Robert P. Stidwill

Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake.

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the αv integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on αv integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn–expressing cells. Surprisingly, 30–50% of the endosomal contents were released into the cytosol of control and also K44A-dyn–expressing cells, and the number of fluid phase–positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection.

Import of adenovirus DNA involves the nuclear pore complex receptor CAN/Nup214 and histone H1

Adenovirus type 2 (Ad2) imports its DNA genome through the nuclear pore complex (NPC) of cells in interphase for viral production. Here we identify the NPC-filament protein CAN/Nup214 as a docking site for incoming Ad2 capsids. Binding to CAN is independent of cytosolic factors. Capsids disassemble at NPCs to free their DNA for import. This process requires binding of nuclear histone H1 to the stably docked capsids and involves H1-import factors, restricting this irreversible process to the proximity of the nucleus. Our results provide a molecular mechanism for disassembly of Ad2 and reveal an unexpected function of histone H1 in virus-mediated DNA import.

The role of the nuclear pore complex in adenovirus DNA entry

Adenovirus targets its genome to the cell nucleus by a multistep process involving endocytosis, membrane penetration and cytoplasmic transport, and finally imports its DNA into the nucleus. Using an immunochemical and biochemical approach combined with inhibitors of nuclear import, we demonstrate that incoming viral DNA and DNA‐associated protein VII enter the nucleus via nuclear pore complexes (NPCs). Depletion of calcium from nuclear envelope and endoplasmic reticulum cisternae by ionophores or thapsigargin blocked DNA and protein VII import into the nucleus, but had no effect on virus targeting to NPCs. Calcium‐depleted cells were capable of disassembling incoming virus. In contrast, inhibitors of cytosolic O‐linked glycoproteins of the NPC blocked virus attachment to the nuclear envelope, capsid disassembly and also nuclear import of protein VII. The data indicate that NPCs have multiple roles in adenovirus entry into cells: they contain a virus‐binding and/or dissociation activity and provide a gateway for the incoming DNA genome into the nucleus.

The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RDG-dependent☆

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to fibronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 micrograms/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.

Heat dissociation and maceration of marine Cnidaria

SummaryThe effect of increased temperature on the tissue integrity of polyps and medusae ofPodocoryne carnea is described. Animals exposed for 10 to 20 min to a temperature of 35°C are easily dissociated into single cells. These dissociated cells round up, form reaggregates and, depending on their origin, regenerate polyp or medusa structures. However, as the exposure time is increased, the dissociated cells gradually lose the ability to reaggregate or to regenerate defined structures. At incubation times exceeding 50 min, the tissue separates into single cells which retain their normalin vivo shapes but which do not form reaggregates. These are termed macerated cells. The ultrastructure and protein profile of macerated cells demonstrate no major changes from those of untreated cells. Both the dissociation and maceration methods are applicable to other cnidarian species for developmental, histological and biochemical studies.

The contributions of microtubules and F-actin to the in Vitro migratory mechanisms of Hydra nematocytes as determined by drug interference experiments

In order to investigate the contributions of microtubules and of F-actin to the in vitro migration mechanisms of Hydra nematocytes we have studied the effects of agents directed against cytoskeletal structures. Disassembly of microtubules by treatment with the drug nocodazole in moving nematocytes resulted in the loss of all locomotory activity within 20 min after the onset of treatment and in the detachment from the substratum after about 30 min. Depolymerization of microtubules by exposure to low temperatures had the same effect but was reversible in this case. Locomoting cells treated with cytochalasin D, which disrupts the actin filaments, stopped movement 2 min after drug administration and detached from the substratum after 15 min. The pattern of F-actin, alpha-tubulin, and tyrosinated tubulin in drug- or cold-treated cells was determined by immunocytochemical techniques and confocal laser scanning microscopy. These patterns and the reactions of the cells to the various drug treatments suggest that both actin filaments and microtubules play a crucial role in nematocyte locomotion. Analysis of the cytoskeletal pattern in drug-treated cells shows that the microtubules which are involved in locomotion are mostly tyrosinated. Furthermore it is suggested that microtubules and actin filaments interact with each other during the locomotion of nematocytes.

A single layer of microtubules is part of a complex cytoskeleton in mature nematocytes of hydra

The architecture of microtubules in mature nematocytes (stinging cells) of Hydra attenuata was investigated in detail by an indirect immunofluorescence study and by scanning and transmission electron microscopy in order to comprehend the function of the cytoskeleton in this extremely complex cell type. Microtubules were detected in all types of nematocytes in the tentacles and were found to be arranged in parallel arrays forming a highly organized basket-like structure around the nematocysts.

Alteration of fibronectin affinity during differentiation modulates the in vitro migration velocities of Hydra nematocytes

In the fresh water Cnidarian Hydra nematocytes differentiate from stem cells in the body column of the polyps and are functional in the tentacles to where they migrate as single cells in an amoeboid fashion. The fluorescent vital stain TROMI (tetramethyl-rhodamine-5/6-maleimide) allows to easily discriminate between cells located in the body column and cells mounted in the tentacles. The two cell populations were found to have different in vitro migration properties. These differences appear to be due largely to a differential attachment to fibronectin. Nematocytes from the tentacles show significantly lower in vitro migration velocities on isolated pieces of the organisms extracellular matrix (the mesoglea) and attach more firmly to fibronectin-coated substrates than cells from the body column. Pretreatment of the mesogleae with antibodies against the cell binding domain of fibronectin or addition of RGD-peptides results in an increase of the average migration velocity of cells from the tentacles and a decreased velocity of the cells from the body column. These findings suggest that (1) modulation of the attachment to fibronectin is decisive for the observed differential migration properties of the two cell populations and (2) the in vitro migration of nematocytes is dependent on subtle and transient interactions of cell surface receptors (most probably integrins) and fibronectin.

Factors effecting manubrium-regeneration in hydromedusae (Coelenterata)

Summary1.Umbrellar fragments of the leptomedusaCampanularia johnstoni with or without parts of the radial canal demonstrate a gradient in the potential for manubrium regeneration and in regeneration time.2.Implantation experiments exclude the manubrium as a source of inhibition or induction in the regeneration of another manubrium. One special case of inhibition appears to be due to competition for a common substrate.3.Medusa fragments consisting of only peripheral umbrella (C-fragments) undergo a considerably different restitution process as compared with fragments including a central portion of the umbrella (A-fragments). Vital stain is seen to disperse in the subumbrellar tissues during this process in C-fragments, whereas vital stain in A-fragments is observed to accumulate and later on is incorporated into the regenerating manubrium.4.The mesogloea of different-sized A-fragments retains a stable form when freed of its adhering cellular components, after a 12–24 h regeneration period; for C-fragments, however, the same result is not observed until 72 to 96 h after their excision.5.InPodocoryne carnea the observed gradients in manubrium regeneration can be abolished when the subumbrellar tissues are separated from the mesogloea by collagenase treatment.6.A model for manubrium regeneration in interradial fragments, based on the influence of tension exerted by the cicatrization process and the counteracting mesogloeal force, is presented and discussed.

The differentiation of cytoskeletal structures in nematocytes of Hydra

Nematocytes of hydra feature a complex cytoskeleton consisting mainly of several bundles of actin filaments and a basket-like structure formed by microtubules. The aim of this study was to establish the sequence of appearance of cytoskeletal elements during nematocyte development using immuno-fluorescence and electron microscopical techniques. Our results are a first step in trying to understand developmental hierarchies and mechanisms which govern the synthesis and assembly of the cytoskeleton in nematocytes. The finger-shaped ‘rods’ around the apex of the capsule are the first detectable elements of the cytoskeleton. Microtubules of the basket structure then follow and later, the actin filaments of microvilli which support the cnidocil. The actin filaments, however, do not show the highly ordered bundling pattern characteristic of filaments in functional nematocytes.