Simon Crawford

NANOSTRUCTURE OF THE DIATOM FRUSTULE AS REVEALED BY ATOMIC FORCE AND SCANNING ELECTRON MICROSCOPY

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of cross‐linking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field‐emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in cross‐section revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 ± 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 ± 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 ± 1.4 nm and that silica particles from the girdle bands were 38.1 ± 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.

Independent Translocation of Two Micronemal Proteins in Developing Plasmodium falciparum Merozoites

ABSTRACT Apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) contains an N-terminal propeptide that is removed prior to the translocation of the mature protein onto the merozoite surface. We localized unprocessed PfAMA1 to the microneme organelles of the intraerythrocytic schizont. The results have suggested that the processed form of PfAMA1 translocates from the microneme compartment independently of another microneme protein, EBA175, which is also involved in the invasion of human erythrocytes.

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity

Prosurvival Bcl-2–like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

Spring constant calibration of atomic force microscope cantilevers of arbitrary shape

The spring constant of an atomic force microscope cantilever is often needed for quantitative measurements. The calibration method of Sader et al. [Rev. Sci. Instrum. 70, 3967 (1999)] for a rectangular cantilever requires measurement of the resonant frequency and quality factor in fluid (typically air), and knowledge of its plan view dimensions. This intrinsically uses the hydrodynamic function for a cantilever of rectangular plan view geometry. Here, we present hydrodynamic functions for a series of irregular and non-rectangular atomic force microscope cantilevers that are commonly used in practice. Cantilever geometries of arrow shape, small aspect ratio rectangular, quasi-rectangular, irregular rectangular, non-ideal trapezoidal cross sections, and V-shape are all studied. This enables the spring constants of all these cantilevers to be accurately and routinely determined through measurement of their resonant frequency and quality factor in fluid (such as air). An approximate formulation of the hydrodynamic function for microcantilevers of arbitrary geometry is also proposed. Implementation of the method and its performance in the presence of uncertainties and non-idealities is discussed, together with conversion factors for the static and dynamic spring constants of these cantilevers. These results are expected to be of particular value to the design and application of micro- and nanomechanical systems in general.

The glucans extracted with warm water from diatoms are mainly derived from intracellular chrysolaminaran and not extracellular polysaccharides

Several recent studies have employed warm-water treatment of diatom cells to extract nominally bound extracellular polymeric substances. Where examined, the dominant neutral sugar in these extracts was glucose. In the present study, we sought to characterize the structure of the glucose-rich polymers in the water extracts of diatoms and to determine the origin of these polymers. The marine diatoms surveyed were Phaeodactylum tricornutum, Cylindrotheca fusiformis, Craspedostauros australis and Thalassiosira pseudonana. A freshwater species, Pinnularia viridis, was also investigated for the dye labelling experiments. Freshly harvested marine diatoms were extracted with water at 30°C for 1 h. Constituent monosaccharide analyses showed that glucose was the dominant neutral sugar (80 – 95 mol% of the total) in the extracts from three marine species, whereas the P. tricornutum extract contained predominantly ribose, galactose and glucose, and was inferred to be enriched in low-molecular-weight components. Linkage analysis of the constituent monosaccharides and proton nuclear magnetic resonance spectroscopy showed that the glucose in these extracts was derived primarily from 1,3-β-D-glucan. Immunocytochemistry with a monoclonal anti-1,3-β-D-glucan antibody confirmed that the glucan was localized in the vacuoles of diatom cells preserved by freeze-substitution. Nearly all diatom cells incubated with a fluorescent dye, DiBAC4(3), during warm water treatment at 30°C or 60°C incorporated the dye, demonstrating that the membrane integrity of the diatoms was compromised and supporting the contention that intracellular glucan was released during the treatment. In light of these data, the extracellular glucans of diatoms reported in some previous studies are re-interpreted as intracellular chrysolaminaran.

PG0026 Is the C-terminal Signal Peptidase of a Novel Secretion System of Porphyromonas gingivalis

Background: Several virulence factors of Porphyromonas gingivalis have a novel C-terminal signal that directs secretion across the outer membrane. Results: The predicted catalytic amino acid of PG0026 was essential for the removal of this signal. Conclusion: PG0026 is a novel C-terminal signal peptidase. Significance: We have identified a novel signal peptidase of a new type of secretion system. Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ∼70–80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.

High Rate of Antibody Secretion Is not Integral to Plasma Cell Differentiation as Revealed by XBP-1 Deficiency

During B cell terminal differentiation, a complex set of transcription factors interact to drive the phenotypic and functional changes leading to the development of Ab-secreting cells (ASCs). The transcription factor X-box binding protein 1 (XBP-1) is an essential part of one of the branches of the unfolded protein response (UPR). The UPR is induced when a cell has to handle large amounts of proteins, as is the case in ASCs. Although XBP-1 was initially also ascribed an indispensable function in plasma cell development, later studies of B cell-specific deletion reported a much milder consequence of XBP-1 deficiency. Our interest was to determine whether XBP-1 was integral for the differentiation of plasma cells. Using both in vitro and in vivo assays, we found efficient generation of ASCs in the absence of XBP-1. ASCs were present at normal frequencies in resting and immunized mice and displayed a pattern of surface markers typical for plasma cells. The absence of XBP-1 resulted in a reduction but not ablation of Ab secretion and the failure to develop the cellular morphology characteristic of ASCs. Thus, XBP-1 deficiency demonstrates that the gene regulatory program controlling plasma cell differentiation can proceed relatively normally in the absence of high rates of Ig secretion.

Blimp-1 controls plasma cell function through the regulation of immunoglobulin secretion and the unfolded protein response

Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.

Porphyromonas gingivalis and Treponema denticola synergistic polymicrobial biofilm development.

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.

Abortive Replication of Influenza Virus in Mouse Dendritic Cells

ABSTRACT The interaction between influenza virus and dendritic cells (DCs) remains poorly defined and controversial. Here we show that influenza virus replication in mouse bone marrow-derived DCs is abortive, despite viral genome transcription and replication occurring for each gene segment and viral hemagglutinin and nucleoprotein, at least, being produced. Electron microscopy reveals that virus assembly, rather than release of virus from the cell surface, is defective.