Contribution of the cytoskeleton to mechanosensitivity reported by dinoflagellate bioluminescence.

The cytoskeleton is crucial to cell mechanics and sensing the extracellular physical environment. The objective of this study was to examine the role of the cortical cytoskeleton in mechanosensitivity in a unicellular protist, the marine dinoflagellate Lingulodinium polyedra, using its intrinsic bioluminescence as a rapid reporter of mechanotransduction. Pharmacological treatments resolved effects due to immediate cytoskeleton disruption from those due to cytoskeletal remodeling during the light to dark phase transition. The cytoskeleton was visualized by confocal laser scanning microscopy of immunohistochemically labeled microtubules and phalloidin labeled F-actin, and mechanosensitivity assessed based on the bioluminescence response to mechanical stimulation measured during the dark phase. Latrunculin B treatment after the transition from the light to dark phase resulted in some disruption of cortical F-actin, no observed effect on the cortical microtubules, and partial inhibition of the bioluminescence response. Treatment with oryzalin, which depolarizes microtubules, completely disrupted the microtubule network and cortical F-actin, and partially inhibited bioluminescence. These results demonstrate that cells retain some mechanosensitivity despite a disrupted cytoskeleton; link mechanosensitivity to intact F-actin; show a close connection between F-actin and microtubules comprising the cortical cytoskeleton; confirm a strong contribution of the actin cytoskeleton to the translocation of scintillons, vesicles containing the luminescent chemistry; and support the role of the actin cytoskeleton in the association of scintillons with the vacuole membrane.

Role of TRP Channels in Dinoflagellate Mechanotransduction.

Transient receptor potential (TRP) ion channels are common components of mechanosensing pathways, mainly described in mammals and other multicellular organisms. To gain insight into the evolutionary origins of eukaryotic mechanosensory proteins, we investigated the involvement of TRP channels in mechanosensing in a unicellular eukaryotic protist, the dinoflagellate Lingulodinium polyedra. BLASTP analysis of the protein sequences predicted from the L. polyedra transcriptome revealed six sequences with high similarity to human TRPM2, TRPM8, TRPML2, TRPP1, and TRPP2; and characteristic TRP domains were identified in all sequences. In a phylogenetic tree including all mammalian TRP subfamilies and TRP channel sequences from unicellular and multicellular organisms, the L. polyedra sequences grouped with the TRPM, TPPML, and TRPP clades. In pharmacological experiments, we used the intrinsic bioluminescence of L. polyedra as a reporter of mechanoresponsivity. Capsaicin and RN1734, agonists of mammalian TRPV, and arachidonic acid, an agonist of mammalian TRPV, TRPA, TRPM, and Drosophila TRP, all stimulated bioluminescence in L. polyedra. Mechanical stimulation of bioluminescence, but not capsaicin-stimulated bioluminescence, was inhibited by gadolinium (Gd3+), a general inhibitor of mechanosensitive ion channels, and the phospholipase C (PLC) inhibitor U73122. These pharmacological results are consistent with the involvement of TRP-like channels in mechanosensing by L. polyedra. The TRP channels do not appear to be mechanoreceptors but rather are components of the mechanotransduction signaling pathway and may be activated via a PLC-dependent mechanism. The presence and function of TRP channels in a dinoflagellate emphasize the evolutionary conservation of both the channel structures and their functions.

A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.

Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.

Fluorescent markers of the endocytic pathway in Zymoseptoria tritici.

Hyphal growth in filamentous fungi is supported by the uptake (endocytosis) and recycling of membranes and associated proteins at the growing tip. An increasing body of published evidence in various fungi demonstrates that this process is of essential importance for fungal growth and pathogenicity. Here, we introduce fluorescent markers to visualize the endocytic pathway in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein (eGFP) to the actin-binding protein fimbrin (ZtFim1), which is located in actin patches that are formed at the plasma membrane and are participating in endocytic uptake at the cell surface. In addition, we tagged early endosomes by eGFP-labelling a Rab5-homologue (ZtRab5) and late endosomes and vacuoles by expressing eGFP-Rab7 homologue (ZtRab7). Using fluorescent dyes and live cell imaging we confirmed the dynamic behavior and localization of these markers. This set of molecular tools enables an in-depth phenotypic analysis of Z. tritici mutant strains thereby supporting new strategies towards the goal of controlling wheat against Z. tritici.

Fluorescent markers for the Spitzenkörper and exocytosis in Zymoseptoria tritici.

Fungal hyphae are highly polarized cells that invade their substrate by tip growth. In plant pathogenic fungi, hyphal growth is essential for host invasion. This makes polarity factors and secretion regulators potential new targets for novel fungicides. Polarization requires delivery of secretory vesicles to the apical Spitzenkörper, followed by polarized exocytosis at the expanding cell tip. Here, we introduce fluorescent markers to visualize the apical Spitzenkörper and the apical site of exocytosis in hyphae of the wheat pathogen Zymoseptoria tritici. We fused green fluorescent protein to the small GTPase ZtSec4, the myosin light chain ZtMlc1 and the small GTPase ZtRab11 and co-localize the fusion proteins with the dye FM4-64 in the hyphal apex, suggesting that the markers label the hyphal Spitzenkörper in Z. tritici. In addition, we localize GFP-fusions to the exocyst protein ZtExo70, the polarisome protein ZtSpa2. Consistent with results in the ascomycete Neurospora crassa, these markers did localize near the plasma membrane at the hyphal tip and only partially co-localize with FM4-64. Thus, these fluorescent markers are useful molecular tools that allow phenotypic analysis of mutants in Z. tritici. These tools will help develop new avenues of research in our quest to control STB infection in wheat.

Fluorescent markers of the microtubule cytoskeleton in Zymoseptoria tritici.

The microtubule cytoskeleton supports vital processes in fungal cells, including hyphal growth and mitosis. Consequently, it is a target for fungicides, such as benomyl. The use of fluorescent fusion proteins to illuminate microtubules and microtubule-associated proteins has led to a break-through in our understanding of their dynamics and function in fungal cells. Here, we introduce fluorescent markers to visualize microtubules and accessory proteins in the wheat pathogen Zymoseptoria tritici. We fused enhanced green-fluorescent protein to α-tubulin (ZtTub2), to ZtPeb1, a homologue of the mammalian plus-end binding protein EB1, and to ZtGrc1, a component of the minus-end located γ-tubulin ring complex, involved in the nucleation of microtubules. In vivo observation confirms the localization and dynamic behaviour of all three markers. These marker proteins are useful tools for understanding the organization and importance of the microtubule cytoskeleton in Z. tritici.

Evidence for shear-induced increase in membrane fluidity in the dinoflagellate Lingulodinium polyedrum.

Fluid shear stress has been demonstrated to affect the structure and function of various cell types. In mammalian cells, it was hypothesized that shear-induced membrane fluidization leads to activation of heterotrimetric G-proteins. The purpose of this study was to determine if a similar mechanism exists in the dinoflagellate Lingulodinium polyedrum, a single-celled eukaryotic aquatic organism that bioluminesces under shear stress. Membrane fluidity changes in L. polyedrum were monitored using the molecular rotor 9-(dicyanovinyl)-julolidine, whose fluorescence intensity changes inversely with membrane fluidity. Dual-staining with 9-(dicyanovinyl)-julolidine and the membrane dye 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate indicates membrane localization. Subjecting L. polyedrum cells to increasing shear stress reversibly decreased 9-(dicyanovinyl)-julolidine fluorescence, while autofluorescence of the cytoplasmic chlorophyll did not change. The relationship between shear stress (0.63 Pa, 1.25 Pa, 1.88 Pa, and 2.5 Pa) and membrane fluidity changes was linear and dose-dependent with a 12% increase in fluidity at 2.5 Pa. To further explore this mechanism a membrane fluidizing agent, dimethyl sulfoxide was added. Dimethyl sulfoxide decreased 9-(dicyanovinyl)-julolidine emission by 41+/-15% and elicited a dose-dependent bioluminescent response at concentrations of 0.2%, 0.5%, 1.0%, and 1.25%. This study demonstrates a link between fluid shear stress and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of dinoflagellates.

Experimental approaches towards interpreting dolphin-stimulated bioluminescence.

Flow-induced bioluminescence provides a unique opportunity for visualizing the flow field around a swimming dolphin. Unfortunately, previous descriptions of dolphin-stimulated bioluminescence have been largely anecdotal and often conflicting. Most references in the scientific literature report an absence of bioluminescence on the dolphin body, which has been invariably assumed to be indicative of laminar flow. However, hydrodynamicists have yet to find compelling evidence that the flow remains laminar over most of the body. The present study integrates laboratory, computational and field approaches to begin to assess the utility of using bioluminescence as a method for flow visualization by relating fundamental characteristics of the flow to the stimulation of naturally occurring luminescent plankton. Laboratory experiments using fully developed pipe flow revealed that the bioluminescent organisms identified in the field studies can be stimulated in both laminar and turbulent flow when shear stress values exceed approximately 0.1 N m-2. Computational studies of an idealized hydrodynamic representation of a dolphin (modeled as a 6:1 ellipsoid), gliding at a speed of 2 m s-1, predicted suprathreshold surface shear stress values everywhere on the model, regardless of whether the boundary layer flow was laminar or turbulent. Laboratory flow visualization of a sphere demonstrated that the intensity of bioluminescence decreased with increasing flow speed due to the thinning of the boundary layer, while flow separation caused a dramatic increase in intensity due to the significantly greater volume of stimulating flow in the wake. Intensified video recordings of dolphins gliding at speeds of approximately 2 m s-1 confirmed that brilliant displays of bioluminescence occurred on the body of the dolphin. The distribution and intensity of bioluminescence suggest that the flow remained attached over most of the body. A conspicuous lack of bioluminescence was often observed on the dolphin rostrum and melon and on the leading edge of the dorsal and pectoral fins, where the boundary layer is thought to be thinnest. To differentiate between effects related to the thickness of the stimulatory boundary layer and those due to the latency of the bioluminescence response and the upstream depletion of bioluminescence, laboratory and dolphin studies of forced separation and laminar-to-turbulent transition were conducted. The observed pattern of stimulated bioluminescence is consistent with the hypothesis that bioluminescent intensity is directly related to the thickness of the boundary layer.

Androgen metabolism in the skin of the rainbow trout (Oncorhynchus mykiss).

Pieces of skin of male and female rainbow trout (Oncorhynchus mykiss) were incubated with testosterone and 11-ketotestosterone as substrates. In immature fish the conversion rate was low. In non-spawning adult males 11-ketotestosterone was reduced to 5α-11KDHT (up to 5.2%). In the fish in spawning condition the 5α-reduction rate was only about 1 to 2%. In the same specimens incubated with testosterone a high 11ß-hydroxylase activity (23.8-25% in the male and 13% in the female skin) was found. Similar sex specific differences were observed for the occurence of 5α-reduced metabolites (about 20% in the male and 13% in the female tissue).

Slow Photic and Chemical Induction of Bioluminescence in the Midwater Shrimp, Sergestes similis Hansen.

The initial luminescent response to photic stimulation of dark-maintained specimens of the midwater shrimp, Sergestes similis Hansen, differed from the conventional counterillumination response. Animals were initially unresponsive to light; bioluminescence was only induced after a latency of 3 min. Maximum intensity was reached after approximately 25 min. During the induction process, light emission from the anterior light organs was frequently observed prior to output from the posterior organ. Once luminescence was induced, responses exhibited the typical fast kinetics of the counterillumination response and changes in light organ output occurred synchronously. Visual input was necessary to maintain this state. Dark readaptation of counterilluminating animals resulted in a return to the slow response kinetics characteristic of untested animals. Because eyestalk ablation or crushing caused immediate production of luminescence in previously untested animals, the slow induction did not involve the ability of the light organs to produce light. Serotonin was effective in stimulating bioluminescence in intact animals; the induction of light emission proceeded at a rate similar to that for photic stimulation. Other putative neurotransmitters, including norepinephrine, acetylcholine, GABA, and L-glutamic acid, did not stimulate bioluminescence. Isolated light organs exhibited high background levels of light emission, which were unchanged by serotonin treatment. However, serotonin was effective in stimulating luminescence in animals with ablated eyestalks. These results suggest a dual control system involved in the induction and maintenance of bioluminescence in S. similis.

In vitro studies on steroid metabolism by gonadal tissues from ambisexual teleosts. II. Conversion of [14C]androstenedione by the heterologous gonadal tissues of the protandric sea bream Pagellus acarne (Risso).

The heterologous gonadal tissues of the protandric sea bream Pagellus acarne were incubated with [14C]androstenedione in vitro. The tissues were taken from two males, an inverting specimen, and a female in early April. Although the testicular tissue regresses in the inverting fish the rate of 11 beta-hydroxylation in the male part of the gonad is at least as high as in the homologous portion of the "pure" male. No difference was found in the enzymatic pattern between the ovarian part of the inverting animal and that of the female. There is, however, a striking distinction in the metabolic activities of the pooled ovarian parts from two males and the homologous tissues from the inverting and female animals. They concern mainly the activities of 11 beta-hydroxylase and 17 beta-HSD. Moreover, ring A-reduction and the ratio of 5 alpha/5 beta-reduced metabolites differ considerably from the corresponding gonadal tissues taken from the inverting and the female specimen. The possible physiological meaning of these findings remains to be elucidated.

Far red bioluminescence from two deep-sea fishes.

Spectral measurements of red bioluminescence were obtained from the deep-sea stomiatoid fishes Aristostomias scintillans (Gilbert) and Malacosteus niger (Ayres). Red luminescence from suborbital light organs extends to the near infrared, with peak emission at approximately 705 nanometers in the far red. These fishes also have postorbital light organs that emit blue luminescence with maxima between 470 and 480 nanometers. The red bioluminescence may be due to an energy transfer system and wavelength-selective filtering.

Cryptic bioluminescence in a midwater shrimp.

The mesopelagic shrimp Sergestes similis emits ventrally directed bioluminescence that closely matches the intensity of downward-directed illumination and is able to rapidly modify its light output to match changes in background intensity. Masking experiments show that the photoreceptors involved are the compound eyes or adjacent tissues. Light emission originates from modified portions of the hepatopancreas and is similar to oceanic light in angular distribution and spectral characteristics. Normally oriented animals respond minimally to upward-directed light.