The Combination of Δ9-Tetrahydrocannabinol and Cannabidiol Suppresses Mitochondrial Respiration of Human Glioblastoma Cells via Downregulation of Specific Respiratory Chain Proteins.

Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ9-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB1, CB2 and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics.

Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically.

The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as "TRFS" probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes' complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.

Selective, Modular Probes for Thioredoxins Enabled by Rational Tuning of a Unique Disulfide Structure Motif.

Specialized cellular networks of oxidoreductases coordinate the dithiol/disulfide-exchange reactions that control metabolism, protein regulation, and redox homeostasis. For probes to be selective for redox enzymes and effector proteins (nM to µM concentrations), they must also be able to resist non-specific triggering by the ca. 50 mM background of non-catalytic cellular monothiols. However, no such selective reduction-sensing systems have yet been established. Here, we used rational structural design to independently vary thermodynamic and kinetic aspects of disulfide stability, creating a series of unusual disulfide reduction trigger units designed for stability to monothiols. We integrated the motifs into modular series of fluorogenic probes that release and activate an arbitrary chemical cargo upon reduction, and compared their performance to that of the literature-known disulfides. The probes were comprehensively screened for biological stability and selectivity against a range of redox effector proteins and enzymes. This design process delivered the first disulfide probes with excellent stability to monothiols yet high selectivity for the key redox-active protein effector, thioredoxin. We anticipate that further applications of these novel disulfide triggers will deliver unique probes targeting cellular thioredoxins. We also anticipate that further tuning following this design paradigm will enable redox probes for other important dithiol-manifold redox proteins, that will be useful in revealing the hitherto hidden dynamics of endogenous cellular redox systems.

Microtubules and motor proteins support zebrafish neuronal migration by directing cargo.

Neuronal migration during development is necessary to form an ordered and functional brain. Postmitotic neurons require microtubules and dynein to move, but the mechanisms by which they contribute to migration are not fully characterized. Using tegmental hindbrain nuclei neurons in zebrafish embryos together with subcellular imaging, optogenetics, and photopharmacology, we show that, in vivo, the centrosome's position relative to the nucleus is not linked to greatest motility in this cell type. Nevertheless, microtubules, dynein, and kinesin-1 are essential for migration, and we find that interference with endosome formation or the Golgi apparatus impairs migration to a similar extent as disrupting microtubules. In addition, an imbalance in the traffic of the model cargo Cadherin-2 also reduces neuronal migration. These results lead us to propose that microtubules act as cargo carriers to control spatiotemporal protein distribution, which in turn controls motility. This adds crucial insights into the variety of ways that microtubules can support successful neuronal migration in vivo.

A cargo model of yolk syncytial nuclear migration during zebrafish epiboly.

In teleost fish, the multinucleate yolk syncytial layer functions as an extra-embryonic signaling center to pattern mesendoderm, coordinate morphogenesis and supply nutrients to the embryo. External yolk syncytial nuclei (e-YSN) undergo microtubule-dependent movements that distribute the nuclei over the large yolk mass. How e-YSN migration proceeds, and the role of the yolk microtubules, is not understood, but it is proposed that e-YSN are pulled vegetally as the microtubule network shortens from the vegetal pole. Live imaging revealed that nuclei migrate along microtubules, consistent with a cargo model in which e-YSN are moved down the microtubules by direct association with motor proteins. We found that blocking the plus-end directed microtubule motor kinesin significantly attenuated yolk nuclear movement. Blocking the outer nuclear membrane LINC complex protein Syne2a also slowed e-YSN movement. We propose that e-YSN movement is mediated by the LINC complex, which functions as the adaptor between yolk nuclei and motor proteins. Our work provides new insights into the role of microtubules in morphogenesis of an extra-embryonic tissue and further contributes to the understanding of nuclear migration mechanisms during development.

Glycine is able to induce both a motility speed in- and decrease during zebrafish neuronal migration.

Various neurotransmitters influence neuronal migration in the developing zebrafish hindbrain. Migrating tegmental hindbrain nuclei neurons (THNs) are governed by depolarizing neurotransmitters (acetylcholine and glutamate), and glycine. In mature neurons, glycine binds to its receptor to hyperpolarize cells. This effect depends on the co-expression of the solute carrier KCC2. Immature precursors, however, typically express NKCC1 instead of KCC2, leading to membrane depolarization upon glycine receptor activation. As neuronal migration occurs in neurons after leaving the cell cycle and before terminal differentiation, we hypothesized that the switch from NKCC1 to KCC2 expression could alter the effect of glycine on THN migration. We tested this notion using in vivo cell tracking, overexpression of glycine receptor mutations and whole mount in situ hybridization. We summarize our findings in a speculative model, combining developmental age, glycine receptor strength and solute carrier expression to describe the effect of glycine on the migration of THNs.

Sequence defined antibodies improve the detection of cadherin 2 (N-cadherin) during zebrafish development.

Cadherin-2 plays a fundamental role during zebrafish CNS and heart morphogenesis. Profiling zCdh2 expression via antibody staining is essential to achieving better understanding of its role during zebrafish development. Generation of recombinant human antibodies to zCdh2 by phage display was used to identify monoclonal antibodies with reduced unspecific binding patterns when compared to available commercial antibodies. Specificity was profiled using flow cytometry of wild type, zCdh2-defective mutant and zCdh2-GFP zebrafish cells. The epitopes recognized by the novel antibodies were mapped to peptides located in the first or second extracellular domains of zCdh2. These antibodies allowed improved observations of the spatial distribution of zCdh2 from imaging of whole mount zebrafish preparations. Since the generated antibodies are sequence defined, they can always be reconstituted from the information stored in the respective computer file, securing future reproducibility of respective experiments. The results further suggest that sequence defined antibodies with specificities thoroughly controlled by flow cytometry and genetic antigen-defective mutants or knockouts can substantially reduce the risk of misleading, false-positive results in whole mount imaging and other assays, and thus can improve the scientific value of such assays.

Neurotransmitter-mediated activity spatially controls neuronal migration in the zebrafish cerebellum.

Neuronal migration during embryonic development contributes to functional brain circuitry. Many neurons migrate in morphologically distinct stages that coincide with differentiation, requiring tight spatial regulation. It had been proposed that neurotransmitter-mediated activity could exert this control. Here, we demonstrate that intracellular calcium transients occur in cerebellar neurons of zebrafish embryos during migration. We show that depolarization increases and hyperpolarization reduces the speed of tegmental hindbrain neurons using optogenetic tools and advanced track analysis optimized for in vivo migration. Finally, we introduce a compound screening assay to identify acetylcholine (ACh), glutamate, and glycine as regulators of migration, which act regionally along the neurons' route. We summarize our findings in a model describing how different neurotransmitters spatially interact to control neuronal migration. The high evolutionary conservation of the cerebellum and hindbrain makes it likely that polarization state-driven motility constitutes an important principle in building a functional brain.

Directional persistence of migrating cells requires Kif1C-mediated stabilization of trailing adhesions.

Directional cell migration requires the establishment and maintenance of long-term differences in structure and function between the front and back of a cell. Here, we show that the microtubule motor Kif1C contributes to persistent cell migration primarily through stabilization of an extended cell rear. Kif1C-mediated transport of α5ß1-integrins is required for the proper maturation of trailing focal adhesions and resistance to tail retraction. Tail retraction precedes and induces changes in migration direction. Stabilization of cell tails through inhibition of myosin II activity suppresses the Kif1C depletion phenotype and results in longer-lived tails and higher directional stability of migrating cells. Taken together, these findings indicate that the maintenance of an extended, tense cell tail facilitates directional migration. We propose a rear drag mechanism for directional persistence of migration whereby the counterforce originating from a well-anchored tail serves to maintain directionality of the force-generating leading edge of the cell.

Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport.

Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ~1.0 µm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.

AcrS/EnvR represses expression of the acrAB multidrug efflux genes in Escherichia coli.

The acrS regulatory gene is located upstream of the acrEF multidrug efflux system genes. However, the roles of AcrS in regulation of drug efflux pumps have not been clearly understood. Here we show that AcrS represses other multidrug efflux genes, acrAB, which encode a major efflux system in Escherichia coli.

Dynamic rearrangement of nucleoporins during fungal "open" mitosis.

Mitosis in animals starts with the disassembly of the nuclear pore complexes and the breakdown of the nuclear envelope. In contrast to many fungi, the corn smut fungus Ustilago maydis also removes the nuclear envelope. Here, we report on the dynamic behavior of the nucleoporins Nup214, Pom152, Nup133, and Nup107 in this "open" fungal mitosis. In prophase, the nuclear pore complexes disassembled and Nup214 and Pom152 dispersed in the cytoplasm and in the endoplasmic reticulum, respectively. Nup107 and Nup133 initially spread throughout the cytoplasm, but in metaphase and early anaphase occurred on the chromosomes. In anaphase, the Nup107-subcomplex redistributed to the edge of the chromosome masses, where the new envelope was reconstituted. Subsequently, Nup214 and Pom152 are recruited to the nuclear pores and protein import starts. Recruitment of nucleoporins and protein import reached a steady state in G2 phase. Formation of the nuclear envelope and assembly of nuclear pores occurred in the absence of microtubules or F-actin, but not if both were disrupted. Thus, the basic principles of nuclear pore complex dynamics seem to be conserved in organisms displaying open mitosis.

Molecular analysis of shower curtain biofilm microbes.

Households provide environments that encourage the formation of microbial communities, often as biofilms. Such biofilms constitute potential reservoirs for pathogens, particularly for immune-compromised individuals. One household environment that potentially accumulates microbial biofilms is that provided by vinyl shower curtains. Over time, vinyl shower curtains accumulate films, commonly referred to as "soap scum," which microscopy reveals are constituted of lush microbial biofilms. To determine the kinds of microbes that constitute shower curtain biofilms and thereby to identify potential opportunistic pathogens, we conducted an analysis of rRNA genes obtained by PCR from four vinyl shower curtains from different households. Each of the shower curtain communities was highly complex. No sequence was identical to one in the databases, and no identical sequences were encountered in the different communities. However, the sequences generally represented similar phylogenetic kinds of organisms. Particularly abundant sequences represented members of the alpha-group of proteobacteria, mainly Sphingomonas spp. and Methylobacterium spp. Both of these genera are known to include opportunistic pathogens, and several of the sequences obtained from the environmental DNA samples were closely related to known pathogens. Such organisms have also been linked to biofilm formation associated with water reservoirs and conduits. In addition, the study detected many other kinds of organisms at lower abundances. These results show that shower curtains are a potential source of opportunistic pathogens associated with biofilms. Frequent cleaning or disposal of shower curtains is indicated, particularly in households with immune-compromised individuals.