline of Annual Research Achievements |
The overall aim of the project is to image large intracellular protein assemblies, such as actin-myosin fibers, with molecular resolution under physiological conditions by high-speed atomic force microscopy (HS-AFM). However, previous HS-AFM scanners ususally have only small scan sizes. In this project phase we developed an ultrawide HS-AFM sample-scanner system able to record large topographic images (40 x 40 micrometer) containing up to 16 megapixels, providing molecular resolution throughout the image frame. With this unique scanner design we are now able for the first time to image even large intracellular structures and entire organelles in a single AFM scan with near molecular resolution aiding the quantitative analysis of such structurally heterogenous samples.
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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
We are currently using the novel ultra-wide HS-AFM scanner to investigate molecular events occurring during ATP-induced actin stress fiber contraction. We can first generate large, high-resolution overview images of large actomyosin structures, such as cortical networks, actin stress fibers or focal adhesions. Subsequently, we can reduce the scan size to image smaller subregions of these organelles with molecular resolution and increased frame rates. Using this approach enabled us to visualize the action of individual myosin motor proteins during stress fiber contraction, which was an important milestone of the project.
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| Strategy for Future Research Activity |
HS-AFM is a powerful technique to visualize individual biomolecules in action in real-time. However, HS-AFM requires physical contact between the probe (AFM tip) and the sample, occasionally leading to sample deformation and destruction. In contrast, Scanning Ion Conductance Microscopy (SICM) is a contactless scanning method and therefore especially suited to fragile biological samples, such as exposed cellular organelles. We are planning to complement our HS-AFM experiments with SICM to obtain additional and complementary structural insight. Furthermore, SICM can also record stiffness distribution of the imaged cellular samples, providing important additional mechanical insight into myosin-driven stress fiber contraction.
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