The tumor suppressor Lgl1 regulates front-rear polarity of migrating cells

Cell migration is a highly integrated, multistep process that plays an important role in physiological and pathological processes. The migrating cell is highly polarized, with complex regulatory pathways that integrate its component processes spatially and temporally.¹ The Drosophila tumor suppressor, Lethal (2) giant larvae (Lgl), regulates apical-basal polarity in epithelia and asymmetric cell division.² But little is known about the role of Lgl in establishing cell polarity in migrating cells. Recently, we showed that the mammalian Lgl1 interacts directly with non-muscle myosin IIA (NMIIA), inhibiting its ability to assemble into filaments in vitro.³ Lgl1 also regulates the cellular localization of NMIIA, the maturation of focal adhesions, and cell migration.³ We further showed that phosphorylation of Lgl1 by aPKCζ prevents its interaction with NMIIA and is important for Lgl1 and acto-NMII cytoskeleton cellular organization.⁴ Lgl is a critical downstream target of the Par6-aPKC cell polarity complex; we showed that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6-aPKCζ in different cellular compartments.⁴ We further showed that aPKCζ and NMIIA compete to bind directly to Lgl1 through the same domain. These data provide new insights into the role of Lgl1, NMIIA, and Par6-aPKCζ in establishing front-rear polarity in migrating cells. In this commentary, I discuss the role of Lgl1 in the regulation of the acto-NMII cytoskeleton and its regulation by the Par6-aPKCζ polarity complex, and how Lgl1 activity may contribute to the establishment of front-rear polarity in migrating cells.     

miRNA-182 and the regulation of the glioblastoma phenotype - toward miRNA-based precision therapeutics

Glioblastoma (GBM) is an incurable cancer, with survival rates of just 14-16 months after diagnosis.¹ Functional genomics have identified numerous genetic events involved in GBM development. One of these, the deregulation of microRNAs (miRNAs), has been attracting increasing attention due to the multiple biologic processes that individual miRNAs influence. Our group has been studying the role of miR-182 in GBM progression, therapy resistance, and its potential as GBM therapeutic. Oncogenomic analyses revealed that miR-182 is the only miRNA, out of 470 miRNAs profiled by The Cancer Genome Atlas (TCGA) program, which is associated with favorable patient prognosis, neuro-developmental context, temozolomide (TMZ) susceptibility, and most significantly expressed in the least aggressive oligoneural subclass of GBM. miR-182 sensitized glioma cells to TMZ-induced apoptosis, promoted glioma initiating cell (GIC) differentiation, and reduced tumor cell proliferation via knockdown of Bcl2L12, c-Met and HIF2A.² To deliver miR-182 to intracranial gliomas, we have characterized Spherical Nucleic Acids covalently functionalized with miR-182 sequences (182-SNAs). Upon systemic administration, 182-SNAs crossed the blood-brain/blood-tumor barrier (BBB/BTB), reduced tumor burden, and increased animal subject survival.^2-4 Thus, miR-182-based SNAs represent a tool for systemic delivery of miRNAs and a novel approach for the precision treatment of malignant brain cancers.     

Sex chromosome evolution: life,death and repetitive DNA

Dimorphic sex chromosomes create problems. Males of many species, including Drosophila, are heterogametic, with dissimilar X and Y chromosomes. The essential process of dosage compensation modulates the expression of X-linked genes in one sex to maintain a constant ratio of X to autosomal expression. This involves the regulation of hundreds of dissimilar genes whose only shared property is chromosomal address. Drosophila males dosage compensate by up regulating X-linked genes 2 fold. This is achieved by the Male Specific Lethal (MSL) complex, which is recruited to genes on the X chromosome and modifies chromatin to increase expression. How the MSL complex is restricted to X-linked genes remains unknown. Recent studies of sex chromosome evolution have identified a central role for 2 types of repetitive elements in X recognition. Helitrons carrying sites that recruit the MSL complex have expanded across the X chromosome in at least one Drosophila species.¹ Our laboratory found that siRNA from an X-linked satellite repeat promotes X recognition by a yet unknown mechanism.² The recurring adoption of repetitive elements as X-identify elements suggests that the large and mysterious fraction of the genome called “junk” DNA is actually instrumental in the evolution of sex chromosomes.     

Multiwalled carbon nanotubes enhance human bone marrow mesenchymal stem cells’ spreading but delay their proliferation in the direction of differentiation acceleration

Investigating the ability of films of pristine (purified, without any functionalization) multiwalled carbon nanotubes (MWCNTs) to influence human bone marrow mesenchymal stem cells’ (hBMSCs) proliferation, morphology, and differentiation into osteoblasts, we concluded to the following: A. MWCNTs delay the proliferation of hBMSCs but increase their differentiation. The enhancement of the differentiation markers could be a result of decreased proliferation and maturation of the extracellular matrix B. Cell spread on MWCNTs toward a polygonal shape with many thin filopodia to attach to the surfaces. Spreading may be critical in supporting osteogenic differentiation in pre-osteoblastic progenitors, being related with cytoskeletal tension. C. hBMSCs prefer MWCNTs than tissue plastic to attach and grow, being non-toxic to these cells. MWCNTs can be regarded as osteoinductive biomaterial topographies for bone regenerative engineering.Cellular interaction with substrate and neighboring cells plays a critical role in osteoblast survival, proliferation, differentiation as well as bone remodeling. Regulated biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration in the stem cell niche. Multiwalled carbon nanotubes (MWCNTs) represent a nanomaterial that has won enormous popularity in nanotechnology, exhibiting extraordinary physicochemical properties and supporting the growth of different kinds of cells.^1-3Simultaneous enhancement of osteoblast cells’ proliferation and differentiation,^4,5 decrease of proliferation rates along with decreased differentiation⁶ or increased differentiation accompanied with decreased proliferation⁷ have been reported. Contradictory results concerning osteoblast cell adhesion, and morphology have also been reported. Osteoblast cell lines on CNTs have been found to elongate but not widen or displayed a spindle-shaped morphology.^8,9 Spreading and surface area covered were reduced.^8-10 On the contrary, Tutak et al.⁷ reported robust spreading on medium roughness CNTs networks.This variable behavior on CNTs is probably due to the various cell types used in these works. It is reported that primary human marrow stromal cells and cell lines use substantially different mechanisms to regulate adhesion and spreading on the substrate.¹¹In a recent work of ours, published in Annals of Biomedical Engineering,¹² it was found that MWCNTs can create an osteogenic environment for human bone marrow mesenchymal stem cells (hBMSCs), even without addition of exogenous factors, representing a suitable reinforcement for bone tissue engineering scaffolds.In the following, we will highlight and discuss some aspects of this work''s results, in the context of literature findings, and provide additional material in order to elucidate issues on the influence of MWCNTs on hBMSCs’ proliferation, morphology, and differentiation into osteoblasts.     

Photoperiodic control of sugar release during the floral transition: What is the role of sugars in the florigenic signal?

Florigen is a mobile signal released by the leaves that reaching the shoot apical meristem (SAM), changes its developmental program from vegetative to reproductive. The protein FLOWERING LOCUS T (FT) constitutes an important element of the florigen, but other components such as sugars, have been also proposed to be part of this signal.^1-5 We have studied the accumulation and composition of starch during the floral transition in Arabidopsis thaliana in order to understand the role of carbon mobilization in this process. In A. thaliana and Antirrhinum majus the gene coding for the Granule-Bound Starch Synthase (GBSS) is regulated by the circadian clock^6,7 while in the green alga Chlamydomonas reinhardtii the homolog gene CrGBSS is controlled by photoperiod and circadian signals.^8,9 In a recent paper¹⁰ we described the role of the central photoperiodic factor CONSTANS (CO) in the regulation of GBSS expression in Arabidopsis. This regulation is in the basis of the change in the balance between starch and free sugars observed during the floral transition. We propose that this regulation may contribute to the florigenic signal and to the increase in sugar transport required during the flowering process.     

Comment on: HMGB1-dependent and -independent autophagy

HMGB1 (high mobility group box 1), a ubiquitously expressed DNA-binding nucleoprotein, has not only been attributed with important functions in the regulation of gene expression but is thought to function as an important damage-associated molecular pattern in the extracellular space. Recently, conditional Hmgb1 deletion strategies have been employed to overcome the perinatal mortality of global Hmgb1 deletion and to understand HMGB1 functions under disease conditions. From these studies, it has become evident that HMGB1 is not required for normal organ function. However, the different conditional ablation strategies have yielded contradictory results in some disease models. With nearly complete recombination in all transgenic mouse models, the main reason for opposite results is likely to lie within different targeting strategies. In summary, different targeting strategies need to be taken into account when interpreting HMGB1 functions, and further efforts need to be undertaken to compare these models side by side.We appreciate the thoughtful analysis on HMGB1-dependent and -independent autophagy by Sun and Tang.¹ However, we disagree with several statements in this review. Sun and Tang write “Mice with hepatocyte-specific deletion of Hmgb1 from Robert Schwabe''s lab are not complete conditional knockout mice; the protein level of HMGB1 in the liver is decreased by about 70%,” as well as “a major difference between Robert Schwabe''s engineered HMGB1 mice and other groups is the tissue-level expression of HMGB1 after knockout.”¹We would like to point out that livers are not solely composed of hepatocytes and that albumin-Cre mediated deletion of target genes in the liver cannot result in complete loss of hepatic mRNA or protein of target genes due to the presence of unrecombined nonparenchymal cells, unless the target gene is exclusively expressed in hepatocytes and/or cholangiocytes. The reduction of hepatic HMGB1 in our studies—reaching 90% and 72% at the mRNA and protein level, respectively—is precisely at the expected level for this conditional strategy, and similar to other studies that employed albumin-Cre for hepatocyte-specific knockout of other target genes.^2-5 Hepatocytes account only for approximately 52% of cells in the liver, with other cell types including Kupffer cells (∼18% of liver cells), hepatic stellate cells (˜8% of liver cells), endothelial cells (∼22% cells of liver cells) and cholangiocytes (<1 % of liver cells) contributing to the remainder.⁶ Accordingly, albumin-Cre-mediated reduction of mRNA and protein levels of target genes (i.e., Hmgb1 and HMGB1 in our study) in the liver cannot exceed the amount of mRNA and protein expressed by hepatocytes and cholangiocytes (which is typically about 70–90%,^2-5 due to higher mRNA and protein levels in hepatocytes than in other hepatic cell types). The high efficacy of our conditional approach is best demonstrated by almost complete loss of HMGB1 expression in the hepatocellular compartment of albumin-Cre mice—as evidenced by loss of HMGB1 expression in all HNF4α-positive cells and in isolated primary hepatocytes—whereas HMGB1 expression is retained in nonparenchymal cells, as demonstrated by costaining for Kupffer cell marker F4/80, endothelial cell marker endomucin, and hepatic stellate cell marker desmin.^7,8 The nearly perfect recombination rate in our mice was further confirmed by experiments that employed Mx1Cre for Hmgb1 deletion, which resulted in almost complete loss of hepatic Hmgb1 mRNA and HMGB1 protein.^7,8 Moreover, our transgenic mice show early postnatal mortality when bred with a germline Cre deleter,⁷ thus reproducing the phenotype of the global HMGB1 knockout.⁹In summary, our transgenic mouse model results in nearly perfect recombination efficiency with virtually complete loss of Hmgb1 mRNA and HMGB1 protein in all targeted cell types, and constitutes a valid tool for the assessment of HMGB1 functions in vivo. Findings from this model need to be taken into account for proper interpretation of the role of HMGB1 in the normal and diseased liver, and cannot be interpreted as a result of incomplete deletion efficiency. Hence, differences in targeting strategies (exons 2–4 by our approach, exons 2–3 in mice from Tang and colleagues) are likely to explain opposite findings, e.g. improvement of ischemia-reperfusion injury in our hands, but aggravation of liver damage in the study by Huang et al.^8,10 Further analysis needs to be performed to determine whether ablation of exons 2–3 versus exons 2–4 leads to complete loss of HMGB1 function.     

Endosomal lipid flippases and their related diseases

 In eukaryotic cell membranes, phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer. For example, the extracellular leaflet of the plasma membrane (PM) is enriched with phosphatidylcholine and sphingomyelin, while the cytosolic leaflet of the PM is enriched with phosphatidylserine (PS) and phosphatidylethanolamine. The asymmetric distribution of PS in the PM is crucial for cell life, since PS in the extracellular leaflet of the PM is recognized as an “eat-me” signal by phagocytes. Inside the cells, a high PS concentration in the cytosolic leaflet of the PM is essential to facilitate various cellular events through the recruitment of signaling molecules such as protein kinase C and Akt.The asymmetric distribution of phospholipids is believed to be generated in part by phospholipid translocases, or “flippases.” The proteins responsible for flippase activity are type IV P-type ATPase (P₄-ATPases). P-type ATPases are multispan transmembrane pumps that use ATP hydrolysis as an energy source. P-type ATPases undergo autophosphorylation of a conserved aspartate residue during the catalytic cycle, hence the designation of “P”-type. P₄-ATPases are unique in that they are phospholipid transporters whereas other types of P-type ATPases are ion transporters.The human genome contains 14 P₄-ATPases, and mutations in some P₄-ATPases cause inherited genetic diseases. For example, mutations in ATP8B1 are associated with intrahepatic cholestasis and also cause hearing loss. Mutations in ATP8A2 are associated with a severe neurological disorder characterized by cerebellar ataxia, mental retardation, and dysequilibrium syndrome (CAMRQ).¹ Despite the accumulating evidence highlighting the physiological importance of P₄-ATPases, how dysfunction of P₄-ATPases causes diseases is poorly understood.In a recent study, we revealed the cellular function of the P₄-ATPase, ATP8A1.² ATP8A1 localizes at recycling endosomes (REs), an organelle that functions in recycling transport of internalized molecules back to the PM, thus defining the amount of proteins at the PM. PS is most concentrated in REs among intracellular organelles and we roughly estimated that 70 and 30% of PS are localized in the cytosolic and the luminal leaflets of RE membranes, respectively.² ATP8A1 generates the asymmetric transbilayer distribution of PS at REs. The knockdown of ATP8A1 halted recycling traffic from REs to the PM. At the mechanistic level, we found that EHD1, a dynamin-like membrane fission protein, lost its RE localization upon ATP8A1 knockdown and EHD1 knockdown also blocked recycling traffic. EHD1 bound PS in vitro and lost its membrane localization in cells that are defective in PS synthesis. Thus, we propose that PS flipping by ATP8A1 recruits EHD1 to RE membranes, thereby regulating the recycling traffic from REs to the PM (Fig. 1). Open in a separate windowFigure 1.Model of flippase-related diseases. Under normal conditions, flippases (e.g., ATP8A1 and ATP8A2) translocate PS to the cytosolic leaflet of RE membranes. PS recruits EHD1 to REs, and then EHD1 participates in the fission of RE membranes to generate transport vesicles that contain cell surface receptors. In flippase-dysfunctional situations, PS levels in the cytosolic leaflet of REs would be low. This impairs the PS/EHD1/membrane traffic axis, leading to a lower abundance of cell surface receptors that are critical for responses to extracellular ligands.ATP8A2 is a tissue-specific ATP8A1 paralogue. We found that a CAMRQ-causative mutation of ATP8A2 (I376M) lost its ATPase and flippase activity toward PS. ATP8A2 is not endogenously expressed in COS-1 cells. Interestingly, the phenotype that was caused by the loss of ATP8A1 in COS-1 cells, was restored by the exogenous expression of wild-type ATP8A2, but not I376M mutant ATP8A2. Moreover, cortical neurons prepared from ATP8A2 knockout mice showed lower abundance of transferrin receptors at the PM. Together, these results indicate that ATP8A2 functions in the recycling traffic in neurons, and that CAMRQ may result from the defect in recycling of important neurological receptor proteins from REs to the PM. One possible candidate protein is very low-density lipoprotein receptor (VLDLR). VLDLR is a receptor for reelin, an extracellular protein that guides neuronal migration in the cerebral cortex and cerebellum. VLDLR circulates between the PM and endosomes (possibly REs) by recycling traffic.³ Significantly, mutations in VLDLR gene are also linked to CAMRQ.^4,5 Therefore, impaired recycling traffic of VLDLR to the PM in neurons with dysfunctional ATP8A2 (I376M) may cause lower expression of VLDLR at the PM, leading to reduced reelin signaling, abnormal neuronal development, and neurological disorder.dATP8B, a P₄-ATPase in Drosophila melanogaster was recently reported to cause an impaired response to cVA pheromone (a sex-specific social cue) and mislocalization of the pheromone receptor in cVA-sensing neurons.⁶ The impaired response to the pheromone in dATP8B mutant was rescued by expressing bovine ATP8A2. Therefore, from insects to mammals, phospholipid flippases may define the localization of neuronal receptors to the PM.Lastly, our findings may explain the phenotype of ATP8A1 knockout mice.⁷ ATP8A1 knockout mice are vital but show deficiencies in hippocampus-dependent learning. Hippocampus-dependent learning involves modification of synaptic strength, and one cellular mechanism for tuning synaptic strength is long-term potentiation (LTP). During LTP, REs supply glutamate receptors to the post-synaptic membrane.⁸ Therefore, we speculate that impaired glutamate receptor traffic from REs to the post-synaptic membranes during LTP may underlie the deficiency in learning in ATP8A1 knockout mice. In agreement with this hypothesis, the dominant-negative form of EHD1 inhibits glutamate receptor traffic during LTP.⁸Many P₄-ATPases are expressed in the Golgi/endosomes and the PM. We expect that they contribute redundantly to the phospholipid asymmetry and membrane traffic through organelles. Simultaneous ablations of P₄-ATPases may dissect their roles and will give more insight into flippase-mediated cellular processes and -related diseases.     

Synergistic effect of antioxidant system and osmolyte in hydrogen sulfide and salicylic acid crosstalk-induced heat tolerance in maize (Zea mays L.) seedlings

Salicylic acid (SA), is a plant hormone with multifunction that is involved in plant growth, development and the acquisition of stress tolerance. Hydrogen sulfide (H₂S) is emerging similar functions, but crosstalk between SA and H₂S in the acquisition of heat tolerance is not clear. Our recent study firstly reported that SA treatment enhanced the activity of L-cysteine desulfhydrase (L-DES), a key enzyme in H₂S biosynthesis, followed by induced endogenous H₂S accumulation, which in turn improved the heat tolerance of maize seedlings.¹ In addition, NaHS, a H₂S donor, enhanced SA-induced heat tolerance, while its biosynthesis inhibitor DL-propargylglycine (PAG) and scavenger hydroxylamine (HT) weakened SA-induced heat tolerance. Also, NaHS had no significant effect on SA accumulation and its biosynthesis enzymes phenylalanine ammonia lyase (PAL) and benzoic-acid-2-hydroxylase (BA2H) activities, as well as significant difference was not observed in NaHS-induced heat tolerance of maize seedlings by SA biosynthesis inhibitors paclobutrazol (PAC) and 2-aminoindan-2-phosph- onic acid (AIP) treatment.¹ Further study displayed that SA induced osmolytes (proline, betaine and trehalose) accumulation and enhancement in activity of antioxidant system in maize seedlings. These results showed that antioxidant system and osmolyte play a synergistic role in SA and H₂S crosstalk-induced heat tolerance of maize seedlings.     

Analysis of LuPME3, a pectin methylesterase from Linum usitatissimum,revealed a variability in PME proteolytic maturation

Pectin methylesterase (PME) catalyzes the de-methylesterification of pectin in plant cell walls during cell elongation.¹ Pectins are mainly composed of α(1, 4)-D-galacturonosyl acid units that are synthesized in a methylesterified form in the Golgi apparatus to prevent any interaction with Ca²⁺ ions during their intracellular transport.² The highly methylesterified pectins are then secreted into the apoplasm³ and subsequently de-methylesterified in muro by PMEs. This can either induce the formation of pectin gels through the Ca²⁺ crosslinking of neighboring non-methylesterified chains or create substrates for pectin-degrading enzymes such as polygalacturonases and pectate lyases for the initiation of cell wall loosening.⁴ PMEs belong to a large multigene family. Sixty­six PME-related genes are predicted in the Arabidopsis genome.¹ Among them, we have recently shown that AtPME3 (At3g14310), a major basic PME isoform in A. thaliana, is ubiquitously expressed in vascular tissues and play a role in adventitious rooting.⁵ In flax (Linum usitatissimum), three genes encoding PMEs have been sequenced so far, including LuPME3, the ortholog of AtPME3. Analysis of the LuPME3 isoform brings new insights into the processing of these proteins.     

A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

We show that a splice variant–derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation.Cyclins are a conserved family of proteins that play a central role in eukaryotic cell division cycle progression, as regulatory subunits of cyclin dependent kinases (CDKs, whose catalytic subunits are homologues of the fission yeast cdc2 protein).¹ CDKs are downstream targets of convergent cascades of regulations at critical points of the cell cycle. M-phase–promoting factor (MPF, formerly maturation promoting factor, reference 21), the factor responsible for M-phase entry and progression in mitosis, has been purified three times by biochemical means (7, 19, 36). MPF from starfish, Xenopus, and carp oocytes has been found to be a heterodimer composed of one molecule of cdc2 and one molecule of cyclin B (CB). B type cyclins are archetypal mitotic cyclins, evolutively and functionally related to fission yeast cdc13p. Among CDKs, the regulation of MPF is by far the best understood today. Cyclin B is required for activity, as well for activation and for inhibition of MPF. The cdc2 monomer has never been found active. Its activation is conferred by the CAK-dependent T161-phosphorylation that requires cyclin B association (4, 28, 33). Inhibition of MPF during S- and G2-phases and also by the DNA replication checkpoint mechanism is achieved by wee1-catalyzed phosphorylation of the tyrosine 15 in cyclin B–bound molecules of cdc2 (9, 22). Cyclin B is also likely required for activation of the protein phosphatase cdc25p that specifically dephosphorylates tyrosine 15 and allows MPF amplification and entry into mitosis (5, 37). Finally, targeted proteolysis of cyclin B by an ubiquitin-dependent pathway is the mechanism by which MPF is inactivated and the cell returns to interphase (8). Therefore, the major part of MPF regulation is accounted for by cyclin B synthesis and proteolysis. This was emphasized in simplified early embryogenesis cycles that are composed of a succession of M- and S-phases without intervening G-phases. Cycles in acellular Xenopus egg extracts are driven by MPF as a basic oscillator, whose periodic activity is scheduled strictly by oscillating abundance of cyclin B (24). Accordingly, during the cleavage period of Xenopus embryogenesis, cdc2 tyrosine 15 is never found phosphorylated (3) and checkpoint mechanisms are downregulated.Site-directed mutagenesis as well as protein crystallization have allowed the mapping of some sequences in cyclins involved in these regulations. Crystal structure of the homologous dimer cdk2–cyclin A showed that the cyclin interacts with the cdk via sequences distributed along the so-called cyclin box, a sequence well conserved among all cyclins (14). In the NH₂ terminus of mitotic cyclins A and B, a destruction box is required to allow ubiquitination of the protein and its targeted proteolysis in anaphase (8). Mutants that are deleted for this box are stable in mitosis, and their overexpression triggers mitotic arrest. Also in the NH₂-terminal region of B type cyclins, a cytoplasmic retention signal (CRS) is presumed to account for differential early prophase localization of nuclear cyclin A and cytoplasmic cyclin B (27). A chimeric cyclin A with the first amino acids of cyclin B remains cytoplasmic until early prophase. Further on, at the beginning of the cyclin box, conserved amino acids in the P-box are thought to be involved in the specific activation of cdc2 by cdc25 (37). Finally, two reports showed that a short COOH-terminal deletion of recombinant cyclins A or B abolished the binding to cdc2 (17, 34), although this region was not found to be directly involved in the physical interaction between cyclin A and cdk2 (14).Here we show that such a COOH-terminal truncation, which removes universally conserved amino acids, is naturally realized in a splice variant of sea urchin cyclin B. Moreover, immunofluorescence experiments suggest this splice variant plays a role in embryogenesis and behaves like a marker of cell lineages in postcleavage embryos.     

Irrepressible,truncated Auxin Response Factors: Natural roles and applications in dissecting auxin gene regulation pathways

The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport.^1,2 We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ),³ which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP’s role in vascular patterning, a previously characterized auxin dependent process.^4,5 Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD⁺ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD⁺ turnover, which makes NAD⁺ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD⁺ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD⁺ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.The cyanoguanidinopyridine GMX1778 (previously known as CHS828) is the active form of the prodrug GMX1777 and has potent antitumor activity in vitro and in vivo against cell lines derived from several different tumor origins (11). The antitumor activity of GMX1778 has been widely studied since its discovery (1, 11, 19-21, 24), but positive identification of the molecular target and the mechanism of action of GMX1778 has been elusive. Here, we demonstrate that GMX1778 exerts its antitumor activity via its potent and selective antagonism of NAD⁺ biosynthesis. GMX1777 is currently being assessed in phase I clinical trials for treatment of patients with refractory solid tumors.The pyridine nucleotide NAD⁺ plays a major role in the regulation of several essential cellular processes (7, 22, 25, 38). In addition to being a biochemical cofactor for enzymatic redox reactions involved in cellular metabolism, including ATP production, NAD⁺ is important in diverse cellular pathways responsible for calcium homeostasis (17), gene regulation (5), longevity (18), genomic integrity (33), and apoptosis (36). Cancer cells exhibit a significant dependence on NAD⁺ for support of the high levels of ATP production necessary for rapid cell proliferation. They also consume large amounts of this cofactor via reactions that utilize poly(ADP) ribosylation, including DNA repair pathways (10, 37, 39).In eukaryotes, the biosynthesis of NAD⁺ occurs via two biochemical pathways: the de novo pathway, in which NAD⁺ synthesis occurs through the metabolism of l-tryptophan via the kynurenine pathway, and the salvage pathway. The NAD⁺ salvage pathway can use either nicotinamide (niacinamide) (NM) or nicotinic acid (niacin) (NA) (via the Preiss-Handler pathway) as a substrate for NAD⁺ production. Saccharomyces cerevisiae species predominantly use NA as the substrate for NAD⁺ biosynthesis, through the deamidation of NM by the nicotinamidase PNC1 (25). However, mammalian cells do not express a nicotinamidase enzyme and use NM as the preferred substrate for the NAD⁺ salvage pathway. The mammalian NAD⁺ biosynthesis salvage pathway using NM is composed of NA phosphoribosyltransferase (NAMPT), which is the rate-limiting and penultimate enzyme that catalyzes the phosphoribosylation of NM to produce nicotinamide mononucleotide (NMN) (27, 29). NMN is subsequently converted to NAD⁺ by NMN adenyltransferases (NMNAT). The gene encoding NAMPT was originally identified as encoding a cytokine named pre-B-cell colony-enhancing factor (PBEF1) (30). NAMPT was also identified as a proposed circulating adipokine named visfatin (thought to be secreted by fat cells) and was suggested to function as an insulin mimetic; however, this role of NAMPT currently remains controversial (8). In mice, NAMPT has been shown to act as a systemic NAD⁺ biosynthetic enzyme that regulates insulin secretion from β cells (28). The molecular structure of NAMPT from human (15), rat (16) and mouse (35) tissue, containing either NMN or the inhibitor APO866, have been determined by X-ray crystallography. These structures revealed that NAMPT is a dimeric type II phosphoribosyltransferase.Here, we report that the anticancer compound GMX1778 is a specific inhibitor of NAMPT in vivo and in vitro and is itself a substrate for the enzyme. Phosphoribosylated GMX1778 inhibits NAMPT as potently as GMX1778 but is preferentially retained within cells. Finally, we have identified a novel anticancer strategy utilizing NA rescue of GMX1778 cytotoxicity to increase the therapeutic index of GMX1777 activity in tumors that are deficient in NA phosphoribosyltransferase 1 (NAPRT1).     

TRPM2 channel deficiency prevents delayed cytosolic Zn2+ accumulation and CA1 pyramidal neuronal death after transient global ischemia

Transient ischemia is a leading cause of cognitive dysfunction. Postischemic ROS generation and an increase in the cytosolic Zn²⁺ level ([Zn²⁺]_c) are critical in delayed CA1 pyramidal neuronal death, but the underlying mechanisms are not fully understood. Here we investigated the role of ROS-sensitive TRPM2 (transient receptor potential melastatin-related 2) channel. Using in vivo and in vitro models of ischemia–reperfusion, we showed that genetic knockout of TRPM2 strongly prohibited the delayed increase in the [Zn²⁺]_c, ROS generation, CA1 pyramidal neuronal death and postischemic memory impairment. Time-lapse imaging revealed that TRPM2 deficiency had no effect on the ischemia-induced increase in the [Zn²⁺]_c but abolished the cytosolic Zn²⁺ accumulation during reperfusion as well as ROS-elicited increases in the [Zn²⁺]_c. These results provide the first evidence to show a critical role for TRPM2 channel activation during reperfusion in the delayed increase in the [Zn²⁺]_c and CA1 pyramidal neuronal death and identify TRPM2 as a key molecule signaling ROS generation to postischemic brain injury.Transient ischemia is a major cause of chronic neurological disabilities including memory impairment and cognitive dysfunctions in stroke survivors.^{1, 2} The underlying mechanisms are complicated and multiple, and remain not fully understood.³ It is well documented in rodents, non-human primates and humans that pyramidal neurons in the CA1 region of the hippocampus are particularly vulnerable and these neurons are demised after transient ischemia, commonly referred to as the delayed neuronal death.⁴ Studies using in vitro and in vivo models of transient ischemia have demonstrated that an increase in the [Zn²⁺]_c or cytosolic Zn²⁺ accumulation is a critical factor.^{5, 6, 7, 8, 9, 10, 11} There is evidence supporting a role for ischemia-evoked release of vesicular Zn²⁺ at glutamatergic presynaptic terminals and subsequent entry into postsynaptic neurons via GluA2-lacking AMPA subtype glutamate receptors (AMPARs) to raise the [Zn²⁺]_c.^{12, 13, 14, 15, 16} Upon reperfusion, while glutamate release returns to the preischemia level,¹⁷ Zn²⁺ can activate diverse ROS-generating machineries to generate excessive ROS as oxygen becomes available, which in turn elicits further Zn²⁺ accumulation during reperfusion.^{18, 19} ROS generation and cytosolic Zn²⁺ accumulation have a critical role in driving delayed CA1 pyramidal neuronal death,^{7, 12, 20, 21, 22} but the molecular mechanisms underlying such a vicious positive feedback during reperfusion remain poorly understood.Transient receptor potential melastatin-related 2 (TRPM2) forms non-selective cationic channels; their sensitivity to activation by ROS via a mechanism generating the channel activator ADP-ribose (ADPR) confers diverse cell types including hippocampal neurons with susceptibility to ROS-induced cell death, and thus TRPM2 acts as an important signaling molecule mediating ROS-induced adversities such as neurodegeneration.^{23, 24, 25, 26} Emergent evidence indeed supports the involvement of TRPM2 in transient ischemia-induced CA1 pyramidal neuronal death.^{27, 28, 29, 30} This has been attributed to the modulation of NMDA receptor-mediated signaling; despite that ROS-induced activation of the TRPM2 channels results in no change in the excitability of neurons from the wild-type (WT) mice, TRPM2 deficiency appeared to favor prosurvival synaptic Glu2A expression and inhibit prodeath extrasynaptic GluN2B expression.³⁰ A recent study suggests that TRPM2 activation results in extracellular Zn²⁺ influx to elevate the [Zn²⁺]_c.³¹ The present study, using TRPM2-deficient mice in conjunction with in vivo and in vitro models of transient global ischemia, provides compelling evidence to show ROS-induced TRPM2 activation during reperfusion as a crucial mechanism determining the delayed cytosolic Zn²⁺ accumulation, CA1 neuronal death and postischemic memory impairment.     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.