Poster: Cytoskeleton: Structure & Function

Abs # 1100: Single molecule analysis of a higher plant myosin responsible for cytoplasmic streaming

Presenter:	Tominaga, Motoki , tominaga@po.crl.go.jp
Authors	Tominaga, Motoki  (A)   Kojima, Hiroaki  (A)   Yokota, Etsuo  (B)   Orii, Hidefumi  (B)   Nakamori, Rinna  (A)   Katayama, Eisaku  (C)   Anson, Michael  (D)   Shimmen, Teruo  (B)   Oiwa, Kazuhiro  (A)
Affiliations:	(A): Kansai Advanced Research Center, Communications Research Laboratory (B): Department of life Science, Graduate School and Faculty of Science, Himeji Institute of Technology (C): Division of Biomolecular Imaging, Institute of Medical Science, The University of Tokyo (D): Division of Physical Biochemistry, National Institute for Medical Research
Web Site:	http://www-karc.crl.go.jp/en/top.html

Cytoplasmic streaming in plant cells is the fastest known biological movement produced by actomyosin interaction. We purified a higher plant myosin responsible for cytoplasmic streaming from cultured tobacco BY-2 cells. This myosin has a heavy chain of 175 kDa and calmodulin as light chains. Sequence analysis showed this 175 kDa myosin shares 60% and 75% sequence identity with MYA1 and MYA2 of Arabidopsis thaliana which are members of class XI myosin. The analysis also showed that this myosin has an a-helical coiled-coil domain leading to dimer formation and has 6 IQ motifs per heavy chain, putative CaM binding domains and its general morphology was similar to myosin V. Electron microscopic observation confirmed this175kDa myosin has dimerized heavy chains each with a long neck and a globular tail as predicted from sequence analysis. Forces and movement developed by single molecules of this myosin were measured by optical trap nanometry. A latex bead coated with one or a small number of myosin molecules was captured with the optical trap and brought into contact with an actin filament fixed on surface of a coverslip. Beads carrying just a single myosin molecule moved processively along actin filaments in 35 nm steps at 7mm/s in the presence of 1mM ATP. This velocity is the fastest among known processive motors. The maximal force measured was less than 0.5pN, much smaller than that produced by skeletal muscle myosin IIs. Dwell-time analysis of single myosin molecules fitted the ATPase kinetics with ADP release being rate-limiting. These results suggest that this plant myosin is highly specialized for the production of fast processive movement with concomitant low force generation.