Subcellular Localization of the TFF Peptides xP1 and xP4 in the Xenopus laevis Gastric/Esophageal Mucosa: Different Secretion Modes Reflecting Diverse Protective Functions.

The TFF peptides xP1 and xP4 from Xenopus laevis are orthologs of TFF1 and TFF2, respectively. xP1 is secreted as a monomer from gastric surface mucous cells and is generally not associated with mucins, whereas xP4 is a typical secretory peptide from esophageal goblet cells, and gastric mucous neck and antral gland cells tightly associated as a lectin with the ortholog of mucin MUC6. Both TFF peptides have diverse protective functions, xP1 as a scavenger for reactive oxygen species preventing oxidative damage and xP4 as a constituent of the water-insoluble adherent inner mucus barrier. Here, we present localization studies using immunofluorescence and immunoelectron microscopy. xP1 is concentrated in dense cores of secretory granules of surface mucous cells, whereas xP4 mixes with MUC6 in esophageal goblet cells. Of note, we observe two different types of goblet cells, which differ in their xP4 synthesis, and this is even visible morphologically at the electron microscopic level. xP4-negative granules are recognized by their halo, which is probably the result of shrinkage during the processing of samples for electron microscopy. Probably, the tight lectin binding of xP4 and MUC6 creates a crosslinked mucous network forming a stabile granule matrix, which prevents shrinkage.

The putative C-type lectin Schlaff ensures epidermal barrier compactness in Drosophila.

The stability of extracellular matrices is in general ensured by cross-linking of its components. Previously, we had shown that the integrity of the layered Drosophila cuticle relies on the presence of a covalent cuticular dityrosine network. Production and composition of this structure remained unstudied. In this work, we present our analyses of the schlaff (slf) gene coding for a putative C-type lectin that is needed for the adhesion between the horizontal cuticle layers. The Slf protein mainly localizes between the two layers called epicuticle and procuticle that separate from each other when the function of Slf is reduced or eliminated paralleling the phenotype of a cuticle with reduced extracellular dityrosine. Localisation of the dityrosinylated protein Resilin to the epicuticle-procuticle interface suggests that the dityrosine network mediates the adhesion of the epicuticle to the procuticle. Ultimately, compromised Slf function is associated with massive water loss. In summary, we propose that Slf is implied in the stabilisation of a dityrosine layer especially between the epicuticle and the procuticle that in turn constitutes an outward barrier against uncontrolled water flow.

The Intimin periplasmic domain mediates dimerisation and binding to peptidoglycan.

Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a ß-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 µM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.

RuvB-like ATPases function in chromatin decondensation at the end of mitosis.

Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

Correlative light and electron microscopy using immunolabeled sections.

In correlative microscopy, light microscopy provides the overview and orientation of the complex cells and tissue, while electron microscopy offers the detailed localization and correlation of subcellular structures. In this chapter we offer detailed high-quality electron microscopical preparation methods for optimum preservation of the cellular ultrastructure. From such preparations serial thin sections are collected and used for comparative histochemical, immunofluorescence, and immunogold staining.In light microscopy histological stains identify the orientation of the sample and immunofluorescence labeling facilitates to find the region of interest, namely, the labeled cells expressing the macromolecule under investigation. Sections, labeled with immunogold are analyzed by electron microscopy in order to identify the label within the cellular architecture at high resolution.

Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease.

We present here the positional cloning of the Danio rerio albino mutant and show that the affected gene encodes Slc45a2. The human orthologous gene has previously been shown to be involved in human skin color variation, and mutations therein have been implicated in the disease OCA4. Through ultrastructural analysis of the melanosomes in albino alleles as well as the tyrosinase-deficient mutant sandy, we add new insights into the role of Slc45a2 in the production of melanin. To gain further understanding of the role of Slc45a2 and its possible interactions with other proteins involved in melanization, we further analyzed the role of the V-ATPase as a melanosomal acidifier. We show that it is possible to rescue the melanization potential of the albino melanosomes through genetic and chemical inhibition of V-ATPase, thereby increasing internal melanosome pH.

Spastic paraplegia mutation N256S in the neuronal microtubule motor KIF5A disrupts axonal transport in a Drosophila HSP model.

Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP-like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.

Serum Response Factor (SRF)-cofilin-actin signaling axis modulates mitochondrial dynamics.

Aberrant mitochondrial function, morphology, and transport are main features of neurodegenerative diseases. To date, mitochondrial transport within neurons is thought to rely mainly on microtubules, whereas actin might mediate short-range movements and mitochondrial anchoring. Here, we analyzed the impact of actin on neuronal mitochondrial size and localization. F-actin enhanced mitochondrial size and mitochondrial number in neurites and growth cones. In contrast, raising G-actin resulted in mitochondrial fragmentation and decreased mitochondrial abundance. Cellular F-actin/G-actin levels also regulate serum response factor (SRF)-mediated gene regulation, suggesting a possible link between SRF and mitochondrial dynamics. Indeed, SRF-deficient neurons display neurodegenerative hallmarks of mitochondria, including disrupted morphology, fragmentation, and impaired mitochondrial motility, as well as ATP energy metabolism. Conversely, constitutively active SRF-VP16 induced formation of mitochondrial networks and rescued huntingtin (HTT)-impaired mitochondrial dynamics. Finally, SRF and actin dynamics are connected via the actin severing protein cofilin and its slingshot phosphatase to modulate neuronal mitochondrial dynamics. In summary, our data suggest that the SRF-cofilin-actin signaling axis modulates neuronal mitochondrial function.

Contact behaviour of setal tips in the hairy attachment system of the fly Calliphora vicina (Diptera, Calliphoridae): a cryo-SEM approach.

The fly Calliphora vicina (Diptera, Calliphoridae) bears attachment pads (pulvilli) covered with setae on their ventral sides. These structures enable attachment to smooth vertical surfaces and ceilings. The contact between the terminal setal tips (spatulae) and various substrates was visualised using various experimental techniques combined with conventional scanning electron microscopy (SEM) and cryo-SEM. The results show that the setal endplates are highly flexible structures that form contact with the surface by bending their tips in the distal direction. With conventional SEM, a comparison of partly attached endplates with unattached endplates demonstrated the presence of a distinct marginal bulge. As observed with cryo-SEM, the bulge continuously disappeared as a larger area of the endplate came into contact. Two explanations of this result are suggested. First, the volume between the bulge, the mid-part of the endplate and the substrate may be filled with a fluid secretion that is released into the contact area in the endplate region. Second, the flexible central part of the endplate may jump into contact with the substrate during contact formation.

Analysis of the BadA stalk from Bartonella henselae reveals domain-specific and domain-overlapping functions in the host cell infection process.

Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin Bartonella adhesin A (BadA) which is modularly constructed and consists of a head, a long and repetitive neck-stalk module with 22 repetitive neck/stalk repeats and a membrane anchor. The BadA head is crucial for bacterial adherence to host cells, binding to several extracellular matrix proteins and for the induction of vascular endothelial growth factor (VEGF) secretion. Here, we analysed the biological role of the BadA stalk in the infection process in greater detail. For this purpose, BadA head-bearing and headless deletion mutants with different lengths (containing one or four neck/stalk repeats in the neck-stalk module) were produced and functionally analysed for their ability to bind to fibronectin, collagen and endothelial cells and to induce VEGF secretion. Whereas a head-bearing short version (one neck/stalk element) of BadA lacks exclusively fibronectin binding, a substantially truncated headless BadA mutant was deficient for all of these biological functions. The expression of a longer headless BadA mutant (four neck/stalk repeats) restored fibronectin and collagen binding, adherence to host cells and the induction of VEGF secretion. Our data suggest that (i) the stalk of BadA is exclusively responsible for fibronectin binding and that (ii) both the head and stalk of BadA mediate adherence to collagen and host cells and the induction of VEGF secretion. This indicates overlapping functions of the BadA head and stalk.

Characterization of NE81, the first lamin-like nucleoskeleton protein in a unicellular organism.

Lamins build the nuclear lamina and are required for chromatin organization, gene expression, cell cycle progression, and mechanical stabilization. Despite these universal functions, lamins have so far been found only in metazoans. We have identified protein NE81 in Dictyostelium, which has properties that justify its denomination as a lamin-like protein in a lower eukaryote. This is based on its primary structure, subcellular localization, and regulation during mitosis, and its requirement of the C-terminal CaaX box as a posttranslational processing signal for proper localization. Our knockout and overexpression mutants revealed an important role for NE81 in nuclear integrity, chromatin organization, and mechanical stability of cells. All our results are in agreement with a role for NE81 in formation of a nuclear lamina. This function is corroborated by localization of Dictyostelium NE81 at the nuclear envelope in human cells. The discovery of a lamin-like protein in a unicellular organism is not only intriguing in light of evolution, it may also provide a simple experimental platform for studies of the molecular basis of laminopathies.

Metabolic priming by a secreted fungal effector.

Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

FraH is required for reorganization of intracellular membranes during heterocyst differentiation in Anabaena sp. strain PCC 7120.

In the filamentous, heterocyst-forming cyanobacteria, two different cell types, the CO(2)-fixing vegetative cells and the N(2)-fixing heterocysts, exchange nutrients and regulators for diazotrophic growth. In the model organism Anabaena sp. strain PCC 7120, inactivation of fraH produces filament fragmentation under conditions of combined nitrogen deprivation, releasing numerous isolated heterocysts. Transmission electron microscopy of samples prepared by either high-pressure cryo-fixation or chemical fixation showed that the heterocysts of a ΔfraH mutant lack the intracellular membrane system structured close to the heterocyst poles, known as the honeycomb, that is characteristic of wild-type heterocysts. Using a green fluorescent protein translational fusion to the carboxyl terminus of FraH (FraH-C-GFP), confocal microscopy showed spots of fluorescence located at the periphery of the vegetative cells in filaments grown in the presence of nitrate. After incubation in the absence of combined nitrogen, localization of FraH-C-GFP changed substantially, and the GFP fluorescence was conspicuously located at the cell poles in the heterocysts. Fluorescence microscopy and deconvolution of images showed that GFP fluorescence originated mainly from the region next to the cyanophycin plug present at the heterocyst poles. Intercellular transfer of the fluorescent tracers calcein (622 Da) and 5-carboxyfluorescein (374 Da) was either not impaired or only partially impaired in the ΔfraH mutant, suggesting that FraH is not important for intercellular molecular exchange. Location of FraH close to the honeycomb membrane structure and lack of such structure in the ΔfraH mutant suggest a role of FraH in reorganization of intracellular membranes, which may involve generation of new membranes, during heterocyst differentiation.

FraC/FraD-dependent intercellular molecular exchange in the filaments of a heterocyst-forming cyanobacterium, Anabaena sp.

The filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients and regulators. In Anabaena sp. strain PCC 7120, inactivation of sepJ or genes in the fraC operon (fraC, fraD and fraE) produce filament fragmentation. SepJ, FraC and FraD are cytoplasmic membrane proteins located in the filament's intercellular septa that are needed for intercellular exchange of the fluorescent tracer calcein (622 Da). Transmission electron microscopy showed an alteration in the heterocyst cytoplasmic membrane at the vegetative cell-heterocyst septa in ΔfraC and ΔfraD mutants. Immunogold labelling of FraD confirmed its localization in the intercellular septa and clearly showed the presence of part of the protein between the cytoplasmic membranes of the adjacent cells. This localization seemed to be affected in the ΔfraC mutant but was not impaired in a ΔsepJ mutant. Intercellular transfer of a smaller fluorescent tracer, 5-carboxyfluorescein (374 Da), was largely impaired in ΔfraC, ΔfraD and double ΔfraC-ΔfraD mutants, but much less in the ΔsepJ mutant. These results show the existence in the Anabaena filaments of a FraC/FraD-dependent intercellular molecular exchange that does not require SepJ.

The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins.

The Escherichia coli Ig-binding (Eib) proteins are trimeric autotransporter adhesins (TAAs) and receptors for IgG Fc. We present the structure of a large fragment of the passenger domain of EibD, the first TAA structure to have both a YadA-like head domain and the entire coiled-coil stalk. The stalk begins as a right-handed superhelix, but switches handedness halfway down. An unexpected ß-minidomain joins the two and inserts a ∼120° rotation such that there is no net twist between the beginning and end of the stalk. This may be important in folding and autotransport. The surprisingly large cavities we found in EibD and other TAAs may explain how TAAs bend to bind their ligands. We identified how IgA and IgG bind and modeled the EibD-IgG Fc complex. We further show that EibD promotes autoagglutination and biofilm formation and forms a fibrillar layer covering the cell surface making zipper-like contacts between cells.

Functional dissection of SiiE, a giant non-fimbrial adhesin of Salmonella enterica.

Salmonella enterica deploys the giant non-fimbrial adhesin SiiE to adhere to the apical side of polarized epithelial cells. The establishment of close contact is a prerequisite for subsequent invasion mediated by translocation of effector proteins of the Salmonella Pathogenicity Island 1 (SPI1)-encoded type III secretion system (T3SS). Although SiiE is secreted into the culture medium, the adhesin is retained on the bacterial envelope in the phase of highest bacterial invasiveness. To dissect the structural requirements for secretion, retention and adhesive properties, comprehensive deletional and functional analyses of various domains of SiiE were performed. We observed that ß-sheet and coiled-coil domains in the N-terminal moiety of SiiE are required for the control of SiiE retention on the surface and co-ordinated release. These results indicate a novel molecular mechanism for the control of surface display of a T1SS-secreted adhesin that acts cooperatively with the SPI1-T3SS.

Electron microscopy techniques to study bacterial adhesion.

Since its introduction 70 years ago electron microscopy has become an invaluable tool for microbiology and the study of bacterial interaction. Technological development over the past decades has enabled researchers to resolve smaller and smaller details in bacterial samples, while new preparation techniques like cryo preparation now allow to investigate bacteria even closer to their natural state. In this chapter we give a brief overview of electron microscopy techniques suitable for the investigation of bacterial adhesion at molecular as well as cellular level and a short outlook on future technologies relevant to the field.

Trimeric autotransporter adhesin-dependent adherence of Bartonella henselae, Bartonella quintana, and Yersinia enterocolitica to matrix components and endothelial cells under static and dynamic flow conditions.

Trimeric autotransporter adhesins (TAAs) are important virulence factors of Gram-negative bacteria responsible for adherence to extracellular matrix (ECM) and host cells. Here, we analyzed three different TAAs (Bartonella adhesin A [BadA] of Bartonella henselae, variably expressed outer membrane proteins [Vomps] of Bartonella quintana, and Yersinia adhesin A [YadA] of Yersinia enterocolitica) for mediating bacterial adherence to ECM and endothelial cells. Using static (cell culture vials) and dynamic (capillary flow chambers) experimental settings, adherence of wild-type bacteria and of the respective TAA-negative strains was analyzed. Under static conditions, ECM adherence of B. henselae, B. quintana, and Y. enterocolitica was strongly dependent on the expression of their particular TAAs. YadA of Y. enterocolitica did not mediate bacterial binding to plasma or cellular fibronectin under either static or dynamic conditions. TAA-dependent host cell adherence appeared more significant under dynamic conditions although the total number of bound bacteria was diminished compared to the number under static conditions. Dynamic models expand the methodology to perform bacterial adherence experiments under more realistic, bloodstream-like conditions and allow dissection of the biological role of TAAs in ECM and host cell adherence under static and dynamic conditions.

The Alg5 ortholog Wollknäuel is essential for correct epidermal differentiation during Drosophila late embryogenesis.

The formation of an extracellular matrix (ECM) presupposes an ordered delivery of its components to ensure its stereotypic architecture. The Drosophila cuticle is an ECM produced by the epidermis at its apical site and is characterized by a layered organization. To understand the mechanisms of cuticle assembly during development, we have investigated early aspects of protein N-glycosylation, i.e. the attachment of a dolichol-linked oligosaccharide to distinct Asn sites of a protein known to be essential for sorting in the secretory pathway. Mutations in the Drosophila alg5 gene wollknäuel (wol) that codes for an enzyme initiating the glucosylation of the dolichol-linked oligosaccharide decrease, as expected, glucosylation and the amounts of N-glycosylated proteins such as the cuticle-organizing factor Knickkopf, without affecting their correct localization. At the same time, the polarity determinants Crumbs and atypical protein kinase C accumulate at the apical plasma membrane in wol deficient embryos. In part, these perturbations may also be caused by the unfolded protein response, which is commonly triggered by ER stress and downsizes transcription and translation in general. In any case, they are associated with the loss of cuticle layering and aberrant apical plasma membrane organization suggesting that glucosylation, either directly or indirectly through controlling protein degradation, is important for the efficient and balanced deployment of the biochemical functions of secreted and membrane-associated proteins during epidermal differentiation.

Does a low-pH microenvironment around phototrophic Fe(II) -oxidizing bacteria prevent cell encrustation by Fe(III) minerals?

Neutrophilic Fe(II) -oxidizing bacteria precipitate positively charged Fe(III) minerals that are expected to sorb to the negatively charged cell surface, leading to encrustation and thus limiting the cells' accessibility to substrates and nutrients. However, electron-microscopy analysis of phototrophic iron-oxidizing Thiodictyon sp. strain F4 cells showed no encrustation, but mineral precipitation at a distance from the cell surface. In situ fluorescence microscopy analysis of F4 cells using a pH-sensitive fluorescent dye revealed a low cell surface pH (6.0 ± 0.1) in contrast to the bulk pH (6.6 ± 0.1). Biogeochemical modeling showed that the pH difference reduces Fe(III) sorption and Fe(III) precipitation rates at the cell surface, therefore directing mineral formation away from the cells. The results from this study therefore suggest that the establishment of a low cell surface pH could provide a mechanism for photoferrotrophs to successfully prevent Fe(III) mineral precipitation on the cell surface.