Minisymposium 23: Cytoskeleton

Abs # 37002: Single molecule analysis of a higher plant myosin responsible for cytoplasmic streaming

Presenter:	Tominaga, Motoki , tominaga@po.crl.go.jp
Authors	Tominaga, Motoki  (A)   Yokota, Etsuo  (B)   Kojima, Hiroaki  (A)   Orii, Hidefumi  (B)   Nakamori, Rinna  (A)   Katayama, Eisaku  (C)   Anson, Michael  (D)   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

Myosins play important functional roles within plant cells in driving actin-based motility such as intracellular vesicle and membrane transport. However mechanical properties of plant myosins have not been well characterized owing to their susceptibility to proteolytic degradation. We separated intact myosins from cultured tobacco BY-2 cells into two isoforms termed 170kDa and 175kDa. Immunoblot analyses revealed that in tobacco plants, 175kDa myosin was expressed in leaves, stems, and roots, except in germinating pollen, while 170kDa myosin was present in all of these. Immunostaining revealed that these myosins localized on different sized vesicular organelles in tobacco BY-2 cells. Here, we focus on the properties of 175kDa myosin because this myosin shows little aggregation even at low ionic strength and so is suitable for single molecule mechanical measurements. Sequence analysis showed this 175 kDa myosin shares 60% and 75% sequence identity with MYA1 and MYA2 respectively of Arabidopsis thaliana which are members of myosin class XI. The analysis also predicted this myosin to have an a-helical coiled-coil domain leading to dimer formation with 6 IQ motifs per heavy chain being putative CaM binding domains, and a general morphology similar to myosin V which is responsible for vesicle transport in neurons. Electron microscopic observation confirmed 175kDa myosin to have dimerized heavy chains each with a long neck and a globular tail. In vitro motility assays showed actin filaments moving on myosin-coated surface with their minus end leading, indicating that 175kDa myosin moves towards the plus end of actin filaments. Forces and movement developed by single molecules of this myosin were measured with 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 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 known for processive motors. Measured maximal force was less than 0.5pN, much smaller than 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.