REVIEW ARTICLE It is all about resolution Meeting report based upon presentations at the 10th International Global BioMillennium 2006 symposium on molecular cell biology (Tbilisi, Georgia) Hermona Soreq 1 and Alik Honigman 2 1 Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel 2 Department of Virology, The Hebrew University of Jerusalem, Israel Introduction Improved resolution in time and space is the hallmark of studies in diverse subfields of the life sciences, including epigenetics, structural biology, and electron tomography. Combined with dynamic imaging of immune cells and with data management of cDNA microarrays, such studies yield reliable synchronization of cell cycle events, with one goal being the develop- ment of specific microRNA and protein signatures of particular tumor cell types, and another being to fol- low the dynamics of neurite growth in the live brains of mice. Meeting report From birth to death of gene products Until recently, epigenetic mechanisms have been equated with the inheritance of chromosomal hetero- chromatin. Evidence that transcriptionally active states of chromatin can also be epigenetically main- tained was presented by A. Francis Stewart (Dresden). Silencing methylations are epigenetically maintained via methyltransferases that associate with proteins that recognize the methylated epitope and propagate the silent state. Together, these observations Keywords alternative splicing; cellular imaging; chromatin; ribosomes; siRNA; ubiquitin Correspondence H. Soreq, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Fax: +972 2 652 0258 Tel: +972 2 658 5109 E-mail: soreq@cc.huji.ac.il (Received 23 August 2006, revised 20 November 2006, accepted 11 December 2006) doi:10.1111/j.1742-4658.2007.05640.x The 2006 Global BioMillennium Conference took place in Tbilisi, Georgia, on 13–17 July 2006. The Conference was focused on key aspects of gene expression processes. Characteristic of state-of-the-art research in the life sciences, the invited lectures spanned approaches in cell biology, gene expression, and protein function. A particular aspect that is special to the BioMillenium series of conferences (this has been the 10th in this series) is the emphasis on new and emerging technologies; the various experts in the subfields that were covered presented what, in their view, should be critical to enabling future progress. Abbreviations DAP, death-associated protein; hnRNP, heterogeneous nuclear ribonucleoprotein; miRNA, micro RNA; MMP, matrix metalloprotease. 924 FEBS Journal 274 (2007) 924–927 ª 2007 The Authors Journal compilation ª 2007 FEBS support a polarization model of chromatin that rein- forces stability. Three post-transcriptional regulation mechanisms were presented. Alternative splicing facilitates large pro- teomic complexity with a limited number of genes, as was discussed by Javier F. Caceres (Edinburgh, UK). The trans-acting factors involved include both serine- arginine (SR) and heterogeneous nuclear ribonucleo- protein (hnRNP) A ⁄ B proteins, with antagonisti c activities influencing different modes of alternative spli- cing in vivo, affecting tissue-specific or developmental regulation of gene expression. Certain SR and hnRNP proteins shuttle continuously between the nucleus and the cytoplasm. Interestingly, hnRNP A1 binds to and is necessary for the processing of a cluster of intronic microRNAs suggested to act as a human oncogene. Nonsense-mediated mRNA decay selectively degrades mRNAs harboring premature termination codons. In humans, nonsense-mediated mRNA is linked to splicing, but in Drosophila and also in Caenorhabditis elegans, it occurs independently of introns. A polarity effect reduces nonsense-mediated mRNA sensitivity to a region close to the 3¢-end of the mRNA. As a next step in the study of the regulation of gene expression, Ada Yonath (Rehovot, Israel) linked ribosomal architecture with antibiotic action. In all known ribosome structures, an internal symmet- rical region connects all functional features. The sym- metry relates the RNA backbone to nucleotide orientation, but shows no sequence homology. This demonstrates the superiority of function over sequence conservation, suggesting that ribosomes evolved by gene fusion. Antibiotics complexed with ribosomes from an archaeon that shares properties with eukaryotes illuminated the structural elements required for therapeutic effectiveness. Structural bio- logy can hence become a key tool for developing novel antibiotics. Post-translational regulation was discussed by Aaron Ciechanover (Haifa, Israel), who argued that the ubiquitin proteolytic system covers the pathway for elucidating the basic mechanisms that are pivotal for drug targeting. Degradation of cellular proteins plays major roles in a multitude of basic pathways during cell life and death, in both health and disease. The ubiquitin–proteasome pathway involves conjugation of multiple ubiquitin moieties to the substrate and subse- quent degradation of the tagged protein, which involves the downstream 26S proteasome complex and unknown mechanisms. The common thread in all of these topics is far greater complexity than was previ- ously perceived, and the need for methods that achieve the maximum resolution. Imaging genomic and cellular properties Different technologies dealing with real-time imaging of molecular events, which has become a major tool in biological research, were presented. Hans J. Tanke (Leiden, the Netherlands) described cellular imaging of telomere localization and dynamics in normal cells and in cancer cells using a fluorescently labeled peptide nucleic acid probe with a sequence complementary to telomeric DNA. Engineering of fusion proteins of telomere-binding proteins with yel- low fluorescent protein and time-lapse imaging were used to follow the dynamic behavior of telomeres, which were shown to interact dynamically with nuclear bodies. Matthias Gunzer (Braunschweig, Germany) studies cell motility and migration of immune cells, which changes considerably when classical liquid cell culture systems are exchanged for more physiologic environ- ments. Mimicking the features of true extracellular tissue can be achieved by embedding the cells in hydra- ted gels of type 1 collagen, or in undisturbed tissues of living animals. Itamar Simon (Jerusalem, Isreal) described a gen- ome-wide analysis of the human cell cycle in primary cells. Combining microarray expression data with pre- cise measurements of the culture synchrony at each time point enables deconvolution of the temporal expression signal, yielding coherent, single cell-based expression patterns. Microarray and complementary fluorescence-activated cell sorting experiments were used to test a number of arrest methods for normal fibroblast cells. Combination of the findings with exist- ing cancer cell cycle data highlights three groups of genes ) those that cycle in both cancer and normal cells; those that cycle only in normal cells; and those that cycle only in cancer cells ) providing new insights into the transformation process. Continuous exchange of macromolecules between the nucleus and cytoplasm is mediated by nuclear pore complexes, macromolecular assemblies that fuse the inner and outer nuclear membrane and form aqueous channels for translocation. Ohad Medalia (Beer-Sheva, Israel) reported on structural analysis of the nuclear pore complex by cryo-electron tomography, exploring transport-active intact nuclei from the slime mold Dictyostelium discoideum. Computerized three-dimen- sional classification and averaging provided a refined structure of the nuclear pore complex, enabling the construction of the trajectories of import complexes using gold-labeled substrates. Nuclear export emerges as the major regulatory mechanism of the nuclear accumulation of STAT2, as H. Soreq and A. Honigman It is all about resolution FEBS Journal 274 (2007) 924–927 ª 2007 The Authors Journal compilation ª 2007 FEBS 925 reported by Hansjo ¨ rg Hauser (Braunschweig, Ger- many). In the absence of interferon, STAT2 perma- nently and rapidly shuttles between the cytoplasm and the nucleus. A region in the C-terminus of STAT2 controls its specific export in the absence of interferon. STAT1 also shuttles in the absence of interferon, but the exchange rate in unstimulated cells is more than 10 times lower. In this context Georgyi Los (Madison, WI, USA) and Marjeta Urh (Madison, WI, USA) described the use of a fusion between a modified halo- genase and target proteins to explore protein functions and dynamics in live cells. Adi Kimchi (Rehovot, Israel) reported on mole- cular networks involved in programmed cell death. She identified gene products, named DAPs (death - associated proteins), that differ substantially in their biochemical properties and intracellular localization. DAP-kinases (DAPk), Ca 2+ ⁄ calmodulin-regulated and Ser ⁄Thr kinases, activate signaling pathways that lead to cell death through membrane blebbing and autophagic cell death. Two closely related kinases, ZIPk and DRP-1, mediate trans-phosphorylation and subsequent functional activation of ZIPk. DAP5 is a translation initiation factor that directs internal ribo- some entry site (IRES)-dependent translation under stress conditions when translation dependent on 5¢ methyl-protected guanosine, designated ‘cap’, is compromised. RNA interference technology serves to knock down various components of the network singly or in combination, and validate these conclu- sions. The linking theme in this session highlighted the need to combine various methodologies in the search for regulatory events. Approaching tumorigenesis The challenge of cross-platform analysis of cancer microarray data was presented by Roland Eils (Heidel- berg), who uses median rank scores and quantile dis- cretization to derive numerically comparable measures of gene expression from different platforms. Applied to six publicly available cancer microarray gene expres- sion datasets from three pairs of studies, this approach was used for examining breast cancer, prostate cancer and acute myeloid leukemia. Two leukemia microarray datasets were further employed to identify important genes with regard to the biology of leukemia. Import- antly, these could be found in an integrated analysis but were missed in the single-set analyses. Cross- platform classification of multiple cancer microarray datasets may yield discriminative gene expression sig- natures generated by different laboratories and micro- array technologies. Reuven Agami (Amsterdam, the Netherlands) dem- onstrated the identification of oncogenic microRNAs using a functional genetic screen. The number of veri- fied human micro (mi)RNAs is still expanding, but only few have been functionally annotated. Agami and colleagues developed a library of vectors expressing the majority of cloned human miRNAs, and created corresponding DNA ‘barcode’ arrays. miR-372 and miR-373 were identified, each permitting proliferation and tumorigenesis of primary human cells, neutralizing p53-mediated CDK2 inhibition. High-throughput for- mats of miRNA screens will be required to search for other cancer-related functions of miRNAs. The emerging approach of dynamic imaging of pro- tease function in cancer cell invasion was covered by Peter Friedl (Wu ¨ rzburg, Germany), who combined confocal and multiphoton microscopic imaging of cell dynamics. During invasive single-cell migration, the cleavage of individual fiber belts occurs at regions of focal pressure, such as fiber insertions at branching pseudopods or nuclear compression zones. These open small degradation tracks, and continuously expanding tubes then become filled with cells and mobile cell masses. Pericellular proteolysis is hence a prerequisite for expansive tumor growth, collective invasion and large-scale tissue reshaping. In contrast, single-cell dis- semination associated with subtle extracellular matrix remodelling is mechanistically independent of protease function and may be rescued by nonproteolytic escape strategies. In this session as well, the complexity of processes and the need for high-resolution strategies were evident. Addressing the brain frontiers Adi Mizrahi (Jerusalem, Israel) reported the use of two-photon microscopy for studying how neuronal dendrites form in adult-born neurons that continuously develop into the olfactory bulb. Lentivirus green fluor- escent protein-labeled neurons were used to directly follow dendritic development in vivo. Within a single day, neuronal morphology changed dramatically, with both formation and retraction of whole dendritic trees. After 10 days, most of the neurons were still migra- ting. After 45 days, most granule neurons had comple- ted migration and showed elaborate, complex dendritic trees. The dynamics of neuronal development in the intact mammalian brain are hence amenable to further study. Elias Michaelis (Lawrence, KS, USA) covered the establishment of the Glud1 transgenic mouse, which overexpresses the mitochondrial enzyme glutamate dehydrogenase 1 (GLUD1), positioned at the second- It is all about resolution H. Soreq and A. Honigman 926 FEBS Journal 274 (2007) 924–927 ª 2007 The Authors Journal compilation ª 2007 FEBS to-last enzymatic step in the pathway for biosynthesis of Glu as a transmitter. Transgenic mice suffer losses in specific neuronal populations, e.g. pyramidal neu- rons of the somatosensory cortex, the CA1 region of the hippocampus, and motoneurons of the spinal cord, and show extensive damage to dendrites and axons of spinal cord motor neurons. Importantly, the GLUD1 hippocampus showed higher expression in transgenic stress response genes, Ca 2+ -regulating genes, and genes whose products control cell survival and neurite growth. Neuronal signaling regulated by intracellular Ca 2+ and oxidative stress is hence associated with raft-like domains in membranes. Luciferase-based reporter bioluminescence assays reported by Keith V. Wood (Madison, WI, USA) offer new methods for measuring such responses, which may become import- ant targets for therapeutic intervention against neuro- degeneration and age-dependent cognitive decline. Anxiety disorders present a major mental health problem. Hermona Soreq (Jerusalem, Israel) discussed anxiety-induced changes in cholinergic neurotransmis- sion that modulate motor control over movement, working memory, and brain-to-body communication through the neuron–immune system interface, modify- ing blood cell composition and platelet production. Importantly, the acetylcholinesterase ACHE gene encodes not one protein, but a combinatorial series of proteins with indistinguishable enzymatic activity but with variant N-termini and C-termini, due to alternat- ive promoter usage and 3¢-alternative splicing. These show distinct nonhydrolytic properties, interact with variant-specific protein partners, and induce inverse signaling cascades. Specifically, causal involvement of both butyrylcholinesterase and acetylcholinesterase in the progression of Alzheimer’s and Parkinson’s dis- eases anticipates future therapeutic needs for drugs targeting specific cholinesterases or the corresponding RNA transcripts. Last, but not least, in this session was Leszek Kacz- marek (Warsaw, Poland), who discussed extracellular proteolysis in neuronal plasticity, learning and mem- ory. C-Fos and its functional form, the AP-1 transcrip- tion factor, emerged in his studies as the best correlate of learning processes, especially of the behavioral information. Matrix metalloproteinases (MMPs) are pivotal for tissue remodeling in the rodent hippocam- pus, where both matrix metalloproteinase protein-9 (MMP-9) protein and its transcript are associated with a subset of dendritic spines bearing asymmetric, excita- tory synapses. Furthermore, functional inactivation of MMP-9 by means of either gene knockout, or specific chemical inhibitors or TIMP-1, delivered by an adeno- virus vector, affected neuronal plasticity and blocked the late phase of long-term potentiation as well as hip- pocampus-dependent learning. Thus, several different proteins were discussed in this session as being relevant for neuronal events characteristic of brain development and aging; however, the gap between cellular and molecular studies appeared to be larger in neuroscience than in tumor-related projects. Acknowledgements We would like to thank the Promega Corporation of Madison, WI, USA for supporting this meeting. Supplementary material The following supplementary material is available online: Doc. S1. Supplementary bibliography. This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corres- ponding author for the article. H. Soreq and A. Honigman It is all about resolution FEBS Journal 274 (2007) 924–927 ª 2007 The Authors Journal compilation ª 2007 FEBS 927 . ARTICLE It is all about resolution Meeting report based upon presentations at the 10th International Global BioMillennium 2006 symposium on molecular cell biology (Tbilisi,. which overexpresses the mitochondrial enzyme glutamate dehydrogenase 1 (GLUD1), positioned at the second- It is all about resolution H. Soreq and A. Honigman 926