Henk K. Koerten

Complete Polarization of Single Intestinal Epithelial Cells upon Activation of LKB1 by STRAD

The LKB1 gene encodes a serine/threonine kinase that is mutated in the Peutz-Jeghers cancer syndrome. LKB1 is homologous to the Par-4 polarity genes in C. elegans and D. melanogaster. We have previously reported the identification and characterization of an LKB1-specific adaptor protein, STRAD, which activates LKB1 and translocates it from nucleus to cytoplasm. We have now constructed intestinal epithelial cell lines in which inducible STRAD activates LKB1. Upon LKB1 activation, single cells rapidly remodel their actin cytoskeleton to form an apical brush border. The junctional proteins ZO-1 and p120 redistribute in a dotted circle peripheral to the brush border, in the absence of cell-cell contacts. Apical and basolateral markers sort to their respective membrane domains. We conclude that LKB1 can induce complete polarity in intestinal epithelial cells. In contrast to current thinking on polarization of simple epithelia, these cells can fully polarize in the absence of junctional cell-cell contacts.

Ultrastructure and Origin of Membrane Vesicles Associated with the Severe Acute Respiratory Syndrome Coronavirus Replication Complex

ABSTRACT The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.

The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR‐family regulator DasR and links N‐acetylglucosamine metabolism to the control of development

Members of the soil‐dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environment, and what controls the signal for the switch to antibiotic production and morphological development? Here we show that high extracellular concentrations of N‐acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N‐acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR‐family regulator DasR, which controls the N‐acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N‐acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6‐phosphate, a central molecule in N‐acetylglucosamine metabolism. Extracellular complementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circumstances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time developmental commitment.

The Production of Recombinant Infectious DI-Particles of a Murine Coronavirus in the Absence of Helper Virus

Abstract We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3′ end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity.

Degradation of calcium phosphate ceramics.

Degradation of three types of sintered calcium phosphate ceramic spheres was investigated in vitro at low pH conditions (LPC) and in an in vivo model, that is, injection into a mouse peritoneal cavity. Degradation was observed under both conditions. The rate of degradation depended on the type of ceramic, with beta-TCP degrading faster than HA and HA degrading faster than FA. Degradation was characterized by dissolution of the necks and the formation of cracks and irregularities in the grains. Intraperitoneal injection of the spheres into a mouse peritoneal cavity led to the formation of foreign body granulomas in which degradation could be observed. The in vivo degradation pattern was similar to that observed in vitro, but longer implantation times resulted in a further degradation. Small fragments rich in Ca and P were present in inclusion bodies. Calcium phosphate crystals sometimes also were observed in mitochondria, many of which were subject to lysis. We observed that ceramic type and implantation period also were related to the number of dead cells in the granulomas. Furthermore, extracellular deposits were seen between cells and ceramic spheres. Ca and P and also Fe were detected in these deposits. The presence of Fe is indicative of a lysosomal origin and thus of exocytotic activity.

Osteoblastic phenotype expression on the surface of hydroxyapatite ceramics

To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow cells and porous HA were implanted subcutaneously and harvested at 3 to 4 weeks postimplantation. De novo bone formation was observed primarily on the HA surface without fibrous tissue interposition. The HA/tissue interface was analyzed by the observations of thin undecalcified histological sections and fractured surfaces of the implants. The observations were done with a light microscope and a scanning electron microscope (SEM) connected to an energy dispersive spectrometer. The interfacial analyses showed the appearance of osteoblastic cells on the HA surface and that the cells had initiated partially mineralized bone (osteoid) formation directly onto the surface. The osteoid matured into fully mineralized bone, resulting in firm bone bonding to the HA surface. Characterization of osteoblastic cells on the surface was done by determining levels of protein and gene expression of bone Gla protein (BGP, a.k.a. Osteocalcin), i.e., immunohistochemistry and in situ hybridization, respectively. The existence of BGP and mRNA in the cytoplasmic area of the cells confirmed that active osteoblast apposition fabricated primary bone on the HA surface. All of these results indicate the importance of the HA surface in supporting osteoblastic differentiation of marrow stromal stem cells, which leads to firm bone bonding.

Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin

Abstract Tape stripping of human stratum corneum is widely used as a method for studying the kinetics and penetration depth of drugs. Several factors can influence the quantity of stratum corneum that is removed by a piece of tape, such as the manner of tape stripping, the hydration of the skin, cohesion between cells, body site and interindividual differences. However, few data are available about the influence of furrows in the human epidermis on the tape-stripping technique. In this study, we investigated the efficacy of tape stripping in removing complete cell layers from the superficial part of the human stratum corneum. A histological section of skin that was tape-stripped 20 times clearly showed nonstripped skin in the furrows, indicating persistent incomplete tape stripping. Replicas of tape-stripped skin surface demonstrated that even after removing 40 tape strips the furrows were still present. We validated the tape-stripping method further with X-ray microanalysis in the mapping mode by scanning electron microscopy, using a TiO 2 -containing compound as a marker. TiO 2 applied to the skin before the tape-stripping procedures was still present after the tenth tape strip, and was specifically located on the rims of the furrows. We emphasize that results from studies using the tape-stripping method have to be viewed from the perspective that cells on one tape strip of the stratum corneum may be derived from different layers, depending on the position of the tape strip in relation to the slope of the furrow, and such results should be interpreted with considerable caution.

Visualization of in vitro percutaneous penetration of mercuric chloride; transport through intercellular space versus cellular uptake through desmosomes

The aim of the present study was to visualize the routes of penetration of mercuric chloride through human skin in vitro at the ultrastructural level, and to find out to what extent intra- and extracellular space contribute to the percutaneous transport of the model compound. Dermatomed human skin was subjected to in vitro mercuric chloride diffusion experiments in a bicompartmental polycarbonate diffusion cell. Upon interrupting a diffusion experiment, skin samples were treated with ammonium sulfide vapour to precipitate the mercury as mercuric sulfide, then processed for transmission electron microscopy. The presence of mercury in the precipitates was verified using X-ray microanalysis. An additional series of experiments involved the immersion of freshly excised human plantar callus in mercuric chloride solutions, followed by the same tissue processing protocol as used for the dermatomed skin samples; the mercury treated callus samples were likewise subjected to transmission electron microscopy. The results indicate that the intercellular route of transport through the stratum corneum predominates, but that after longer transport times, apical corneocytes tend to take up material, leading to a bimodal distribution of mercury: in the apical region of the stratum corneum there is mercury both in- and outside the cells; in the medial and proximal region intercellular transport prevails. There were no signs of a discontinuity in the in-depth distribution of mercury in that an almost unmountable barrier would exist in the lower region of the stratum corneum, as suggested by e.g. Sharata and Burnette [1,2]. However, there was evidence of the presence of two types of cells: apical corneocytes, which tend to take up mercuric ions relatively easily, and medial and proximal corneocytes, which are less capable of doing so. Interestingly, the results furthermore indicated that intracellular mercury uptake in the apical, squamous region of the stratum corneum occurred preferentially via the desmosomes. This was also the case in the callus immersion studies. These results suggest that the desmosomes may serve to channel material into corneocytes, especially in the squamous region where the desmosomes are beginning to disintegrate and hence the cellular lipid envelopes are leaky. A reservoir function for the apical zone of the stratum corneum is being suggested.

Structure of the E. coli signal recognition particle bound to a translating ribosome

The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane. It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 Å structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.

New Aspects of the Skin Barrier Organization

In the superficial layer of the skin, the stratum corneum (SC), the lipids form two crystalline lamellar phases with periodicities of 6.4 and 13.4 nm (long-periodicity phase). The main lipid classes in SC are ceramides, free fatty acids and cholesterol. Studies with mixtures prepared with isolated ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases, and the presence of ceramide 1 is crucial for the formation of the long-periodicity phase. This observation and the broad-narrow-broad sequence of lipid layers in the 13.4-nm phase led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains on both sides of a broad layer with a crystalline structure. This model is referred to as ‘the sandwich model’. While the presence of free fatty acids does not substantially affect the lipid lamellar organization, it is crucial for the formation of the orthorhombic sublattice, since the addition of free fatty acids to cholesterol/ceramide mixtures results in transition from a hexagonal to a crystalline lipid phase. Studies examining lipid organization in SC derived from dry or lamellar X-linked ichthyosis skin revealed that in native tissue the role of ceramide 1 and free fatty acids is similar to that observed with mixtures prepared with isolated SC lipids. From this we conclude that the results obtained with lipid mixtures can be used to predict the SC lipid organization in native tissue.