Vaccinia-like cytoplasmic replication of the giant Mimivirus.

Poxviruses are considered to be unique among all DNA viruses, because their infection cycle is carried out exclusively in the host cytoplasm. Such an infection strategy is of interest, because it necessitates generation of elaborate factories in which viral replication and assembly are promoted. By using diverse imaging techniques, we show that the infection cycle of the largest virus currently identified, the Acanthamoeba polyphaga Mimivirus, similarly occurs exclusively in the host cytoplasm. We further show that newly synthesized mRNAs accumulate at discrete cytoplasmic sites that are distinct from the sites where viral replication occurs, and this is observed in vaccinia infection. By revealing substantial physiologic similarity between poxviruses and Mimivirus and thus, implying that an entirely cytoplasmic viral replication might be more common than generally considered, these findings underscore the ability of DNA viruses to generate large and elaborate replication factories.

DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death.

Death-associated protein kinase (DAPk) and DAPk-related protein kinase (DRP)-1 proteins are Ca+2/calmodulin-regulated Ser/Thr death kinases whose precise roles in programmed cell death are still mostly unknown. In this study, we dissected the subcellular events in which these kinases are involved during cell death. Expression of each of these DAPk subfamily members in their activated forms triggered two major cytoplasmic events: membrane blebbing, characteristic of several types of cell death, and extensive autophagy, which is typical of autophagic (type II) programmed cell death. These two different cellular outcomes were totally independent of caspase activity. It was also found that dominant negative mutants of DAPk or DRP-1 reduced membrane blebbing during the p55/tumor necrosis factor receptor 1-induced type I apoptosis but did not prevent nuclear fragmentation. In addition, expression of the dominant negative mutant of DRP-1 or of DAPk antisense mRNA reduced autophagy induced by antiestrogens, amino acid starvation, or administration of interferon-gamma. Thus, both endogenous DAPk and DRP-1 possess rate-limiting functions in these two distinct cytoplasmic events. Finally, immunogold staining showed that DRP-1 is localized inside the autophagic vesicles, suggesting a direct involvement of this kinase in the process of autophagy.

Hyaluronan in the pericellular coat: an additional layer of complexity in early cell adhesion events.

Cell adhesion is a multistage process whereby specific surface receptors interact with the corresponding ligands on the extracellular matrix or on neighboring cells. These complex interactions involve a wide variety of cellular molecules including transmembrane and cytoskeletal components, scaffolding proteins, and a wide variety of signaling enzymes. In this article we discuss recent data characterizing the involvement of the pericellular hyaluronan coat in early stages of cell-matrix adhesion. In particular, we address the mechanisms underlying the transition from hyaluronan- to integrin-mediated adhesion, and the role of the actin cytoskeleton in the "inside-out" regulation and maintenance of the pericellular hyaluronan coat.

Mechanical interplay between invadopodia and the nucleus in cultured cancer cells.

Invadopodia are actin-rich membrane protrusions through which cells adhere to the extracellular matrix and degrade it. In this study, we explored the mechanical interactions of invadopodia in melanoma cells, using a combination of correlative light and electron microscopy. We show here that the core actin bundle of most invadopodia interacts with integrin-containing matrix adhesions at its basal end, extends through a microtubule-rich cytoplasm, and at its apical end, interacts with the nuclear envelope and indents it. Abolishment of invadopodia by microtubules or src inhibitors leads to the disappearance of these nuclear indentations. Based on the indentation profile and the viscoelastic properties of the nucleus, the force applied by invadopodia is estimated to be in the nanoNewton range. We further show that knockdown of the LINC complex components nesprin 2 or SUN1 leads to a substantial increase in the prominence of the adhesion domains at the opposite end of the invadopodia. We discuss this unexpected, long-range mechanical interplay between the apical and basal domains of invadopodia, and its possible involvement in the penetration of invadopodia into the matrix.

Latrunculin B effects on trabecular meshwork and corneal endothelial morphology in monkeys.

To determine the mechanism of latrunculin B (LAT-B)-induced decrease in outflow resistance and the effect of LAT-B on the cornea, structural changes of the trabecular meshwork (TM) and the corneal endothelium following LAT-B were studied in the live monkey eye. LAT-B (0.5 microM) and vehicle were administered by anterior chamber exchange and infusion with cationized and non-cationized gold solution in opposite eyes. The eyes were fixed by infusing Ito's solution and enucleated. Anterior segments were quadrisected and embedded in Epon-Embed 812. Morphology of the TM and the corneal endothelium was studied by light and electron microscopy. LAT-B-induced morphological changes in the TM included: (1) loss of microfilament integrity in cells, especially in TM cells on the collagen beams; (2) development of numerous cytoplasmic projections of the sub-canalicular cells (SUB); (3) reorganization of intermediate filaments in Schlemm's canal inner wall (IW) cells; (4) massive 'ballooning' of the juxtacanalicular (JXT) region, leading to a substantial expansion of the space between the IW of Schlemm's canal and the trabecular collagen beams; and (5) retention of extracellular matrix (ECM), trapped between the SUB cell layer and IW cells. No detrimental effects on tight junctions, giant vacuoles, and cell-cell and cell-ECM adhesions were observed. Endocytosis of gold particles was not affected. Morphology of the corneal endothelium of the LAT-B-treated eye was unchanged. In conclusion, TM changes in the LAT-B-treated eye suggest that the expansion of the JXT space may account for the decrease in outflow resistance induced by latrunculins. The outflow-effective concentration of LAT-B administered intracamerally does not significantly affect the corneal endothelium.

Functional and structural reversibility of H-7 effects on the conventional aqueous outflow pathway in monkeys.

To determine the mechanism of H-7-induced outflow resistance decrease, the reversibility of H-7 effects on outflow pathway was studied physiologically and morphologically in live monkey eyes. Total outflow facility was measured by two-level constant pressure perfusion before (baseline measurement) and after (post-drug measurement) anterior chamber (AC) exchange with 300microM H-7 or vehicle in opposite eyes of eight monkeys. H-7 was then removed by AC exchange with drug-free vehicle in both eyes, followed by a 2.5hr waiting period, after which outflow facility was measured again with (Group 2; n=4) or without (Group 1; n=4) another preceding drug-free AC exchange. For morphological study, five monkeys were initially perfused similarly to Group 1 in physiology, but the facility measurement beginning 2.5hr after drug removal was either omitted or replaced by gold solution infusion. Following baseline measurement, two of the five monkeys received H-7 or vehicle in opposite eyes, while three monkeys received H-7 in both eyes 2.5hr apart, contributing one H-7-treated 'recovery' eye and one H-7-treated 'acute' eye. After perfusion, both eyes of all five monkeys were studied by light and electron microscopy. Outflow facility during post-drug measurement in the H-7-treated eye was significantly increased by two-fold. However, the facility increase was reduced when measured beginning 2.5hr after drug removal, with the reduction being greater in Group 1. 'Recovered' outflow facility after drug removal gradually increased again under continuous AC infusion with drug-free vehicle. Morphologically, major changes in and around Schlemm's canal (SC) in the H-7-treated 'acute' eye included protrusion of the entire inner wall (IW) into SC, relaxation of the IW cells and reorganization of the IW cytoskeleton. The changes in IW cells and juxtacanalicular region of the H-7-treated 'recovery' eye were non-uniform, with areas resembling the vehicle-treated eye ('contracted areas') and areas resembling the H-7-treated 'acute' eye ('relaxed areas'). The average junction-to-junction distances in the IW cells of the H-7-treated 'recovery' eye were intermediate between the vehicle-treated eye and the H-7-treated 'acute' eye. In conclusion, H-7's effect on outflow facility seems reversible, but AC exchange or continuous infusion with drug-free vehicle can re-elevate the 'recovered' outflow facility. Major morphological changes in the TM immediately after H-7 include IW protrusion, cellular relaxation and cytoskeleton reorganization. The decrease in 'relaxed areas' in the TM, in conjunction with the reversed outflow facility, 2.5hr after drug removal suggests that cellular relaxation in the TM is the structural basis for H-7-induced increase in outflow facility.