Single-molecule fluorescence tracer based on quantum dots reveals the stepping mode of myosin motor

There are nearly one hundred different molecular motors in a eukaryotic cell, each with different mechanisms of action, which are adapted to their unique physiological functions. Among them, myosin is a widely-existing motor protein, which plays an important role in endocytosis, protein secretion, vesicle transport, and maintenance of Golgi morphology. Toshio Yanagida, a research group at Osaka University in Japan, used a single-molecule fluorescent tracer technique to observe and analyze the step pattern of myosin VI on the cytoskeleton. The relevant results were published in CELL.

So Nishikawa et al. constructed an insect cell-baculovirus expression vector of myosin and integrated HaloTag and 6×His-tag into an expression vector. The recombinant protein was purified by nickel column and HaloTag PEG-Biotin ligand was added to biotinylation of myosin. Finally, streptavidin conjugated Quantum Dots (SA-QDs) were used to quantify the binding of myosin by quantum dots using the binding properties of biotin-avidin (Fig. 1 and Fig. 2). . For the tracing of the quantum dot-labeled myosin along the microfilament skeleton, total internal reflection fluorescence microscopy (TIRFM) and Olympus epifluorescence microscopy were used to acquire images with EMCCD, and data analysis was performed with related software. The experimental results show that quantum dot labeling does not produce steric hindrance to myosin movement; compared with green fluorescent protein (GFP), quantum dot fluorescent probes have better temporal and spatial resolution. The researchers revealed the molecular mechanism of the transition between the two steps of myosin motor size, which has important theoretical significance for understanding the mechanism of motor protein.

Figure 1 Marking myosin head with quantum dots and observing its step behavior

Figure 2 Marking the two heads of myosin with different color quantum dots and observing their step behavior

Source of the document:

Nishikawa S, Arimoto I, Ikezaki K, Sugawa M, Ueno H, Komori T, Iwane AH, Yanagida T. Switch between large hand-over-hand and small inchworm-like steps in myosin VI. Cell 2010;142(6): 879-88.

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