A protocol for single molecule imaging and tracking of processive myosin motors

Figures

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  Fig. 1. Sketch of the biotinylated myosin (bio-HMM-5B) structure. The picture shows the two myosin heads (the motor units, blue), the six calmodulin molecules bound to the light-chain binding domains (green), the dimerization domain (purple), and the C-terminus (red) with biotin tags (yellow).

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  Fig. 2. Sketch of the flow chamber. A glass coverslip, smeared with nitrocellulose, is attached onto a microscope slide through double sticky tape stripes to form a flow-cell of about 20 μl volume. Solutions are flown from one side of the chamber with a pipette and sucked from the other side through a filter paper to create a flow in the arrow direction.

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  Fig. 3. Sketch of the gliding assay. Successive layers of nitrocellulose (blue), biotinylated BSA (BSA, brown; biotin, yellow), and streptavidin (red) are adhered on a coverslip surface. Biotinilated myosin bio-HMM-5B is then attached on top of the streptavidin layer. The surface is passivated through a final layer of biotinylated BSA.

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  Fig. 4. Sketch of the single molecule motility assay. Fluorescently-labelled actin filaments are attached on the nitrocellulose-coated coverslip surface through a layer of inactivated NEM-myosin II. Single biotinylated myosin-5B molecules attached to a streptavidinated QD walk along actin in the presence of ATP.

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  Fig. 5. The gliding assay. Image of fluorescent actin filaments bound to myosin in the in vitro gliding assay.

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  Fig. 6. The single molecule motility assay. Images of single QDs (red) moving along fluorescent actin filaments (green) bound to the coverslip in the single molecule motility assay. C.