Poster: Cytoskeleton structure & dynamics

Abs # 675: Calcium regulation mechanisms of higher plant myosin XI responsible for cytoplasmic streaming

Presenter:	Tominaga, Motoki , tominaga@po.crl.go.jp
Authors	Tominaga, Motoki  (A)   Yokota, Etsuo  (B)   Nakamori, Rinna  (A)   Shimmen, Teruo  (B)   Kazuhiro, Oiwa  (A) (B)
Affiliations:	(A): Kansai Advanced Research Center, Communication Research Laboratory (B): Graduate School of Science, Himeji Institute of Technology
Web Site:	http://www-karc.crl.go.jp/en/index.html

The motor responsible for cytoplasmic streaming is a member of Myosin Class XI, which has dimerized heavy chains each with light chain binding domains containing 6 IQ motifs and its general morphology is similar to myosin V. We have reported that myosin XI purified from tobacco cultured BY-2 cells moves processively on an actin filament with 35nm steps at high velocity (7μm/sec) (Tominaga et al. EMBO J. 22, 1263-1272, 2003). Increase in intra-cellular Ca²⁺ concentration in plant cells ceases their cytoplasmic streaming through the inhibition of motile activity of myosin. The molecular mechanism of this Ca²⁺ regulation still remains unknown. We have examined this mechanism using in vitro motility assays. When myosin XI was exposed to Ca²⁺ at concentrations higher than pCa5.5 (Ca²⁺ treatment), light chains (calmodulin) are dissociated from the neck domains of the myosin and affinity of this myosin to actin filament decreased. The sliding movement of actin filaments was observed even at < pCa 5.5, when surface densities of myosin were increased to 500 molecules/μm² or when ATP concentration was decreased to less than 10 μM. These results suggested that the detachment of light chains from the neck domains of myosin XI leads to the reduction of its duty ratio. We determined the elasticity of the neck domains of a myosin molecule by measuring the rotation of a short actin filament attached to a single myosin molecule in the absence of ATP. The Ca²⁺ treatment decreased the degree of erratic rotation of an actin filament, indicating that removal of light chains makes myosin neck stiffer. These results indicate that molecular mechanism of Ca²⁺ regulation of myosin XI is the dissociation of the light chains leading to reduction of processivity of myosin XI.