Biofilm, starring

headlines fertilization chamber), and male spe-9 mutants can successfully copulate with and ejaculate into wild-type hermaphrodites. However, despite documented intimate interaction between gametes, spe-9 mutant sperm are not recognized by wild-type eggs. Cloning revealed that the spe-9 gene contains a signal sequence and 10 epidermal growth factor (EGF)-like repeats, a motif found in many proteins involved in extracellular functions. The structure of spe-9 is similar to the Notch/ LIN-12/GLP-1 class of ligands, suggesting that the protein encoded by spe-9 is on the sperm surface and functions as a ligand for an egg receptor. The finding that two spe-9 mutant alleles contain point mutations in one of the EGF-like repeats supports this hypothesis. However, whether the protein encoded by spe-9 is expressed by spermatozoa, is localized to the sperm membrane or even interacts with the egg have yet to be determined. While more-conclusive experiments are required to evaluate spe-9 function, this system provides a much needed genetic component to fertilization research.

A time and a place POMBO, A. et al. (1998) Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle EMBO J. 17, 1768–1778 In the past few years, numerous subcompartments have been identified within the mammalian cell nucleus. The function of these nuclear compartments has been enigmatic. One key question to address their function is whether their position within the nucleus is random or is related to functions they might exert. A strong argument for nonrandom localization of nuclear bodies comes now from the study of transcription-factor distribution. The transcription factors PTF and Oct1 are involved in the activation of genes encoding snRNAs and are activators of RNA polymerases II and III. The two proteins are distributed in local concentrations throughout the nucleoplasm but are also found in prominent nuclear domains in late G1 phase and S phase. The domain is typically near the nucleolus, contains RNA polymerase II and active sites of transcription and was therefore named OPT (for Oct1/PTF-transcription) domain. OPT domains do not contain components of the splicing machinery, which is interesting in the light of the fact that the Oct1/PTF target genes are known not to contain introns. Most importantly, the OPT domain appears preferentially associated with chromosomes 6 and 7, and specifically a 30-Mb region of chromosome 6 was mapped to associate frequently with the domain. These observations suggest that the position of the OPT domain with respect to chromosomes is not random but is rather the result of a transcriptional hot-spot of genes activated by Oct1 and PTF. Together with the facts that the nucleolus forms around transcriptionally highly active ribosomal genes and that another nuclear body, the coiled body, has been reported to be associated preferentially with several snRNA genes, it can now be speculated that nuclear bodies might represent sites of high levels of trends in CELL BIOLOGY (Vol. 8) July 1998

transcriptional activity of particular genes. This is an intriguing and testable concept that once again confirms the emerging view that nuclear structure is crucial for the orchestration of nuclear functions.

Nuclear waste disposal SHIMIZU, N., ITOH, N., UTIYAMA, H. and WAHL, G. (1998) Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase J. Cell Biol. 140, 1307–1320 Certain cancer cells accumulate aberrant chromosomal structures, such as circular, autonomously replicating double-minute chromosomes (DMs). The acentric DMs arise at high frequency in cells that have been treated with cytostatic drugs and thus are thought to be responsible for drug-resistant tumours. Wahl and collaborators now present a morphological characterization of a process termed micronucleation that functions in the removal of such aberrant structures from the nucleus. Interestingly, in synchronized tissue-culture cells, the DMs are included into small, membraneenclosed structures that arise from the nuclear envelope during S phase, when the nuclear envelope is intact. This mechanism is a viable alternative to the previously observed nuclear envelope condensation during postmitotic nuclear reassembly that also can result in micronucleus formation. The S-phase-specific micronucleation process seems to be enhanced by treating cells with replication inhibitors, offering a strategy for how to extrude DMs from tumour cells and thus increase their sensitivity to cytostatic drugs. The fact that these micronuclei only form in the presence of DMs predicts that the cell must employ a mechanism to recognize acentric DNA and initiate a series of events that ultimately lead to inclusion of the deviant DNA into a nuclear-envelope-enclosed vesicle. The micronuclei must then be targeted for either extrusion from the cell or internal degradation. Further studies aimed at understanding the signals that induce micronucleation and the molecular mechanism of this important cellular event should unravel an interesting signal-transduction pathway.

Biofilm, starring… DAVIES, D. G., PARSEK, M. R., PEARSON, J. P., IGLEWSKI, B. H., COSTERTON, J. W. and GREENBERG, E. P. (1998) The involvement of cell-to-cell signals in the development of a bacterial biofilm Science 280, 295–298 Most bacteria develop a mechanism for adhering to solid surfaces to establish complex communities known as biofilms. These consist of differentiated mushroom- and

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headlines pillar-like structures separated by water-filled spaces. In some cases, such as the biofilms that form on the surface of plant roots, biofilms are beneficial in preventing fungal development on the plant. In other cases, such as medical implants or the lungs of most cystic fibrosis patients, biofilms formed on the surfaces of these devices and organs are deadly because they lead to resistance to antibiotics and biocides. Davies et al. have discovered an intercellular signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), whose synthesis is directed by the gene product of lasI and is necessary for the differentiation of Pseudomonas aeruginosa from individual cells into complex multicellular structures. When the cell density is sufficiently high, gene activation termed ‘quorum sensing and response’ is triggered by this intercellular signal. Bacteria mutant for this signal form flat and undifferentiated biofilms that are sensitive to the biocide sodium dodecyl sulfate. However, the mutant bacteria develop into normal biofilms when the synthetic molecule is added back to the culture. These findings suggest that inhibition of the intercellular signal involved in biofilm development could help resolve many problems in both industry and medicine. Taking a broader view, one might hope to find similar molecules in other cell–cell interactions that direct organogenesis or initiation of cancer.

Cytoskeletal organization by ‘bilingual’ Rho COOK, T. A., NAGASAKI, T. and GUNDERSEN, G. G. (1998) Rho guanosine triphosphatase mediates the selective stabilisation of microtubules induced by lysophosphatidic acid J. Cell Biol. 141, 175–185 Although most microtubules are dynamic structures, with half-lives of a few minutes, often a minority are much more stable, with half-lives in excess of one hour, and can be identified by virtue of posttranslational modifications to their tubulin monomers (e.g. detyrosination, acetylation). By using antibodies specific to detyrosinated (glu) tubulin, Cook et al. show that cells treated with the mitogenic lipid lysophosphatidic acid (LPA) rapidly stabilize a subpopulation of their microtubules. Treatment with C3 toxin (which ADP ribosylates the small GTPase Rho, inactivating it) eliminates the effect, whereas microinjection of constitutively active Rho (val14 Rho) leads to microtubule stabilization in the absence of LPA treatment, indicating that the response is mediated by this well-known signalling intermediate. Rho plays a fundamental role in organizing the actin cytoskeleton, and its activation following LPA treatment has previously been shown to trigger the production of actin stress fibres. However, LPA-induced stabilization of microtubules is not dependent on this other Rhomediated effect, suggesting that the two are independent of one another. Following LPA application, only a subset of the microtubule population is stabilized – those that are not stabilized show no change in their dynamics. Interestingly, the

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stabilized microtubules are always those adjacent to the plasma membrane, which supports previous results showing that Rho is isoprenylated in vivo and translocates to the plasma membrane upon activation. Thus, these data are the first evidence for selective microtubule stabilization and strongly implicate Rho in coordinating both the actin and microtubule cytoskeletons.

Virus-inducible gene transcription WATHELET, M. G., LIN, C. H., PAREKH, B. S., RONCO, L. V., HOWLEY, P. M. and MANIATIS, T. (1998) Virus infection induces the assembly of coordinately activated transcription factors on the IFN-␤ enhancer in vivo Mol. Cell 1, 507–518 Wathelet et al. reveal a unique example of the coordinate activation of multiple transcriptional activator proteins and their highly cooperative assembly into a transcriptional enhancer complex in virus-induced cells. The interferon (IFN)-stimulated response element (ISRE) present in the promoter of IFN-inducible genes is recognized by the protein complex ISGF-3. A subset of IFNinducible genes can also be directly activated by virus infection. In some of these genes, the ISRE sequence differs slightly, which might account for its ability to respond to IFN and/or virus infection. The virus-inducible enhancer of the gene encoding IFN-␤, by contrast, is a complex enhancer containing at least four positive-regulatory domains: PRDI–PRDIV. PRDII is a binding site for NF-␬B, while PRDIV is recognized by the ATF-2/c-Jun heterodimer. The PRDIII-I (or P31) sequence is recognized by IRF-1, a protein implicated in IFN-␤ gene expression. Using electrophoretic mobility-shift assays, Wathelet et al. identified a virus-activated factor (VAF) complex and demonstrated, with specific antibodies, that the virusinducible transcription factors IRF-3 and IRF-7 and the coactivators p300 and CBP are part of VAF. Virus infection led to phosphorylation of IRF-3 and possibly IRF-7 and, subsequently, the formation of VAF. CAT assays revealed that VAF binds with high affinity to the ISRE of IFN- and virus-inducible genes but not to the ISRE of genes that are induced solely by IFN. Thus, different signal-transduction pathways can converge on a single enhancer element by inducing the assembly of distinct transcriptional activator complexes. Protein–DNA crosslinking experiments in vivo showed that VAF forms a multicomponent transcriptional enhancer complex with two other virus-inducible transcription factors, ATF-2/c-Jun and NF-␬B. Indeed, reporter gene studies suggested that VAF binds weakly, yet cooperatively, to the IFN-␤ enhancer. The authors also demonstrated that not only is IRF-1 dispensable for virus induction but it cannot substitute for IRF-3 and IRF-7 in the formation of a virus-activated multicomponent enhancer complex. The combinatorial assembly of transcription factors on enhancers, such as the IFN-␤ enhancer, provides a mechanism to maintain diversity and specificity of signal transduction as well as to allow the integration of different signalling pathways. trends in CELL BIOLOGY (Vol. 8) July 1998