Plants exhibit an ultimate case of the intracellular motility involving rapid organelle trafficking and continuous streaming of the endoplasmic reticulum (ER). Although it was long assumed that the ER dynamics is actomyosin-driven, the responsible myosins were not identified, and the ER streaming was not characterized quantitatively. Here we developed software to generate a detailed velocity-distribution map for the GFP-labeled ER. This map revealed that the ER in the most peripheral plane was relatively static, whereas the ER in the inner plane was rapidly streaming with the velocities of up to ∼3.5 μm/sec. Similar patterns were observed when the cytosolic GFP was used to evaluate the cytoplasmic streaming. Using gene knockouts, we demonstrate that the ER dynamics is driven primarily by the ER-associated myosin XI-K, a member of a plant-specific myosin class XI. Furthermore, we show that the myosin XI deficiency affects organization of the ER network and orientation of the actin filament bundles. Collectively, our findings suggest a model whereby dynamic three-way interactions between ER, F-actin, and myosins determine the architecture and movement patterns of the ER strands, and cause cytosol hauling traditionally defined as cytoplasmic streaming.myosin XI | actin filament | cytoplasmic streaming | velocity distribution map | Arabidopsis thaliana C ytoplasmic streaming, defined as an extensive intracellular motility in plants, was first described in 1774 (1). It is thought that unidirectional actin filament (AF) bundles and organelleassociated myosin XI, a plant-specific class of myosin motors, cause bulk flow in the cell (reviewed in refs. 2-4). Some myosin XI were indeed reported to slide along AFs in vitro (5, 6). Using immunocytochemical analyses (7-11) and fluorescent proteinlabeled myosins (12, 13), myosin XI have been shown to be associated with the particulate organelles. Recent analyses using gene knockouts and dominant-negative inhibition demonstrated that several class XI myosins have overlapping functions in the rapid movement of organelles (14-18). Among these, myosin XI-K was found to play the most prominent role in the movement of Golgi bodies, peroxisomes, and mitochondria. Studies have also concluded that none of these three organelles fits the paradigm of cytoplasmic streaming, raising the question of what drives this conspicuous process (14, 15).The endoplasmic reticulum (ER), an organelle present in all eukaryotic cells, harbors the largest reservoir of cellular membranes. Cytoskeleton-dependent remodeling of the ER network, which consists of cisternae and interconnected membrane tubes, is well known in both animals and plants (reviewed in refs. 19-22). In addition, dynamic streaming of the plant ER has been observed in subperipheral cytoplasm and transvacuolar strands (23-25). However, the molecular mechanism underlying the ER streaming is not known, although it was reported that the 175 kDa myosin XI was localized on the ER of tobacco cell culture BY-2 (26).Unlike the movement of the part...

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In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells.

Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants.