Plant and Cell Physiology, 1995, Vol. 36, No. 8 1541-1547
© 1995
Biochemical, Immunochemical and Immunohistochemical Identification of Myosin Heavy Chains in Cultured Cells of Catharanthus roseus
1 Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City Hyogo, 678-12 Japan
2 Biological Laboratory, Hitotsubashi University Naka 2-1, Kunitachi, Tokyo, 186 Japan
In the pollen tubes of the lily Lilium longiflorum, myosin, composed of 170-kDa heavy chains is responsible for the intracellular transport of organelles [Yokota and Shimmen (1994) Protoplasma 177: 153]. Polypeptides of 170 kDa with similar antigenicity to this pollen-tube myosin have also been found in other angiosperm cells [Yokota et al. (1995) Protoplasma 185: 178]. To clarify the role of this type of myosin in cytoplasmic streaming, we prepared partially purified myosin fraction from cultured cells of Catharanthus roseus by co-precipitation with F-actin. In a motility assay in vitro with this fraction, rhodamine-phalloidin-labeled F-actin moved with an average velocity of 10.7 µm s-1. This sliding velocity was similar to that of the cytoplasmic streaming observed in intact cultured cells. Antibodies raised against the 170-kDa heavy chain of pollen-tube myosin recognized only a single polypeptide of 170 kDa in this partially purified fraction. The same polypeptide was also identified by these antibodies in a crude extract of proteins from cultured cells. The myosin-specific fluorescence was concentrated around the nuclei and was associated with particles of various sizes. Dual localization using antibodies against myosin and against actin revealed that these particles were preferentially co-localized with actin filaments. On the other hand, no component of the crude extract or of the partially purified myosin fraction cross-reacted with antibodies against heavy chains of myosin II from animal cells.
These results suggest that the 170-kDa polypeptide is the myosin heavy chain and that this myosin generates the motive force for cytoplasmic streaming in cultured cells of Catharanthus roseus.
(Received March 28, 1995; Accepted September 14, 1995)
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