6

Abstract


The outer membrane of gram-negative bacteria is a unique asymmetric membrane bilayer that is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders gram-negative bacteria more resistant to antibiotics than their gram-positive counterparts. LPS comprises three regions: lipid A, core oligosaccharide, and O-antigen polysaccharide. In this talk, I will present our ongoing efforts on understanding various bacterial outer membranes and their interactions with outer membrane proteins, including (1) construction of a model of an E. coli R1 (core) O6 (antigen) LPS molecule using the CHARMM36 lipid and carbohydrate force fields and simulations of various E. coli R1.O6 LPS bilayers; (2) modeling of E. coli R2, R3, R4, and K12 cores and other O-antigens and their bilayer simulations; (3) development of LPS Modeler in CHARMM-GUI; (4) modeling and simulation of E. coli outer membranes with phospholipids in the inner leaflet and LPS in the outer leaflet as well as OmpLA in the outer membrane; (5) modeling and simulation of BamA in the E. coli outer membrane; (6) other ongoing outer membrane – protein simulations in other bacteria.

 

 

 

6

Abstract


Climate change, extreme weather patterns, and pests/pathogens threaten sustainable agronomic crop production. Global circulation models predict future climates with increased ambient temperatures and an enhanced frequency of severe weather events, including drought, drenching rains, severe flooding, and heat waves. Consequently, climate change will expose plants to increasing episodes of abiotic and biotic stress that are expected to negatively impact global agriculture. Atmospheric carbon dioxide concentrations may double during the current century. Plants have complex responses to both atmospheric carbon dioxide and to abiotic/biotic stress but it is unclear how these two processes interact at the whole plant and cellular levels. An integrated multiple omics analyses were used to unravel a holistic understanding of crop responses in the face of climate change. Our studies provide preliminary evidence that atmospheric CO2 concentrations have the potential to modify plant responses to abiotic and biotic stress.

 

10

Abstract


Multicellular organization of plant cells/tissues shows rather supracellular nature than simple multicellular one, e.g. a group of cells behaves as a single cell (a simplasmic domain). In this phenomenon, plasmodesmata (PD) play a pivotal role as the intercellular channels through which both nutrients and signaling molecules are rapidly exchanged. The signaling molecules include not only micro-molecules such as ions, nutrient molecules and phytohormones, but also macro-molecules such as transcription factors and RNAs including mRNA and small noncoding RNAs. Although symplasmic domain requires dynamic exchange of intracellular molecules, the opening and closure of PD should be finely controlled. However, regulatory machineries of PD are not much understood. Typically, two callose-dependent and independent pathways control plasmodesmal gating. Callose is a β-1,3-glucan polysaccharide accumulated at cell wall, specifically at PD region and has been proposed to regulate the size exclusion limit (SEL) of PD. High levels of callose, generated by β-1,3-glucan synthases, result in a reduction in PD SEL, whereas removal of callose, by PD-localized β-1,3-glucanases, results in an increase in PD SEL. Previously, PD regulation by callose deposition was considered as a coarse control. However, recent finding suggested that PD can be finely regulated by callose. Here, we present that the operation of an auxin-mediated bi-stable PD switch that down-regulates PD permeability to control auxin back-diffusion in order to allow development of the auxin gradient required for the hypocotyl tropic response. We also present on-going research projects related to PD regulation.

 

9

Abstract


Plants are regularly facing multiple types of abiotic stresses, altering their fitness and lowering their yield. Both temporary and more long-lasting types need to be studied if we are to develop crops capable of facing ever-degrading agricultural conditions. Traditional approaches of forward and reverse genetics have proven (and are still proving) their value in the identification of specific candidates in the studied phenomenon. However techniques from the “omics” era have brought means of covering genome-wide analyses to not only target specific genes, but also to apprehend more integrated mechanisms as a whole. In this context, the combination of complementary approaches has been explored to further decipher the regulatory networks underlying long-term and short-term physiological changes allowing a plant to cope with drought stress and with uranium soil contamination. This work has also been the opportunity to develop new bioinformatic tools for the comparison of highly heterogeneous datasets. In particular, we have evaluated the possibility of using stochastic methods to incorporate pairwise distances calculations into existing analysis approaches for binary and weighted (soft thresholded) networks.

 

8

Abstract


In plants, rises in cytosolic Ca2+ concentration ([Ca2+]cyt) occur in response to both biotic and abiotic stimuli. These rises can, depending on the stimulus, display the form of a single transient or repetitive Ca2+ oscillations and are commonly designated as “Ca2+ signatures”. Generation and shaping of [Ca2+]cyt signatures depends on fine-tuning of Ca2+ influxes and effluxes occurring at both the plasma membrane (PM) and membranes of the different subcellular compartments. The opening of Ca2+-permeable influx channels in response to a stimulus will release Ca2+ into the cytosol and cause the generation of a Ca2+ spike, while activity of Ca2+ efflux transporters (H+-Ca2+ antiporters and Ca2+-ATPases) will return the [Ca2+]cyt to resting concentrations. In order to understand how the cytosolic Ca2+ dynamics are generated and shaped in different cell types, two issues need to be addressed: i) the study of how do organelles participate in these processes and ii) the possibility to perform single cell Ca2+ analyses in complex tissues and organs, in natural context and in close-to physiological conditions.

 

6

Abstract


In response to drought stress, many genes with various functions are induced at transcriptional level. Their gene products have been shown to function in drought stress tolerance and responses. There are complex regulatory networks in stress-responsive gene expression; ABA-dependent and ABA-independent. In one of the ABA-independent pathways, a cis-acting element (DRE/CRT) and its binding proteins, DREBs, are important regulatory factors in stress-responsive gene expression. In the ABA-dependent pathway, the ABRE cis-acting element and bZIP transcription factors (AREB/ABF) function as major regulatory factors. Genes for key enzymes involved in ABA biosynthesis and metabolism, and signal transduction pathways upstream of the AREB transcription factors in drought stress response have been identified. We are now interested not only in cellular signaling but also in intercellular signaling or long distance signaling in stress response. We have shown that ATP-binding cassette (ABC) transporter genes are involved in ABA transport in stress response. Based on genome analysis, we have recently identified sORFs encoding small peptides that are involved in drought stress response, and analyzed their functions in regulation of stress and ABA response. We have been developing a high-throughput phenotyping system for detailed temporal and spatial growth analysis under mild water stress conditions. We used the phenotyping system for the evaluation of water-use-efficiency of ABCG25 ox plants and compare it with wild-type Arabidopsis. Drought-inducible Arabidopsis genes were applied for molecular breeding of drought tolerant crops in collaboration with International crop research institutes. I will report the progress of evaluation of drought tolerance of transgenic lines in dry research fields.

 

10

Abstract


Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant ion channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. But what do we know about the molecular mechanism of plant excitability in general and the ion channel bases for the Venus flytrap action potential in detail? Charles Darwin over 100 years ago recognized that the Venus flytrap Dionaea muscipula living on nutrient poor soil is capturing animals. When small animals visit the trap surface and touch the trigger hairs the trap gets excited and after firing two action potentials closes. Trying to escape the encaged prey keeps on exciting the capture organ and thereby glands covering the inner trap surface trigger secretion of a digestive fluid. During prey decomposition the animal-derived nutrients are ingested. Although the concept of botanical carnivory is known since Darwin’s time, due to the entire lack of genomic information, the molecular processes providing for animal feeding remain still unknown. To bridge that gap, we sequenced the genome together with transcriptome expressed in different organs of Dionaea and assembled a backbone transcriptome of the carnivorous plant. Given that with Dionaea leaves only the bi-lobed tip but not the petiole develop into a sophisticated capture organ, we focused on trap genes that become active upon contact with the animal victim. Special attention we gave to trigger hairs and glands engaged with i) generation of the action potential, ii) secretion of hydrolases, and iii) uptake of nutrients extracted from the digested animal. Serving the latter function we spotted ion channels and transporter. Following expression of the Dionaea gland-expressed nutrient transporter genes in Xenopus oocytes, ion selective voltage changes and currents were recorded. Our studies indicate that Dionaea glands operate selective, high capacity channels and transporters to provide nutrients and osmotic potential while the feeding on a decomposing victim. During the seminar the molecular nature and mechanism of the hunting cycle of the most exciting green carnivore will be discussed.

 

9

Abstract


Salinity stress brings about serious negative impacts on the growth and productivity of plants. Detrimental effects of high salt concentrations are multifactorial, however, they are attributable to two major factors: “ion toxicity” that inhibits various metabolic reactions and “osmotic stress” that reduces water influx into roots. In this seminar, current results regarding the mechanisms to circumvent Na+ toxicity and water loss in salt-stressed rice (Oryza sativa) and barley (Hordeum vulgare) plants will be presented. As for the protection mechanism from Na+ toxicity, we investigated physiological and molecular functions of HKT1 transporters in rice (OsHKT1s). Characterization of oshkt1;5 mutant rice plants revealed that OsHKT1;5 mediates Na+ unloading from xylem in roots during salinity stress to prevent Na+ transfer to young leaf blades. Together with the results of the immuno-staining and 22Na+-imaging, our analyses also indicated novel functions of OsHKT1;5 in the removal of Na+ in phloem of basal nodes and xylem of leaf sheaths to protect leaf blades. Contribution of the OsHKT1;4 transporter-mediated Na+ exclusion in rice plants in the vegetative growth period will also be discussed. To dissect the response to osmotic stress, we analyzed the root hydraulic conductivity (Lpr) that is predominantly constituted by the activity of water channel proteins called aquaporins in salt-stressed barley plants. Lpr measurements of barley seedlings using the pressure chamber revealed a novel regulatory mechanism of water flow in response to high salt environments, which appears to function in “non-salt sensitive” cultivars but not in “salt-sensitive” cultivars of barley.

 

8

Abstract


Stomatal apparatus consists of a pair of kidney-shaped guard cells and plays a central role in gas exchange of angiosperms. Due to its essentiality in diverse physiological aspects, size of stomatal aperture is finely regulated against environmental and interior stimuli. Abscisic acid (ABA) is a major regulator that induces closure at the onset of drought stress and has drawn attention of plant physiologists for a long time. In 1990’s it was demonstrated that guard cells possess at least two classes of ABA receptors. Recently, it was suggested that ABA functions to prime signaling of other plant hormones. These suggest diverse function of ABA receptors in guard cells. In 2009 PYR/PYL/RCAR family proteins that is comprised of 14 members were identified as a class of ABA receptor. In this study we examined the involvement of several PYR/PYL/RCAR ABA receptors in (1) differential functions between closure induction and opening inhibition and (2) priming methyljasmonate-induced stomatal closure. Our study suggests that ABA receptors share diverse actions of ABA to control stomata.

 

6

Abstract


One-dimensional nanostructures are ideal building blocks for functional nanoscale assembly. Peptide based nanofibers have a high potential in building smart hierarchical structures due to their tunable structures at the single residue level and their ability to reconfigure themselves dynamically in response to environmental stimuli. We observed that silk-elastin-like peptide polymer (SELP) self-assembles into amyloid nanofibers on a mica substrate when nanomechanical force was applied as an external stimuli. Time lapse lateral force microscopy revealed that mechanical stretching of a single or multiple SELP molecules is a key molecular event for amyloid nucleation. The mechanically induced nucleation allows for positional and directional control of amyloid assembly in vitro, which we demonstrate by creating a single nanofiber at a predetermined site. At the single molecule level, DNA condensation by cationic peptides was investigated using optical tweezers. Force-extension curves showed characteristic force plateaus and hysteresis during stretching and relaxation cycles. Upon environmental changes such as concentrations, pH, and divalent cations, force profiles changed very dynamically indicating that mechanical properties of DNA:cationic peptide complex are regulated at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored DNA complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.

 

10

Abstract


Calcium functions in a plethora of crucial processes in eukaryotes, but how specificity of the second messenger Ca2+ is achieved remains incompletely understood. Plant guard cells that form stomatal pores for gas exchange provide a powerful system for in depth investigation of Ca2+-signaling mechanisms. Stomatal closing stimuli including the hormone abscisic acid (ABA) and elevated CO2 enhance (prime) cytoplasmic Ca2+-sensitivity. However, the underlying genes and mechanisms mediating Ca2+-sensitivity priming are unknown. We have now found that a mutant in calcium-activated protein kinases leads to disruption of ABA-activation of S-type anion channels and ABA-induced stomatal closing. [Ca2+]cyt-sensitivity is constitutively primed in a newly isolated mutant without ABA, revealing a mechanism that tightly controls specificity in Ca2+ signaling. We have also found how these proteins regulate Ca2+ sensitivity. Surprisingly, disruption of Ca2+-independent mechanisms abrogates Ca2+-activation of anion channels in guard cells, indicating an interdependence of the Ca2+-dependent and Ca2+-independent signaling pathways. These newly characterized mechanisms describe a signaling network explaining how specificity and robustness within Ca2+ signaling is achieved in a single cell type.

 

9

Abstract


Both intrinsic and extrinsic micro-environmental factors influence pathway function in individual cancer cells, which results in establishing tumor heterogeneity. We have been developing imaging methods to allow simultaneous quantification of RNAs and proteins at the single cell level. First, we established single molecule fluorescent in situ hybridization (smFISH) technology to quantify individual transcripts. Oncogene Her2 mRNA particle counts are closely related to DNA copy number data in a variety of breast cancer cells. The unexpected nuclear Her2 mRNA aggregates are resolved using super-resolution structural illumination microscopy (SR-SIM). Next, we established “immuno-smFISH,” combining immunocytochemistry and smFISH for the simultaneous co-imaging of protein and RNA, and applied it to time-lapse analyses of Her2 mRNA expression and phosphoAkt protein levels in Her2-positive breast cancer single cells treated with the HER-family tyrosine kinase inhibitor Lapatinib. Nuclear morphometries are also analyzed by measuring the size, intensity, aspect ratio, perimeter, roundness, and circularity of DAPI-stained nuclei, whose differences might cause expression level changes. Our imaging methods provide information about the association between transcription level, cellular localization, and protein expression in individual cells, and would be applied to pinpoint target cancer cells of aberrant signaling and subsequent end-point gene expression in human tumor biopsy samples and xenograft tissues.

 

8

Abstract


Osteoarthritis (OA) is a prevalent disease affecting majority of aged human, yet its effective treatment is currently absent. We have previously shown that Nkx3.2-mediated constitutive activation of NF-kB functions to maintain chondrocyte viability. Besides, Barx1 has been implicated for its role in development of gut, spleen and craniofacial skeletons, but its precise molecular function in chondrocyte has been poorly understood. Here we show that Barx1 can induce chondrocyte apoptosis by abrogating Nkx3.2-mediated NF-kB activation. Interestingly, we also observed remarkable decrease and increase in Nkx3.2 and Barx1 expression, respectively, both in mouse OA models and in human OA patients. Consistent with these, in vivo experiments employing intra-articular infection of Lentivirus modifying Nkx3.2 or Barx1 expression revealed that OA cartilage destruction can be suppressed by Nkx3.2, whereas Barx1 aggravates it. Further, either Nkx3.2 overexpression or Barx1 knockdown is capable of rendering regeneration of damaged cartilage in mouse DMM-OA model. Lastly, we demonstrate that OA progression is decelerated and accelerated in Nkx3.2 transgenic and knockout mice, respectively. Thus, these results indicate that Nkx3.2-Barx1 crosstalk plays a critical role in OA pathogenesis

 

6

Abstract


Adaptation to environmental stresses is critical for determining plant growth and productivity. Plant responses to environmental stresses are governed by complex molecular and biochemical signal transduction processes, which coordinately act to determine tolerance or sensitivity at the whole-plant level. To understand plant stress adaptation, genes that are required for stress tolerance have been isolated in our lab. Selected from the repertoire of genes studied, I will present work on the biological functions of several key stress defense protein players: Gigantea (GI), YUCCA6, and a WD40-repeat protein (HOS15).

 

10

Abstract


Recently, nanotechnology has been rapidly emerging in biomedical and biotechnological applications, including drug/gene delivery carriers, disease diagnosis, and cancer therapy. While organic bio-nanomaterials such as liposomes and biodegradable polymers are still playing key roles in nanomedicine because of their safety in the human body, nanotube shapes of materials are attractive vehicles for drug/gene delivery because of their hollow and porous structures and facile surface functionalization. The inner void can take up a large amount of drug, and the open ends of pores serve as gates that can control the release of drug/gene. The nanotube materials can be differentially functionalized between the inner and outer surfaces, which can provide a platform of multifunctionality into the nanoparticle. In addition, since hollow structures can isolate and protect drug/gene payload in the inner voids from the environment, they can transport the payloads safely into target without enzymatic and hydrolytic degradation of biological payload or aggregation of nanomaterials caused by many hydrophobic drug molecules during the delivery. In this presentation, the recent developments of nanotubular structured materials are discussed, which will show a large potential at the nano-bio interfaces for nanomedicine applications including controlled drug delivery, biosensors, bioseparations, and catalytic reactions in nanoscale containers. Further importantly, there is also a growing concern that unusual and unexpected toxicity may arise from unusual complex shapes, higher reactivity, and accessibility to cells, which man-made materials possess in the nanoscale. Therefore, it is essential to establish a new toxicology study that deviates from or modifies conventional methods. General toxicity and biodistribution of the nanotubes will be discussed. At the end, conductive polymer nanotube patch will be shortly discussed as a transdermal drug delivery system.

 

9

Abstract


One-dimensional nanostructures are ideal building blocks for functional nanoscale assembly. Peptide based nanofibers have a high potential in building smart hierarchical structures due to their tunable structures at the single residue level and their ability to reconfigure themselves in response to environmental stimuli. As an external stimulus, we applied mechanical force using atomic force microscopy (AFM). Under nanomechanical force silk-elastin-like peptide polymer (SELP) self-assembles into amyloid nanofibers through dynamic conformational changes on a mica substrate. Time lapse lateral force microscopy revealed that mechanical stretching of a single or multiple SELP molecules is a key molecular event for amyloid nucleation. The mechanically induced nucleation allows for positional and directional control of amyloid assembly in vitro, which we demonstrate by creating single nanofibers at predetermined sites. At the single molecule level, DNA condensation by cationic peptides was investigated using optical tweezers. Force-extension curves showed characteristic force plateaus and hysteresis during stretching and relaxation cycles. Upon environmental changes such as concentrations, pH and divalent cations, force profiles changed significantly indicating that mechanical properties of DNA:cationic peptide complex are dynamically regulated. Implications from our single molecule studies for designing efficient carriers for gene therapy will be discussed.

 

8

Abstract


As sedentary organisms, higher plants have evolved unique strategies in cell-to-cell and long-distance communications, which allowed them to orchestrate physiological and developmental processes between cells, tissues, and organs that are remotely located. Here, plasmodesmata play the essential role not only in mediating intercellular communication but also in coupling the local communication with the long-distance signaling. As highly dynamic channels, plasmodesmata undergo various types of spatiotemporal regulations in response to myriad extracellular challenges as well as internal signals throughout the plant life. However, molecular and genetic components underlying the crosstalk between plasmodesmal control and cellular signaling pathways are not fully understood. One of the main research focuses of my laboratory is on unraveling how various signaling pathways are integrated into the regulation of plasmodesmata. In my talk, I will walk the audience through our key studies illustrating how hormonal signaling pathways are interlinked with the regulation of plasmodesmata-mediated cell-to-cell coupling. I will present novel regulatory circuits that link salicylic acid and auxin signaling pathways to the control of plasmodesmata. I will also discuss a comprehensive genetic framework underlying the control of plasmodesmal permeability in the context of plant responses to both biotic and abiotic stresses as well as the potential impacts of engineering plasmodesmal control in improving plant health and overall fitness.