Natsumaro Kutsuna

Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells.

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.

Cobtorin target analysis reveals that pectin functions in the deposition of cellulose microfibrils in parallel with cortical microtubules

Cellulose and pectin are major components of primary cell walls in plants, and it is believed that their mechanical properties are important for cell morphogenesis. It has been hypothesized that cortical microtubules guide the movement of cellulose microfibril synthase in a direction parallel with the microtubules, but the mechanism by which this alignment occurs remains unclear. We have previously identified cobtorin as an inhibitor that perturbs the parallel relationship between cortical microtubules and nascent cellulose microfibrils. In this study, we searched for the protein target of cobtorin, and we found that overexpression of pectin methylesterase and polygalacturonase suppressed the cobtorin-induced cell-swelling phenotype. Furthermore, treatment with polygalacturonase restored the deposition of cellulose microfibrils in the direction parallel with cortical microtubules, and cobtorin perturbed the distribution of methylated pectin. These results suggest that control over the properties of pectin is important for the deposition of cellulose microfibrils and/or the maintenance of their orientation parallel with the cortical microtubules.

Differential Expression Control and Polarized Distribution of Plasma Membrane-Resident SYP1 SNAREs in Arabidopsis thaliana

Membrane trafficking to the plasma membrane (PM) is a highly organized process which enables plant cells to build up their bodies. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) genes, which encode the proteins involved in membrane trafficking, are much more abundant in the Arabidopsis genome than in that of any other eukaryote. We have previously shown that a large number of SNARE molecules in the Arabidopsis cell are localized predominantly on the PM. In the present study, in order to elucidate the physiological function of each PM-localized SNARE, we analyzed the spatiotemporal expression profiling of nine SYP1s that are resident in the PM of Arabidopsis, and used the information thus acquired to generate transgenic Arabidopsis plants expressing green fluorescent protein-fused Qa-SNAREs under control of their authentic promoters. Among the nine SYP1s, only SYP132 is expressed ubiquitously in all tissues throughout plant development. The expression patterns of the other SYP1s, in contrast, are tissue specific, and all different from one another. A particularly noteworthy example is SYP123, which is predominantly expressed in root hair cells during root development, and shows a focal accumulation pattern at the tip region of root hairs. These results suggest that SYP132 is involved in constitutive membrane trafficking to the PM throughout plant development, while the other SYP1s are involved in membrane trafficking events such as root formation or tip growth of root hair, with some redundancy.

Specific Enrichment of miRNAs in Arabidopsis thaliana Infected with Tobacco mosaic virus

Abstract RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.

Insights into the Localization and Function of the Membrane Trafficking Regulator GNOM ARF-GEF at the Golgi Apparatus in Arabidopsis

This work presents a model of the plant intracellular trafficking system developed by analyzing the subcellular localization of GNOM ARF-GEF, which is crucial for auxin transport-mediated plant development. The data show that GNOM is associated with Golgi body, rather than endosomes as previously believed, and redefine our notions about the identity and function of recycling endosomes in plants. GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants.

An Atypical Tubulin Kinase Mediates Stress-Induced Microtubule Depolymerization in Arabidopsis

BACKGROUND As sessile organisms, plants adapt to adverse environmental conditions by quickly adjusting cell physiology and metabolism. Transient depolymerization of interphase microtubules is triggered by various acute stresses and biotic interactions with pathogenic organisms. Although rapid remodeling of plant microtubule arrays in response to external stresses is an intriguing phenomenon, the underlying molecular mechanisms and the advantages of this response to plant performance are poorly understood. RESULTS A domain with weak homology to the slime mold actin-fragmin kinase in the Arabidopsis mitogen-activated protein kinase phosphatase PROPYZAMIDE-HYPERSENSITIVE 1 (PHS1) is a Mn2+-dependent kinase. This atypical kinase domain phosphorylates Thr349 of α-tubulin at the longitudinal interdimer interface, thereby generating a polymerization-incompetent isoform, and effectively depolymerizes microtubule arrays when ectopically expressed in plant or animal cells. The intrinsic tubulin kinase activity is normally suppressed by the phosphatase activity of PHS1 but is unmasked immediately after osmotic stress. In the phs1 null mutant, stress-induced microtubule depolymerization does not occur. CONCLUSIONS The rapid and reversible modification of tubulin subunits by PHS1-mediated phosphorylation enables dynamic remodeling of the plant microtubule cytoskeleton in response to external stimuli. Suppression of the potent tubulin kinase activity by the juxtaposed phosphatase domain tightly controls this stress-activated microtubule regulator.

β-Alanyl-L-Histidine Rescues Cognitive Deficits Caused by Feeding a High Fat Diet in a Transgenic Mouse Model of Alzheimer's Disease

Our goal in this study was to determine whether or not feeding young (4 months old) Alzheimers disease model transgenic mice with a high fat diet (HFD), consisting of 32% fat, is capable of causing cognitive decline and whether treatment with β-alanyl-L-histidine (carnosine) is capable of reducing these effects. Carnosine is an endogenous antioxidant and antiglycating agent that is abundantly present in the brain and muscle tissues of vertebrates. After 8 weeks of feeding with HFD, we observed a significant decline in the contextual memory in transgenic mice fed with HFD as compared to transgenic mice fed with a normal diet as well as to normal diet-wild type mice. Treatment with carnosine at a dose of 5 mg/day for 6 weeks was effective in preventing cognitive decline, as the transgenic group fed with HFD and treated with carnosine displayed a level of cognition comparable to controls. No differences in senile plaque load were observed between all groups. However, we observed an increase in the expression of RAGE in blood vessels as well as increased microglial activation in the hippocampus of animals fed with HFD, effects that were reversed when treated with carnosine. Given these results, there is a possibility that inflammation and cerebrovascular abnormalities might be the cause of cognitive decline in this model.

Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

BackgroundPlant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs) and actin microfilaments (MFs). Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown.ResultsTime-sequential changes in MF-distribution were monitored by live imaging of tobacco BY-2 cells stably expressing the GFP-actin binding domain 2 (GFP-ABD2) fusion protein, which vitally co-stained with the endocytic tracer, FM4-64, that labels the cell plate. During cytokinesis, MFs accumulated near the newly-separated daughter nuclei towards the emerging cell plate, and subsequently approached the expanding cell plate edges. Treatment with an actin polymerization inhibitor caused a decrease in the cell plate expansion rate, which was quantified using time-lapse imaging and regression analysis. Our results demonstrated time-sequential changes in the contribution of MFs to cell plate expansion; MF-disruption caused about a 10% decrease in the cell plate expansion rate at the early phase of cytokinesis, but about 25% at the late phase. MF-disruption also caused malformation of the emerging cell plate at the early phase, indicative of MF involvement in early cell plate formation and expansion. The dynamic movement of endosomes around the cell plate was also inhibited by treatment with an actin polymerization inhibitor and a myosin ATPase inhibitor, respectively. Furthermore, time-lapse imaging of the endoplasmic reticulum (ER) revealed that MFs were involved in ER accumulation in the phragmoplast at the late phase.ConclusionBy expression of GFP-ABD2 and vital staining with FM4-64, the dynamics of MFs and the cell plate could be followed throughout plant cytokinesis in living cells. Pharmacological treatment and live imaging analysis also allowed us to quantify MF contribution to cell plate expansion during cytokinesis. Our results suggest that MFs play significant roles in cell plate formation and expansion via regulation of endomembrane dynamics.

Acceleration of Vacuolar Regeneration and Cell Growth by Overexpression of an Aquaporin NtTIP1;1 in Tobacco BY-2 Cells

Aquaporin is a water channel that increases water permeability through membranous structures. In plants, vacuoles are essential organelles that undergo dynamic volume changes during cell growth. To understand the contribution of aquaporins to plant cell growth, we developed a transgenic tobacco BY-2 cell line overexpressing the tonoplast intrinsic protein (TIP), gammaTIP. Vacuolar membranes of isolated vacuoles from gammaTIP-overexpressing cells showed higher water permeation activities than those from wild-type cells. We then examined the role of gammaTIP in vacuolar regeneration of evacuolated tobacco BY-2 protoplasts (miniprotoplasts). Vacuolar regeneration from thin to thick tube-network vacuoles and subsequent development of large vacuoles was accelerated in miniprotoplasts of this cell line. A parallel increase in the rate of cell expansion indicated a tight relationship between vacuolar development and cellular volume increases. Interestingly, overexpression of tobacco gammaTIP also enhanced cell division. Thus, increased vacuolar aquaporin activity may accelerate both cell expansion and cell division by increasing water permeability through the vacuolar membrane.

Chapter 3 New Insights into Plant Vacuolar Structure and Dynamics

The plant vacuole is a multifunctional organelle and is essential for plant development and growth. The most distinctive feature of the plant vacuole is its size, which usually occupies over 80-90% of the cell volume in well-developed somatic cells, and is therefore highly involved in cell growth and plant body size. Recent progress in the visualization of the vacuole, together with developments in image analysis, has revealed the highly organized and complex morphology of the vacuole, as well as its dynamics. The plant vacuolar membrane (VM) forms not only a typically large vacuole but also other structures, such as tubular structures, transvacuolar strands, bulbs, and sheets. In higher plant cells, actin microfilaments are mainly located near the VM and are involved in vacuolar shape changes with the actin-myosin systems. Most recently, microtubule-dependent regulation of vacuolar structures in moss plant cells was reported, suggesting a diversity of mechanisms regulating vacuolar morphogenesis.