No abstract

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...

SUMMARYManual evaluation of cellular structures is a popular approach in cell biological studies. However, such approaches are laborious and are prone to error, especially when large quantities of image data need to be analyzed. Here, we introduce an image analysis framework that overcomes these limitations by semiautomatic quantification and clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density are quantified by measurement of newly-developed, robust metric parameters from microscopic images. Thereafter, the microscopic images are classified without supervision by clustering based on the metric patterns. Clustering allows us to collectively investigate the large number of cytoskeletal structure images without laborious inspection. Application of this framework to images of GFP-actin binding domain 2 (GFP-ABD2)-labeled actin cytoskeletons in Arabidopsis guard cells determined that microfilaments (MFs) are radially oriented and transiently bundled in the process of diurnal stomatal opening. The framework also revealed that the expression of mouse talin GFP-ABD (GFP-mTn) continuously induced MF bundling and suppressed the diurnal patterns of stomatal opening, suggesting that changes in the level of MF bundling are crucial for promoting stomatal opening. These results clearly demonstrate the utility of our image analysis framework.

Despite the absence of a conspicuous microtubule-organizing centre, microtubules in plant cells at interphase are present in the cell cortex as a well oriented array. A recent report suggests that microtubule nucleation sites for the array are capable of associating with and dissociating from the cortex. Here, we show that nucleation requires extant cortical microtubules, onto which cytosolic gamma-tubulin is recruited. In both living cells and the cell-free system, microtubules are nucleated as branches on the extant cortical microtubules. The branch points contain gamma-tubulin, which is abundant in the cytoplasm, and microtubule nucleation in the cell-free system is prevented by inhibiting gamma-tubulin function with a specific antibody. When isolated plasma membrane with microtubules is exposed to purified neuro-tubulin, no microtubules are nucleated. However, when the membrane is exposed to a cytosolic extract, gamma-tubulin binds microtubules on the membrane, and after a subsequent incubation in neuro-tubulin, microtubules are nucleated on the pre-existing microtubules. We propose that a cytoplasmic gamma-tubulin complex shuttles between the cytoplasm and the side of a cortical microtubule, and has nucleation activity only when bound to the microtubule.