Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.

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-γ. 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.

Adherens‐type junctions (AJs) are major subcellular targets for tyrosine specific protein phosphorylation [Volberg et al. (1991) Cell Regul., 2, 105–120]. Here we report on the apparent effect of such phosphorylation events on the assembly and integrity of AJs. We show that incubation of MDCK cells with potent inhibitors of tyrosine‐specific phosphatases (PTP), namely H2O2 and vanadate, leads to a dramatic increase in AJ‐associated phosphotyrosine which was apparent already within 2–5 min of treatment and progressed upon further incubation. Examination of H2O2 vanadate treated cells at later time points indicated that intercellular AJs rapidly deteriorated, concomitantly with a marked increase in the number and size of vinculin and actin containing focal contacts. In parallel, major changes were observed in cell structure and topology, as revealed by electron microscopy. These were manifested by rapid rounding‐up of the cells followed by reorganization of the cell monolayer. Other intercellular junctions, including desmosomes and tight junctions, visualized by staining with desmoplakin and ZO‐I antibodies, were not significantly affected. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we have carried out reciprocal experiments in which Rovs Sarcoma virus (RSV) transformed chick lens cells, expressing high levels of pp60src kinase, were treated with inhibitors of tyrosine kinases, (tyrphostins). We show that following such treatment, intercellular AJs which were deteriorated in the transformed cells, were reformed. Based on these observations, we propose that specific tyrosine phosphorylation of AJ components is involved in the downregulation of these cellular contacts.

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.electron tomography | nucleocytoplasmic large DNA viruses | poxviruses | viral factories W ith the single exception of poxviruses, all currently known DNA viruses carry out replication and transcription either entirely or partially within host nuclei. The overwhelming bias to nucleus-centered viral transactions is rationalized by the fact that the nucleus provides the elaborate machinery required for these processes. Moreover, by concentrating essential factors into particular intranuclear sites as well as by enabling spatiotemporal regulation of viral replication and transcription, the nuclear environment considerably enhances the efficiency of these processes (1). Such underlying benefits render the entirely cytoplasmic infection cycle of poxviruses all of the more intriguing (2, 3). Nucleus-centered viral transactions present, however, remarkable hurdles associated with the prerequisite of transporting viral genomes from their entry sites at the cell periphery to and into the nucleus. Specifically, the cellular milieu is refractory to motion of DNA molecules because of the high viscosity of the cytosol and the dense molecular sieve generated by the cytoskeleton (4). Moreover, the nuclear envelope imposes a severe barrier for both entry and exit of long DNA molecules (1).The hurdles associated with genome trafficking are particularly high for nucleocytoplasmic large DNA viruses (NCLDV), which include the eukaryote-infecting families Poxviridae, Phycodnaviridae, Iridoviridae, and Asfarviridae, all characterized by DNA genomes longer than 100 kbp (5). After entry, genomes of phycodnaviruses (6), iridoviruses (7), and the African swine fever virus (8) are released into the cytoplasm at the host cell periphery and then, are shuttled toward and transported into the nucleus where initial replication cycles occur. Viral DNA is subsequently delivered to specific cytoplasmic factories in which all transactions required for viral assembly take place. The factors that attenuate the barriers imposed by the constrained DNA motion and thus, enabl...

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