| 研究課題/領域番号 |
20H03218
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| 研究機関 | 金沢大学 |
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研究代表者 |
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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | High-speed AFM / Bioimaging / SPM / actin / cytoskeleton / myosin |
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| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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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| 今後の研究の推進方策 |
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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