Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

HERG channel and cancer: A mechanistic review of carcinogenic processes and therapeutic potential.

The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.

Synthesis and cytotoxic activities of novel copper and silver complexes of 1,3-diaryltriazene-substituted sulfonamides.

In this study, a series of 10 novel copper (II) and silver complexes of 1,3-diaryltriazene-substituted sulfonamides was synthesised. All the synthesised ligands and their metal complexes were assessed for in vitro cytotoxicity against human colorectal adenocarcinoma (DLD-1), cervix carcinoma (HeLa), breast adenocarcinoma (MDA-MB-231), colon adenocarcinoma (HT-29), endometrial adenocarcinoma (ECC-1), prostate cancer (DU-145 and PC-3), normal embryonic kidney (HEK-293), normal prostate epithelium (PNT-1A), and normal retinal pigment epithelium (ARPE-19) cells. Most of the metal complexes from the series showed to be more active against all cancerous cells than the uncomplexed 1,3-diaryltriazene-substituted sulfonamides, and lower cytotoxic effects observed on normal cells. Most of the Cu (II) and Ag (I) metal complexes from the presented series showed high cytotoxic activity against HeLa cells with IC50 values ranging from 2.08 to >300 µM. Specifically, compound L3-Ag showed one of the highest cytotoxicity against all cancer cell lines with IC50 values between 3.30 to 16.18 µM among other tested compounds.

Microtubules play a role in trafficking prevacuolar compartments to vacuoles in tobacco pollen tubes.

Fine regulation of exocytosis and endocytosis plays a basic role in pollen tube growth. Excess plasma membrane secreted during pollen tube elongation is known to be retrieved by endocytosis and partially reused in secretory pathways through the Golgi apparatus. Dissection of endocytosis has enabled distinct degradation pathways to be identified in tobacco pollen tubes and has shown that microtubules influence the transport of plasma membrane internalized in the tip region to vacuoles. Here, we used different drugs affecting the polymerization state of microtubules together with SYP21, a marker of prevacuolar compartments, to characterize trafficking of prevacuolar compartments in Nicotiana tabacum pollen tubes. Ultrastructural and biochemical analysis showed that microtubules bind SYP21-positive microsomes. Transient transformation of pollen tubes with LAT52-YFP-SYP21 revealed that microtubules play a key role in the delivery of prevacuolar compartments to tubular vacuoles.

Novel thiazolidinone-containing compounds, without the well-known sulphonamide zinc-binding group acting as human carbonic anhydrase IX inhibitors.

A small collection of 26 structurally novel thiazolidinone-containing compounds, without the well-known sulphonamide zinc-binding group, were synthesised and tested in enzyme inhibition assays against the tumour-associated hCA IX enzyme. Inhibition constants in the lower micromolar region (KI < 25 µM) have been measured for 17 of the 26 compounds. Even though the KI values are relatively weak, the fact that they do not contain a sulphonamide moiety suggests that these compounds do not interact with the active site zinc ion. Therefore, docking studies and molecular dynamics simulations have been performed to suggest binding poses for these structurally novel inhibitors.

The Microtubule-Associated Protein CLASP Sustains Cell Proliferation through a Brassinosteroid Signaling Negative Feedback Loop.

The capacity for sustained cell division within the plant meristem is a critical determinant of organ structure and performance. This capacity is diminished in mutants lacking the microtubule-associated protein CLASP and when brassinosteroid signaling is increased. Here, we discovered that CLASP is both targeted by and promotes activity of the brassinosteroid pathway in Arabidopsis root apical meristems. We show that enhanced brassinosteroid signaling reduces CLASP transcript and protein levels, dramatically shifts microtubule organization, and reduces the number of cells in the meristem. In turn, CLASP, which tethers sorting nexin 1 vesicles to microtubules, sustains brassinosteroid signaling by fostering retrieval of endocytosed BRI1 receptors to the plasma membrane. clasp-1 null mutants have dampened brassinosteroid (BR)-mediated transcriptional activity and responses. Global transcript profiling confirmed the collapse of cell-cycle activity in clasp-1 and identified CLASP-mediated hormone crosstalk. Together, these findings reveal an unprecedented form of negative feedback supporting meristem homeostasis.

The Microtubule-Associated Protein IQ67 DOMAIN5 Modulates Microtubule Dynamics and Pavement Cell Shape.

The dynamic arrangement of cortical microtubules (MTs) plays a pivotal role in controlling cell growth and shape formation in plants, but the mechanisms by which cortical MTs are organized to regulate these processes are not well characterized. In particular, the dynamic behavior of cortical MTs is critical for their spatial organization, yet the molecular mechanisms controlling MT dynamics remain poorly understood. In this study, we used the puzzle piece-shaped pavement cells of Arabidopsis (Arabidopsis thaliana) leaves as a model system in which to study cortical MT organization. We isolated an ethyl methanesulfonate mutant with reduced interdigitation of pavement cells in cotyledons. This line carried a mutation in IQ67 DOMAIN5 (IQD5), which encodes a member of the plant-specific IQ motif protein family. Live-cell imaging and biochemical analyses demonstrated that IQD5 binds to MTs and promotes MT assembly. MT-depolymerizing drug treatment and in vivo MT dynamics assays suggested that IQD5 functions to stabilize MTs. Hence, our findings provide genetic, cell biological, and biochemical evidence that IQD5 regulates MT dynamics that affect MT organization and subsequent cell shape formation.

Azosemide is more potent than bumetanide and various other loop diuretics to inhibit the sodium-potassium-chloride-cotransporter human variants hNKCC1A and hNKCC1B.

The Na+-K+-2Cl- cotransporter NKCC1 plays a role in neuronal Cl- homeostasis secretion and represents a target for brain pathologies with altered NKCC1 function. Two main variants of NKCC1 have been identified: a full-length NKCC1 transcript (NKCC1A) and a shorter splice variant (NKCC1B) that is particularly enriched in the brain. The loop diuretic bumetanide is often used to inhibit NKCC1 in brain disorders, but only poorly crosses the blood-brain barrier. We determined the sensitivity of the two human NKCC1 splice variants to bumetanide and various other chemically diverse loop diuretics, using the Xenopus oocyte heterologous expression system. Azosemide was the most potent NKCC1 inhibitor (IC50s 0.246 µM for hNKCC1A and 0.197 µM for NKCC1B), being about 4-times more potent than bumetanide. Structurally, a carboxylic group as in bumetanide was not a prerequisite for potent NKCC1 inhibition, whereas loop diuretics without a sulfonamide group were less potent. None of the drugs tested were selective for hNKCC1B vs. hNKCC1A, indicating that loop diuretics are not a useful starting point to design NKCC1B-specific compounds. Azosemide was found to exert an unexpectedly potent inhibitory effect and as a non-acidic compound, it is more likely to cross the blood-brain barrier than bumetanide.

CLASP promotes stable tethering of endoplasmic microtubules to the cell cortex to maintain cytoplasmic stability in Arabidopsis meristematic cells.

Following cytokinesis in plants, Endoplasmic MTs (EMTs) assemble on the nuclear surface, forming a radial network that extends out to the cell cortex, where they attach and incorporate into the cortical microtubule (CMT) array. We found that in these post-cytokinetic cells, the MT-associated protein CLASP is enriched at sites of EMT-cortex attachment, and is required for stable EMT tethering and growth into the cell cortex. Loss of EMT-cortex anchoring in clasp-1 mutants results in destabilized EMT arrays, and is accompanied by enhanced mobility of the cytoplasm, premature vacuolation, and precocious entry into cell elongation phase. Thus, EMTs appear to maintain cells in a meristematic state by providing a structural scaffold that stabilizes the cytoplasm to counteract actomyosin-based cytoplasmic streaming forces, thereby preventing premature establishment of a central vacuole and rapid cell elongation.

Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide.

Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M-1 s-1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

Fungal biofilm inhibition by piperazine-sulphonamide linked Schiff bases: Design, synthesis, and biological evaluation.

We report the synthesis of some new piperazine-sulphonamide linked Schiff bases as fungal biofilm inhibitors with antibacterial and antifungal potential. The biofilm inhibition result of Candida albicans proposed that the compounds 6b (IC50 = 32.1 µM) and 6j (IC50 = 31.4 µM) showed higher inhibitory activity than the standard fluconazole (IC50 = 40 µM). Compound 6d (MIC = 26.1 µg/mL) with a chloro group at the para position was found to be the most active antibacterial agent of the series against Bacillus subtilis when compared with the standard ciprofloxacin (MIC = 50 µg/mL). Compound 6j (MIC = 39.6 µg/mL) with an OH group at the ortho position showed more potent antifungal activity as compared to that of the standard fluconazole (IC50 = 50 µM) against C. albicans. Thus, the synthesized compounds 6a-k were found to be potent biofilm inhibitors as well as active antibacterial and antifungal agents. The molecular docking study of the synthesized compounds against the secreted aspartyl protease (SAP5) enzyme of C. albicans exhibited good binding properties. The in silico ADME properties of the synthesized compounds were also analyzed and showed their potential to be developed as potential oral drug candidates.

A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control.

KinG Is a Plant-Specific Kinesin That Regulates Both Intra- and Intercellular Movement of SHORT-ROOT.

Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR.

Dinitroaniline herbicide resistance in a multiple-resistant Lolium rigidum population.

BACKGROUND: The pre-emergence dinitroaniline herbicides (such as trifluralin and pendimethalin) are vital to Australian no-till farming systems. A Lolium rigidum population collected from the Western Australian grain belt with a 12-year trifluralin use history was characterised for resistance to dinitroaniline, acetyl CoA carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides. Target-site resistance mechanisms were investigated. RESULTS: This L. rigidum population exhibited 32-fold resistance to trifluralin, as compared with the susceptible population. It also displayed 12- to 30-fold cross-resistance to other dinitroaniline herbicides (pendimethalin, ethalfluralin and oryzalin). In addition, this population showed multiple resistance to commonly used post-emergence ACCase- and ALS-inhibiting herbicides. Two target-site α-tubulin gene mutations (Val-202-Phe and Thr-239-Ile) previously documented in other dinitroaniline-resistant weed species were identified, and some known target-site mutations in ACCase (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg) and ALS (Pro-197-Gln/Ser) were found in the same population. An agar-based Petri dish screening method was established for the rapid diagnosis of resistance to dinitroaniline herbicides. CONCLUSION: Evolution of target-site resistance to both pre- and post-emergence herbicides was confirmed in a single L. rigidum population. The α-tubulin mutations Val-202-Phe and Thr-239-Ile, documented here for the first time in L. rigidum, are likely to be responsible for dinitroaniline resistance in this population. Early detection of dinitroaniline herbicide resistance and integrated weed management strategies are needed to maintain the effectiveness of dinitroaniline herbicides. © 2017 Society of Chemical Industry.

Disruption of actin filaments in Zea mays by bisphenol A depends on their crosstalk with microtubules.

Bisphenol A (BPA) is a widespread environmental pollutant, reportedly harmful to living organisms. In plant cells, BPA was shown to disrupt microtubule (MT) arrays and perturb mitosis, but its effects on filamentous actin (F-actin) have not been explored. Here we studied the effects of BPA on actin filaments (AFs) in meristematic root tip and leaf cells of Zea mays, by fluorescent labeling and confocal microscopy. Considering the typical dynamic interaction between MTs and AFs, the effects on these two essential components of the plant cytoskeleton were correlated. It was found that BPA disorganized rapidly AFs in a concentration- and time-dependent manner. The fine filaments were first to be affected, followed by the subcortical bundles, resulting in rod- and ring-like conformations. The observed differences in sensitivity between protodermal and cortex cells were attributed to the deeper location of the latter. Depolymerization or stabilization of MTs by relevant drugs (oryzalin, taxol) revealed that AF susceptibility to BPA depends on MT integrity. Developing leaves required harder and longer treatment to be affected by BPA. Ontogenesis of stomatal complexes was highly disturbed, arrangement of AFs and MT arrays was disordered and accuracy of cell division sequence was deranged or completely arrested. The effect of BPA confirmed that subsidiary cell mother cell polarization is not mediated by F-actin patch neither of preprophase band organization. On the overall, it is concluded that AFs in plant cells constitute a subcellular target of BPA and their disruption depends on their crosstalk with MTs.

The Xanthomonas effector XopL uncovers the role of microtubules in stromule extension and dynamics in Nicotiana benthamiana.

Xanthomonas campestris pv. vesicatoria type III-secreted effectors were screened for candidates influencing plant cell processes relevant to the formation and maintenance of stromules in Nicotiana benthamiana lower leaf epidermis. Transient expression of XopL, a unique type of E3 ubiquitin ligase, led to a nearly complete elimination of stromules and the relocation of plastids to the nucleus. Further characterization of XopL revealed that the E3 ligase activity is essential for the two plastid phenotypes. In contrast to the XopL wild type, a mutant XopL lacking E3 ligase activity specifically localized to microtubules. Interestingly, mutant XopL-labeled filaments frequently aligned with stromules, suggesting an important, yet unexplored, microtubule-stromule relationship. High time-resolution movies confirmed that microtubules provide a scaffold for stromule movement and contribute to stromule shape. Taken together, this study has defined two populations of stromules: microtubule-dependent stromules, which were found to move slower and persist longer, and microtubule-independent stromules, which move faster and are transient. Our results provide the basis for a new model of stromule dynamics including interactions with both actin and microtubules.

Identification of insulin-sensitizing molecules acting by disrupting the interaction between the Insulin Receptor and Grb14.

Metabolic diseases are characterized by a decreased action of insulin. During the course of the disease, usual treatments frequently fail and patients are finally submitted to insulinotherapy. There is thus a need for innovative therapeutic strategies to improve insulin action. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter that specifically binds to the activated insulin receptor (IR) and inhibits its tyrosine kinase activity. Molecules disrupting Grb14-IR binding are therefore potential insulin-sensitizing agents. We used Structure-Based Virtual Ligand Screening to generate a list of 1000 molecules predicted to hinder Grb14-IR binding. Using an acellular bioluminescence resonance energy transfer (BRET) assay, we identified, out of these 1000 molecules, 3 compounds that inhibited Grb14-IR interaction. Their inhibitory effect on insulin-induced Grb14-IR interaction was confirmed in co-immunoprecipitation experiments. The more efficient molecule (C8) was further characterized. C8 increased downstream Ras-Raf and PI3-kinase insulin signaling, as shown by BRET experiments in living cells. Moreover, C8 regulated the expression of insulin target genes in mouse primary hepatocytes. These results indicate that C8, by reducing Grb14-IR interaction, increases insulin signalling. The use of C8 as a lead compound should allow for the development of new molecules of potential therapeutic interest for the treatment of diabetes.

Design and Antiproliferative Evaluation of Novel Sulfanilamide Derivatives as Potential Tubulin Polymerization Inhibitors.

A series of sulfanilamide-1,2,3-triazole hybrids were designed by a molecular hybridization strategy and evaluated for antiproliferative activity against three selected cancer cell lines (MGC-803, MCF-7 and PC-3). The detailed structure-activity relationships for these sulfanilamide-1,2,3-triazole hybrids were investigated. All these sulfanilamide-1,2,3-triazole hybrids exhibited moderate to potent activity against all cell lines. In particular 4-methyl-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)benzenesulfonamide (11f) showed the most potent inhibitory effect against PC-3 cells, with an IC50 value of 4.08 µM. Furthermore, the tubulin polymerization inhibitory activity in vitro of compound 11f was 2.41 µM. These sulfanilamide hybrids might serve as bioactive fragments for developing more potent antiproliferative agents.

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis.

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co-localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site-specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild-type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild-type Arabidopsis. Thus, we conclude that the PM-associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity-dependent manner, rather than its subcellular translocation.