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Control of actin moving trajectory by patterned poly(methylmethacrylate) tracks
1Kansai Advanced Research Center, Communications Research Laboratory, Kobe, Japan. suzuki@crl.go.jp
Abstract:
Poly(methylmethacrylate) (PMMA), a photoresist polymer, was found to be useful for immobilizing heavy meromyosin (HMM) molecules while retaining their abilities to support the movement of actin filaments. PMMA substrate was spin-coated on a coverslip, and various shapes of PMMA tracks, such as straight lines, concentric circles, and alphabetical letters, were fabricated by UV photolithography. An observation by a Tapping mode atomic force microscope (AFM) shows that the typical circular tracks were 1-2 microns wide and about 200 nm high. In in vitro motility assay, a solution of HMM molecules was applied to immobilize the molecules on the tracks by adsorption, and movement of actin filaments labeled with tetramethylrhodamine-phalloidin were observed in the presence of ATP by using an epifluorescence microscope and an image-intensified CCD camera. Actin filaments were seen to move precisely only on the PMMA tracks, and their traces drew the exact shapes of the tracks. The mean velocity of actin movement on the PMMA was 4.5 mm/s at 25 degrees C, and it was comparable to that on a conventionally used nitrocellulose film.
Insights
Poly(methylmethacrylate) (PMMA) effectively immobilizes heavy meromyosin (HMM) for actin filament motility assays. Actin filaments precisely follow PMMA tracks, demonstrating a new method for studying molecular movement.
Area of Science:
- Biophysics
- Materials Science
- Cell Biology
Background:
- Heavy meromyosin (HMM) is crucial for actin filament movement.
- Immobilizing HMM on specific substrates is essential for studying motility.
- Poly(methylmethacrylate) (PMMA) is a photoresist polymer with potential for biomolecule immobilization.
Purpose of the Study:
- To evaluate PMMA as a substrate for immobilizing HMM molecules.
- To investigate the precise movement of actin filaments on patterned PMMA tracks.
- To compare actin motility on PMMA with conventional substrates.
Main Methods:
- Fabrication of PMMA tracks (lines, circles, letters) using UV photolithography.
- Immobilization of HMM molecules onto PMMA tracks via adsorption.
- In vitro motility assay observing rhodamine-phalloidin labeled actin filaments using epifluorescence microscopy.
- Atomic Force Microscopy (AFM) to characterize PMMA track dimensions.
Main Results:
- Actin filaments moved precisely along the fabricated PMMA tracks, mirroring their shapes.
- PMMA successfully immobilized HMM, retaining its motor function.
- The mean velocity of actin movement on PMMA was 4.5 mm/s at 25°C.
- AFM confirmed track dimensions (1-2 microns wide, ~200 nm high).
Conclusions:
- PMMA is a suitable material for creating patterned substrates for HMM-based in vitro motility assays.
- This technique allows for precise control and observation of actin filament movement along defined paths.
- PMMA offers a viable alternative to traditional nitrocellulose films for actin motility studies.