Uli Schwarz

Autolytic enzymes and cell division of Escherichia coli

Abstract The shape of most bacteria is determined by a defined macromolecule which encloses the cell completely. Therefore, in this case, morphogenesis can be studied by analyzing the biosynthesis of this defined type of molecule, a sacculus, which is made up from the polymer murein. The sacculus can be isolated in pure form and can be examined under the electron microscope. We took advantage of this fact to study some topological aspects of murein biosynthesis in Escherichia coli. The growth of the structural determinant of the cell wall, the sacculus, was found to be carried out by several functionally different systems. One is involved in cell elongation, others in cell division; these systems were revealed by their differential sensitivity to penicillin. By use of this antibiotic, we were able to localize the site of action of murein hydrolases which are directly involved in bacterial morphogenesis; the topology and timing of their action are correlated with DNA replication. This procedure revealed zonal growth of the sacculus during cell division. Thus, this structural element of the cell wall is enlarged exactly as predicted by the replicon hypothesis.

Structural Design and Biomechanics of Friction-Based Releasable Attachment Devices in Insects

Abstract Design of attachment devices in insects varies enormously in relation to different functional loads. Many systems, located on different parts of the body, involve surfaces with particular frictional properties. Such systems evolved to attach parts of the body to each other, or to attach an insect to the substratum by providing fast and reversible attachment/detachment. Among these systems, there are some that deal with predefined surfaces, and others, in which one surface remains unpredictable. The first type of system occurs, for example, in wing-locking devices and head-arresting systems and is called probabilistic fasteners. The second type is mainly represented by insect attachment pads of two alternative designs: hairy and smooth. The relationship between surface patterns and/or mechanical properties of materials of contact pairs results in two main working principles of the frictional devices: mechanical interlocking, or maximization of the contact area. We give an overview of the functional design of two main groups of friction-based attachment devices in insects: probabilistic fasteners and attachment pads.

Chemical composition of the attachment pad secretion of the locust Locusta migratoria

This study is the first attempt to characterise the chemical composition of the secretion of the smooth pads of the locust Locusta migratoria and to relate this to the composition of the cuticle coverage of the pads and the wings. Gas-chromatography and mass-spectrometry (GC-MS) were the principal techniques used for the characterization of these materials. Secretion droplets were visualised and quantified with the aid of diverse microscopic techniques. The chemical composition of prints is shown to differ from the cuticle coverage, in particular, with respect to the fatty acid distribution: in the secretion, saturated and unsaturated fatty acids with chain lengths between C(16) and C(20) in both the free form and as glycerides predominate, whereas cuticle coverage contains waxes of long-chained fatty-acids bound to long-chain primary alcohols. The second important difference is the significant amount of glucose and other saccharides found in methanolyzates of the pad fluid. A considerable amount of the amino acids (up to 53%) was detected in the non-volatile portion of the fluid. Data obtained from the shock-freezing, carbon-platinum coating and replica preparation show that the secretory droplets contain nano-droplets on their surfaces. The results lead us to suggest that the pad secretion is an emulsion consisting of lipidic nano-droplets dispersed in an aqueous liquid. According to the chemical composition of the secretion, a high-viscosity of the fluid may be suggested. Presumably, the fluid is a kind of a coupling agent, promoting and strengthening adhesion between otherwise incompatible materials by providing the proximity of contact for intermolecular forces.

Induction of axonal growth by heterophilic interactions between the cell surface recognition proteins Fll and Nr-CAM/Bravo

F11 and Nr-CAM/Bravo are two axon-associated glycoproteins belonging to different subgroups of the immunoglobulin superfamily. In this report we have investigated the interaction of both proteins using neurite outgrowth and binding assays. Antibody blocking experiments demonstrate that neurite extension of tectal cells on immobilized F11 is mediated by Nr-CAM/Bravo. Binding studies further reveal a direct heterophilic interaction between F11 and Nr-CAM/Bravo. This activity can be mapped to the amino-terminal second or third immunoglobulin-like domain within F11 with domain-specific monoclonal antibodies and deletion mutant proteins expressed on COS cells. Furthermore, perturbation experiments with domain-specific monoclonal antibodies demonstrate that this region is required for adhesion and neurite extension.

Oriented axon outgrowth from avian embryonic retinae in culture

The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged varied with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explanation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.

Topologically restricted appearance in the developing chick retinotectal system of Bravo, a neural surface protein: experimental modulation by environmental cues.

A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance.

Guidance and topographic stabilization of nasal chick retinal axons on target-derived components in vitro

We studied mechanisms underlying the generation of topographic order within the developing chick retinotectal connection by combining the recently introduced stripe assay with a novel membrane protein fractionation technique. Our experiments show a preference of temporal and nasal retinal fibers for growing on cell membranes prepared from their proper target area. In addition, membrane preparations from posterior tectum were found to prolong substantially the survival of nasal neurites in vitro. We conclude that tropic as well as trophic interactions contribute to the generation of topographic maps during embryogenesis, in our case to the homing of nasal axons within the posterior tectum.

Mechanisms in the development of retinotectal projections in the chick embryo studied by surgical deflection of the retinal pathway

Retinotopic analysis of the pathways of normal and aberrant retinal axons within the tectum of developing chick embryos was performed by selective labeling of retinal axons with a fluorescent dye, rhodamine-B isothiocyanate. To produce aberrant retinal axons, the presumptive optic chiasma was surgically disorganized at the 3rd day of incubation. At the 11th and 13th days of incubation, more than half of the operated embryos exhibited several aberrant retinal axons which reached ectopic parts of the tectum. The pathways of these aberrant axons within the tectum depended on the position of their initial invasion into the tectum at the diencephalotectal junction, and not on their position of origin within the retina. The aberrant retinal axons did not show any sign of correction of their pathways toward their normal sites of innervation within the tectum. As development proceeded, elimination of the aberrant retinal axons occurred. By the 16th day of incubation, almost all operated embryos lacked aberrant retinal axons and although the total number of axons often appeared reduced, a nearly normal topography of retinotectal projections was established. These findings indicate that the initial invasion of the retinal axons into the tectum is conducted predominantly by nonspecific mechanisms and, thereafter, a selective maintenance of appropriate retinal axons occurs.

Synergistic Neurite-outgrowth Promoting Activity of Two Related Axonal Proteins, Bravo/Nr-CAM and G4/Ng-CAM in Chicken Retinal Explants

In the developing chicken retina, optic fibres migrating to the tectum express on their surfaces several cell adhesion molecules, including Bravo/Nr‐CAM and G4/Ng‐CAM. We have previously described differential distribution along the retinotectal projection and differential modulation by environmental cues for Bravo and G4 and here we further compare the characteristics of these immunoglobulin superfamily molecules. From day 6 of embryonic development (E6) to day 20 (E20), Bravo and G4 were found to coexist in the retinal optic fibre layer. However, while G4 staining was confined to that layer, as development proceeded Bravo staining spread to the plexiform layers and some radial structures of the retina. G4 displayed a dose‐dependent neurite‐outgrowth promoting activity for E6 retinal explants, while Bravo did not support neurite growth. Surprisingly, when the retinal explants were grown on mixtures of the two molecules, a much more vigorous growth of neurites was seen, revealing a synergistic effect. We propose that Bravo and G4, as well as other axonal surface molecules, affect axonal growth in different ways when they are present in combination than when they are alone.

Differential impact of semaphorin 3E and 3A on CNS axons.

During the development of the central nervous system (CNS), the correct wiring of outgrowing neurites is mediated by antagonistic mechanisms. Aberrant growth is prevented by repulsive factors such as semaphorins. Expression of the ligands Sema3A and ‐3E and the receptors neuropilin Npn‐1, ‐2a and ‐2b in the chick visual system were analyzed by RT‐PCR. Whereas Sema3A and its major receptor Npn‐1 were abundant, Sema3E and Npn‐2 isoform expression was highly restricted and developmentally regulated. Peak expression occurred during retinal axon innervation of the tectum. Functional in vitro assays with recombinant proteins revealed a topography‐specific growth cone collapsing activity of Sema3A for tectal axons. Interestingly, whereas tectal axons collapsed in a topographic‐specific manner only in the presence of Sema3A, retinal axons responded only to Sema3E. The collapsing activity was intracellularly mediated by cGMP. For a detailed analysis of neuronal responses to sempahorins, time lapse video recording was performed. When tectal and retinal axons were pre‐exposed to brain‐derived neurotrophic factor (BDNF), a protective effect was evident only in the case of retinal axons. Our results suggest a molecular mechanism whereby ingrowth of retinal axons into the tectum can be regulated by Sema3E/BDNF modulation without disturbing tectal axon growth out of the tectum mediated by Sema3A.