Diversity and dynamics of the Spitzenkörper in growing hyphal tips of higher fungi

Summary The Spitzenkörper, located in the apex of growing hyphae of septate fungi, has been portrayed previously as a spheroid complex containing a cluster of apical (secretory) vesicles which sometimes encloses a differentiated core area. With the aid of computer-enhanced video microscopy and phase-contrast optics, we studied 32 fungi in the Ascomycetes, Deuteromycetes, Hyphomycetes, Basidiomycetes, and Agonomycetes. The Spitzenkörper appeared as a highly dynamic and pleomorphic multicomponent complex capable of changing shape, size, and position within the hyphal apex during growth. The main theme of this study is to demonstrate two kinds of morphological diversity/variation in Spitzenkörper from diverse fungi: (a) inherent diversity — Spitzenkörper features characteristic of particular fungi, and (b) dynamic pleomorphism — gradual or rapid changes in size, shape, and position of the Spitzenkörper within a single hyphal tip. Several components associated with the Spitzenkörper were identified: (a) vesicle cluster, (b) vesicle cloud, (c) differentiated core region(s) within the Spitzenkörper, (d) apical granules, (e) cytoplasmic filaments. Eight morphological patterns of Spitzenkörper organization are described in the higher fungi based on the shape and distribution of their components. An additional (ninth) pattern was recognized in the chytridiomyceteAllomyces macrogynous from recent work by others. All these patterns appeared to be conserved at the genus level. In all patterns but one, a core region was observed by light microscopy. The Spitzenkörper not only exhibited spontaneous dynamic pleomorphism but also reacted to stress conditions (light, mechanical, and electrical fields). These reactions include migration of the Spitzenkörper back into the subapical zone and/or disassembly of its components. The understanding and conceptualization of this dynamic complex is problematic and should remain flexible enough to encompass the diversity of Spitzenkörper patterns and the dynamic pleomorphism of this specialized apical apparatus which appears to drive hyphal tip growth in the higher fungi.Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

The Spitzenkörper: a signalling hub for the control of fungal development?

In Aspergillus nidulans , asexual development culminates in the formation of a multicellular conidiophore that bears spores known as conidia. Although several factors required for conidiophore formation have been characterized, the mechanisms underlying the transition from vegetative growth to development remain obscure. However, the recent characterization of two of these factors, FlbB and FlbE, provides important new insight. Notably, these studies suggest that the Spitzenkörper (i.e. apical body), which is known to regulate hyphal morphogenesis, might also serve as a signalling hub that co-ordinates development transitions.     

The cytoplasmic organization of hyphal tip cells in the fungusAllomyces macrogynus

Summary Light and transmission electron microscopy were used to examine hyphal tip cells of the fungusAllomyces macrogynus (Chytridiomycetes). A well defined apical body, i.e., Spitzenkörper, was observed at the extreme apex of hyphal cells. This distinctive, spherical cytoplasmic region consisted of a granular matrix devoid of ribosomes and most organelles. To our knowledge this is the first report describing such a structure in hyphae of an aseptate fungus. Vesicles (45–65 nm diameter) were concentrated in the peripheral cytoplasm of the apex, while relatively few were observed within the Spitzenkörper. Filasomes, spherical patches of dense fibrillar material containing a microvesicle core, were abundant in the apical regions near the plasma membrane. Microtubules traversed the Spitzenkörper at various angles and were in close association with the plasma membrane. Microfilaments were observed as individual elements in the cytoplasm or were organized into bundles. Individual microfilaments were frequently in close association with the plasma membrane, vesicles and microtubules. In the immediate subapical region mitochondria, multivesicular bodies, microbodies, Golgi equivalents and nuclei were abundant.Abbreviations CW cell wall - F filasome - M mitochondria - N nucleus - PM plasma membrane - TEM transmission electron microscopy     

The tubulin cytoskeleton and its sites of nucleation in hyphal tips ofAllomyces macrogynus

Summary The tubulin cytoskeleton in hyphal tip cells ofAllomyces macrogynus was detected with an -tubulin monoclonal antibody and analyzed with microscopic and immunoblot techniques. The -tubulin antibody identified a 52 kilodalton polypeptide band on immunoblots. Immunfluorescence data were collected from formaldehyde-and cryofixed hyphae. Both methods provided similar images of tubulin localization. However, cryofixation yielded more consistent labeling and did not require detergent extraction or cell-wall lytic treatments. Tubulin was primarily localized as microtubules observed in the peripheral and central cytoplasmic regions and in mitotic spindles. Cytoplasmic microtubules were oriented parallel to the cells' longitudinal axis, with central microtubules more often varied in their alignment, and emanated from a region in the hyphal apex resulting in an apical zone of bright fluorescence. A thin layer of microtubules appearing as bands of fluorescence encircled many nuclei. Discrete spots of fluorescence were also associated with nuclei. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins that may be involved in the regulation of microtubule nucleation, stained centrosomes but not apical regions of hyphae. Nocodazole was used to depolymerize the microtubule network and reveal its regions of origin. A hocodazole concentration of 0.01 g/ ml (3.3× 10^–8M) provided a 70 to 75% inhibition of hyphal tip growth and was used throughout this study. The number of cells having an apical zone of fluorescence declined by 15 min of exposure. This zone was present in only a few cells after 60 min. After 30 min, the central cytoplasm consisted of small microtubule fragments and nuclear-associated spots. A small number of peripheral microtubules and nuclear-associated spots persisted throughout nocodazole treatments. Spindle microtubules were restored by 30 min after removal of nocodazole. This was followed by the reappearance of the apical zone of fluorescence and then by central and peripheral cytoplasmic microtubules. Apical fluorescence coincided with the presence of a Spitzenkörper. The results suggest that the Spitzenkörper and centrosome function as centers of microtubule nucleation and organization during hyphal tip growth in this fungus.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IB incubation buffer - LN₂ liquid nitrogen - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate buffered saline - PIPES 1,4-piperazinedietha-nesulfonic acid - PFB PIPES fixation buffer - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SPB spindle pole body - TEM transmission electron microscopy - YpSs yeast extract-inorganic phosphate-soluble starch     

Spitzenkörper, vacuoles, ring-like structures, and mitochondria of Phanerochaete velutina hyphal tips visualized with carboxy-DFFDA, CMAC and DiOC6(3)

Growth and organelle morphology in the wood rotting basidiomycete fungus Phanerochaete velutina were examined in Petri dishes, on agar-coated slides, and in submerged cultures, using DIC, fluorescence and four-dimensional (4-D; x,y,z,t) confocal microscopy, with several fluorescent probes. Phanerochaete is ideal for this work because of its fast growth, robustness, and use in a wide range of other studies. The probe carboxy-DFFDA, widely used for labelling vacuoles, has no effect either on hyphal tip extension or colony growth at the concentrations usually applied in labelling experiments. Carboxy-DFFDA labels the vacuoles and these form a tubular reticulum in hyphal tip cells. The probe also labels extremely small vesicles (punctate fluorescence) in the apex of tip cells, the Spitzenkörper, and short tubules that undergo sequences of characteristic movements and transformations to produce various morphologies, including ring-like structures. Their location and behaviour suggest that they are a distinct group of structures, possibly a subset of vacuoles, but as yet to be fully identified. Regular incursions of tubules extending from these structures and from the vacuolar reticulum into the apical dome indicate the potential for delivery of material to the apex via tubules as well as vesicles. Such structures are potential candidates for delivering chitin synthases to the apex. Spitzenkörper behaviour has been followed as hyphal tips with linear growth encounter obstacle hyphae and, as the hydrolysis product of carboxy-DFFDA only accumulates in membrane-enclosed compartments, it can be inferred that the labelled structures represent the Spitzenkörper vesicle cloud. Mitochondria also form a reticular continuum of branched tubules in growing hyphal tips, and dual localisation with DiOC₆(3) and CMAC allows this to be distinguished from the vacuolar reticulum. Like vacuolar tubules, mitochondrial tubules also span the septa, indicating that they may also be a conduit for intercellular transport.     

Bevölkerungswachstum: Bildung ist die Lösung

Population growth – education is the answer Over the last decades world population has been constantly growing by some 80 million per year. Whereas the growth rate as well as the fertility rate have been cut by half since the 1970th, population growth will continue well over mid‐century. As the developed countries have completed the demographic transition from high mortality and fertility rates to low ones, population growth is fading out there or has already been reversed into decline. In the least developed countries mortality has fallen as well, whereas fertility decline has stalled. Therefor population growth is very high making the solution of the widespread problems in this part of the world more and more difficult. One obvious way out of this trap would be a better education that could open new development perspectives. A positive side effect is that educated women have much less offspring than their counterparts who never went to school.     

Knöllchenbakterien: Helfer der Landwirtschaft

Knöllchenbakterium – the “microbe of the year” In 2015, the VAAM selected Knöllchenbakterium as “microbe of the year”. Knöllchenbakterium ist a collective term for a number of different bacterial species that are able to establish a root nodule symbiosis with legumes. During nodule development the bacteria differentiate into bacteroids that are confined by an additional membrane. These organelle‐like structures are now called symbiosomes, whose task is to fix molecular nitrogen for the benefit of the plant. In return, the plant has to supply all nutrients. The symbiotic interaction is initiated by a specific signal exchange. The first signals are flavonoids secreted by the plant. This leads to the activation of the bacterial nod genes. The Nod proteins synthesize and secrete Nod factors: modified and fatty acid‐carrying oligosaccharide. They serve as a specific signal to the plant and induce nodule formation. Besides this core signaling, a number of extracellular components, e.g. exopolysaccharides, lipopolysaccharides and secreted proteins influence the symbiotic interaction very specific for each individual system.     

The pre-Bötzinger oscillator in the mouse embryo

Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-Bötzinger complex (preBötC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preBötC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation.     

The scaffold protein JIP3 functions as a downstream effector of the small GTPase ARF6 to regulate neurite morphogenesis of cortical neurons

The small GTPase ADP-ribosylation factor 6 (ARF6) plays crucial roles in a wide variety of cell functions. To better understand the molecular mechanisms of ARF6-mediated signaling and cellular functions, we sought new ARF6-binding proteins in the mouse brain. We identified the signaling scaffold protein JNK-interacting protein 3 (JIP3), which is exclusively expressed in neurons, as a downstream effector of ARF6. Overexpression of a unique dominant negative mutant of ARF6, which was unable to interact with JIP3, and knockdown of JIP3 in mouse cortical neurons stimulated the elongation and branching of neurites. These results provide evidence that ARF6/JIP3 signaling regulates neurite morphogenesis.

Structured summary

MINT-7892698: PIP5K gamma 661 (uniprotkb:O70161) physically interacts (MI:0915) with Arf6 (uniprotkb:P62331) by anti tag coimmunoprecipitation (MI:0007)MINT-7892333, MINT-7892573, MINT-7892594, MINT-7892629, MINT-7892644, MINT-7892522, MINT-7892716: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by anti tag coimmunoprecipitation (MI:0007)MINT-7892509: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892770: Arf6 (uniprotkb:P62331) binds (MI:0407) to JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892755: Arf6 (uniprotkb:P62331) binds (MI:0407) to JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892289, MINT-7892314: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892353, MINT-7892615, MINT-7892657, MINT-7892672, MINT-7892549, MINT-7892738: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by anti tag coimmunoprecipitation (MI:0007)     

Hepatitis C virus infection and the risk of Sjögren or sicca syndrome: a meta‐analysis

Previous studies have suggested an association between hepatitis C virus (HCV) infection and the development of Sjögren's syndrome (SS), also known as sicca syndrome. The main objective of this study was to summarize the existing evidence and quantitatively evaluate the association between hepatitis C virus infection and SS/sicca syndrome by performing a meta‐analysis of observational studies. MEDLINE and PubMed (January 1980–August 2013) were searched to identify relevant studies in English. Outcomes were calculated and are reported as odds risk (OR) and 95% CIs based on a random‐effects model. Heterogeneity was assessed with I² statistics. Quality assessment was performed with the Newcastle–Ottawa scale. Based on meta‐analysis of five cross‐sectional and five cohort studies, a significant positive relationship between HCV infection and development of SS/sicca syndrome was found, the pooled random effects OR being 3.31 (95% CI, 1.46–7.48; P < 0.001). In subset analyses, the studies that used European diagnostic criteria showed a higher summary OR than did studies that adopted other diagnostic criteria. When the data were stratified by source of controls, significant associations were also observed when healthy people (OR = 9.44; 95% CI = 2.67–33.40; P = 0.204) or subjects with hepatitis B virus infection (OR = 6.57; 95% CI = 1.21–35.57; P = 0.5) were used as controls, but not when the controls were hospital‐based (OR = 0.99; 95% CI = 0.61–1.61; P = 0.169). In summary, the findings suggest that HCV infection is associated with SS/sicca syndrome. The observed increased risk in studies in which European diagnostic criteria and healthy controls were used and the decreased risk in studies with hospital‐based controls may be attributable to selection bias or other unknown factors.     

The oldest marine vertebrate fossil from the volcanic island of Iceland: a partial right whale skull from the high latitude Pliocene Tjörnes Formation

Extant baleen whales (Cetacea, Mysticeti) are a disparate and species‐rich group, but little is known about their fossil record in the northernmost Atlantic Ocean, a region that supports considerable extant cetacean diversity. Iceland's geographical setting, dividing North Atlantic and Arctic waters, renders it ideally situated to shed light on cetacean evolution in this region. However, as a volcanic island, Iceland exhibits very little marine sedimentary exposure, and fossil whales from Iceland older than the late Pleistocene are virtually unknown. Here, we present the first fossil whale found in situ from the Pliocene Tjörnes Formation (c. 4.5 Ma), Iceland's only substantial marine sedimentary outcrop. The specimen is diagnosed as a partial skull from a large right whale (Mysticeti, Balaenidae). This discovery highlights the Tjörnes Formation as a potentially productive fossil vertebrate locality. Additionally, this find indicates that right whales (Eubalaena) and bowhead whales (Balaena) were sympatric, with broadly overlapping latitudinal ranges in the Pliocene, in contrast to the modern latitudinal separation of their living counterparts.     

The evolution of self-incompatibility: a molecular voyeur's perspective

This review summarises current understanding of the evolution of self-incompatibility inferred from DNA sequence analysis. Self-incompatibility in many plant families is controlled by a single, highly polymorphicS-locus which, in the Solanaceae, encodes an allelic series of stylar ribonucleases known as the S-RNases. PCR approaches are a convenient way to examine the diversity of S-RNase sequences within and between wild populations of a self-incompatible species and provide a unique view into the species' current and historic population structure. Similar molecular appoaches have also been used to show that S-RNases are involved in self-incompatibility in families other than the Solanaceae. A model for the evolution of ribonuclease-based self-incompatibility systems is discussed.     

Preparation and Enantioselectivity Binding Studies of a New Chiral Cobalt(II)porphyrin‐Tröger's Base Conjugate

A new bis[cobalt(II)porphyrin]‐Tröger's base conjugate was studied as a potential receptor for methyl esters of several amino acids. The conjugate was prepared as racemate, and then resolved via preparative high‐performance liquid chromatography (HPLC) on a chiral column. The high affinity to lysine, histidine, and proline methyl esters was found by complexation studies followed by UV‐Vis spectroscopy. The studies of pure enantiomers, followed by UV‐Vis and electronic circular dichroism spectroscopy, revealed the highest enantioselectivity for lysine methyl ester. Chirality 26:361–367, 2014. © 2014 Wiley Periodicals, Inc.     

The tachykinin tale: molecular recognition in a historical perspective

Crystallography, mutational mapping and crosslinking are but a few of the experimental techniques that have helped to elucidate the underlying principles of molecular recognition between macromolecules and to improve our understanding of the evolution of the structure-activity relationship (SAR). While this development has been particularly successful for small and rigid ligands and substrates that bind to larger hydrophilic biomolecules, our understanding of membrane-embedded proteins is still rather limited. This review uses the example of the neuropeptide family of tachykinins and their G-protein coupled receptors (GPCR) to present how complementary experimental strategies over the past decades have nourished and modified conceptual models of the structural requisites of molecular recognition and function. Given the little we know, the pertinent question is how we proceed from here.     

The Rhaetian flora of Rögla,northern Scania,Sweden

Abstract: Rögla is the northernmost locality yielding Mesozoic plant fossils in Scania, southern Sweden, and is one of the northernmost Rhaetian assemblages in Europe. The assemblage consists of over 500 specimens collected 50–60 years ago, of which 139 yielded identifiable plant remains referable to 15 plant species; another 19 specimens are tentatively assigned to four species because of their fragmentary preservation. The flora includes sphenophytes, ferns, cycads, bennettitaleans, seed ferns of uncertain alliance, conifers and some leaf remains that are tentatively assigned to ginkgophytes based on their epidermal anatomy. The species‐level composition of the assemblage is consistent with a Rhaetian age and is similar to well‐known floras from nearby Höganäs and Bjuv, except for the absence of cycads belonging to Nilssonia, which are very common in most other Scanian floras. The fossil assemblage is interpreted to derive from multi‐storey vegetation occupying moist habitats on a coastal plain. Strong affinities are evident with the coeval floras of Jameson Land, Greenland, reinforcing the concept of a distinctive North Atlantic floristic sub‐province at the close of the Triassic.     

A structural perspective on the interactions of TRAF6 and Basigin during the onset of melanoma: A molecular dynamics simulation study

Metastatic melanoma is the most fatal type of skin cancer. The roles of matrix metalloproteinases (MMPs) have well been established in the onset of melanoma. Basigin (BSG) belongs to the immunoglobulin superfamily and is critical for induction of extracellular MMPs during the onset of various cancers including melanoma. Tumor necrosis factor receptor‐associated factor 6 (TRAF6) is an E3‐ligase that interacts with BSG and mediates its membrane localization, which leads to MMP expression in melanoma cells. This makes TRAF6 a potential therapeutic target in melanoma. We here conducted protein‐protein interaction studies on TRAF6 and BSG to get molecular level insights of the reactions. The structure of human BSG was constructed by protein threading. Molecular‐docking method was applied to develop the TRAF6‐BSG complex. The refined docked complex was further optimized by molecular dynamics simulations. Results from binding free energy, surface properties, and electrostatic interaction analysis indicate that Lys340 and Glu417 of TRAF6 play as the anchor residues in the protein interaction interface. The current study will be helpful in designing specific modulators of TRAF6 to control melanoma metastasis.     

Ultrastructure of the foregut and associated glands of Calicotyle kröyeri (Monogenea: Monopisthocotylea)

The mouth, pharynx and oesophagus of Calicotyle are lined by syncytial epithelia, and there are numerous unicellular glands associated with the oesophagus. An infolding of unmodified external tegument lines the mouth cavity and is connected by discrete cytoplasmic processes to subjacent perikarya. It contains two types of secretory body and its luminal surface is invested with a finely filamentous coating. The pharynx and oesophagus are lined by irregularly-folded epithelia that are interconnected by a septate desmosome. Membranous inclusions distinguish the pharynx epithelium and there is a well developed basal lamina for insertion of the pharyngeal muscles. The oesophagus epithelium is perforated by the openings of the oesophageal glands. These lie in the surrounding parenchyma and produce a dense, membrane-bound secretion which is conveyed by duct-like extensions of the glands to the oesophagus lumen. The ducts are supported in places by microtubules and are anchored to the oesophageal epithelium by septate desmosomes. A septate desmosome also marks the junction between the epithelium and the gut caeca.     

The interferon-inducible IFI16 gene inhibits tube morphogenesis and proliferation of primary, but not HPV16 E6/E7-immortalized human endothelial cells

Immunohistochemical analysis has demonstrated that the human IFI16 gene, in addition to the hematopoietic tissues, is highly expressed in endothelial cells and squamous stratified epithelia. In this study, we have developed a reliable HSV-derived replication-defective vector (TO-IFI16) to efficiently transduce IFI16 into primary human umbilical vein endothelial cells (HUVEC), which are usually poorly transfectable. HUVEC infection with TO-IFI16 virus suppressed endothelial migration, invasion and formation of capillary-like structures in vitro. In parallel, sustained IFI16 expression inhibited HUVEC cell cycle progression, accompanied by significant induction of p53, p21, and hypophosphorylated pRb. Further support for the involvement of these pathways in IFI16 activity came from the finding that infection with TO-IFI16 virus does not impair the in vitro angiogenic activity and cell cycle progression of HUVEC immortalized by HPV16 E6/E7 oncogenes, which are known to inactivate both p53 and pRb systems. This use of a reliable viral system for gene delivery into primary human endothelial cells assigns a potent angiostatic activity to an IFN-inducible gene, namely IFI16, and thus throws further light on antiangiogenic therapy employing IFNs.