Cell biology

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Cell biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in cell biology.

mark to observe cytoskeletal movement and turnover. The movement of microtubules in mitotic spindles, actin in migrating cells, and microtubule associated proteins was analyzed.

Current Opinion in Cell Biology 1999, 11:1–9

Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast. Straight A, Sedat JW, Murray AW: J Cell Biol 1998, 142:687-694. • Significance: Genetic analysis of Saccharomyces cerevisiae has revealed overlapping functions for kinesin-related proteins in mitotic spindle function. This study shows that three of these microtubule motors, Cin8p, Kip19, and Kip3p actually have distinct roles in specific microtubule-based movements in mitosis. Findings: Time-lapse microscopy of cells expressing fluorescent tubulin and centromere markers was used for kinetic analysis of mitosis in yeast cells with mutations in Cin8p, Kip1p, and Kip3p. Cells with cin8D mutations lacked the initial rapid phase of anaphase B, cells with kip1D mutations showed a decrease in the rate of the slow phase of spindle elongation, and cells with kip3D mutations exhibited longer spindles with defects in spindle breakdown at the end of mitosis.

http://biomednet.com/elecref/0955067401100001 © Elsevier Science Ltd ISSN 0955-0674 Contents (chosen by) 1 Cytoskeleton (Julie Canman and Clare M Waterman-Storer) 2 Cell regulation (Serge Roche and Giulio Superti-Furga) 3 Nucleus and gene expression (Elisa Izaurralde, Rein Aasland and Peter Verrijzer) 5 Membranes and sorting (Karl Matter) 5 Membrane permeability (Paul A Slesinger) 6 Cell-to-cell contact and extracellular matrix (Martin Pfaff) 8 Cell differentiation (Eric A Miska) 8 Cell multiplication (Robert A Sclafani) • ••

of special interest of outstanding interest

Cytoskeleton Selected by Julie Canman and Clare M Waterman-Storer University of North Carolina, Chapel Hill, NC, USA

Dynamics of axonal microtubules regulate the topology of new membrane insertion into the growing neurites. Zakharenko S, Popov S: J Cell Biol 1998, 143:1077-1086. • Significance: The site of new membrane insertion in growing axons has been controversial. This study elegantly demonstrates that membrane is transported from the cell body and inserted into the plasma membrane at the growth cone. Findings: Using a vital membrane dye locally applied at the cell soma, the movement of membrane vesicles down the axon to the growth cone was monitored. Only at the growth cone did the dye become visible in the plasma membrane, indicating membrane insertion. Inhibiting microtubule dynamic instability inhibited vesicle insertion, but not transport. Inducing microtubule depolymerization after inhibition of dynamic instability induced membrane insertion, indicating that microtubule shortening is required for membrane insertion. Fluorescent speckle microscopy, a method to visualize the dynamics of proteins assemblies in living cells. WatermanStorer C, Desai A, Bulinski JC, Salmon ED: Curr Biol 1998, 8:1227-1230. •• Significance: The movement and turnover of fluorescently labeled cytoskeletal proteins has previously been monitored in living cells using the cumbersome methods of laser photobleaching or photoactivation of fluorescence. This paper presents a simple way to monitor fluorescent cytoskeletal movements in large areas and thick regions of live cells that will make this type of analysis available to a broad range of scientists. Findings: By introducing into cells a very low level of fluorescent cytoskeletal protein relative to the total cellular pool of that protein, cytoskeletal structures acquired a speckled appearance in high resolution images captured with a sensitive camera, as a result of stochastic incorporation of labeled and unlabeled subunits. The speckle pattern was used as a fiduciary

Roles of rho-associated kinase in cytokinesis; mutations in rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments. Yasui Y, Amano M, Nagata K, Inagaki N, Nakamura H, Saya H, Kaibuchi K, Inagaki M: J Cell Biol 1998, 143:1249-1258. • Significance: The Rho family of small GTPases are signalling molecules known to affect the dynamics of the actin cytoskeleton. This is the first in vivo demonstration that a Rho effector acts on intermediate filaments during cytokinesis. Findings: Glial fibrillary associated proteins (GFAPs) that were mutated at Rho-kinase phosphorylation sites were expressed in intermediate-filament-negative cells. These mutants prevented disassembly of GFAPs at the cleavage furrow and led to unequal segregation of GFAPs at cytokinesis and unusual cellular morphology at the midzone after furrow completion. Drosophila Polo kinase is required for cytokinesis. Carmena M, Riparbelli M, Minestini G, Tavares A, Adams R, Callaini G, Glover D: J Cell Biol 1998, 143:659-671. • Significance: This paper is the first to identify a regulatory kinase involved in midzone complex formation and cleavage furrow positioning during cytokinesis. Findings: Spermatocyte development was investigated in polo mutant flies to learn the role that polo may play in meiosis. By immunofluorescence, midzone complex and furrow components were found to be mislocalized and a normal midzone complex was not found despite normal cyclin-B degradation. Thus, polo plays a role in cytokinesis during meiosis. Cleavage planes in frog eggs are altered by strong magnetic fields. Denegre J, Valles J, Lin K, Jordan W, Mowry K:Proc Natl Acad Sci USA 1998, 95:14729-14732. • Significance: This is the first paper to demonstrate that magnetic fields have an effect on cleavage plane positioning during cell division. Findings: A strong static magnetic field — either parallel or antiparallel to the anteroventral axis — was applied to Xenopus embryos undergoing cleavage. In parallel fields, the first and

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second cleavages occurred normally, however, the third cleavage plane was parallel to rather than along the normal horizontal cleavage plane. Likewise, in anti-parallel fields, the second plane was misoriented by 90° in the same direction as the magnetic field. A novel direct interaction of endoplasmic reticulum with microtubules. Klopfenstein D, Kappeler F, Haui H-P: EMBO J 1998, 17:6168-6177. • Significance: It is well established that endoplasmic reticulum (ER) membrane is associated with microtubules in cells. This paper reports the first integral membrane protein that can bind the ER to microtubules. Findings: A reversibly palmitoylated type-II integral membrane protein of the ER, p63, was overexpressed in cells. This led to a change in the distribution of ER and bundling of microtubules. In vitro, p63 was shown to bind to microtubules via its cytoplasmic tail.

Cell regulation Selected by Serge Roche Centre de Recherche de Biochimie Macromoléculaire, CNRS, University of Montpellier, France

Bifurcation of lipid and protein kinase signals of PI3Kγγ to the protein kinases PKB and MAPK. Bonveda T, Pirola L, Bulgarelli-Leva G, Rubio I, Wetzcker R, Wymann MP: Science 1998, 282:293-296. •• Significance: Type I phosphatidylinositol 3 kinases (PI 3-Ks) were previously reported to have a dual enzymatic activity including a lipid phosphoinositide kinase and a protein serine kinase; however the relevance of the protein serine kinase activity was unknown. This paper now provides strong evidence of a role for this activity in the Ras/ mitogen activated protein kinase (MAP kinase) signaling pathway. This pathway does not involve the lipid phosphatidylinositol 3-kinase activity. Findings: PI 3-Kγ can regulate MAP kinase activation as induced by receptors coupled to heterotrimeric G proteins. PI 3-Kγ hybrids were generated that no longer showed any lipid kinase activity but retained protein kinase activity. In transient transfection assays, PI 3-K mutants with lipid kinase activity induced the activation of the downstream kinase Akt, whereas protein kinase mutants without lipid kinase activity were responsible for the in vivo MAP kinase activation. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. De Rooij J, Zwartkuis FJT, Verheijen MHG, Cool RH, Nijman SMB, Wittinghofer A, Bos JL: Nature 1998, 396:474-477. •• Significance: cAMP was originally thought to be a second messenger that solely activates protein kinase A (PKA) but De Rooij et al. now identify a new target for this cyclic nucleotide called Epac. Epac is a guanine-nucleotide-exchange factor for the small Ras-like GTPase Rap1 that may play an important function in Rap1-dependent signaling events such as cell growth control. Findings: Rap1 was identified as a negative regulator of Ras during cell transformation. Rap1 can be activated by cAMP in vivo but this was independent of PKA. The cAMP target gene product called Epac was subsequently cloned and characterised. Epac was identified by searching data bases for sequence homology both to guanine-nucleotide-exchange factor and to the cAMP-binding site. Epac showed a cAMP binding site and a guanine exchange factor domain for Rap1. Indeed, Epac activated Rap1 activity in a cAMP-dependent manner both in vitro and in vivo.

β regulates cyclinD1 proteolyGlycogen synthase kinase-3β sis and subcellular localization. Diehl JA, Cheng M, Roussel MF, Sherr CJ. Genes Dev 1998, 12:3499-3511. AND

Cyclin D expression is controlled post-transcriptionally via a Phosphatidylinositol 3-kinase/Akt dependent pathway. Muise-Helmericks RC, Leighton Grimes H, Bellacosa A, Malstrom SE, Tsichlis PN, Rosen N. J Biol Chem 1998, 45:29864-29872. • Significance: Induction of cyclin D1 expression is an important event for G1 to S phase entry and is activated by various extracellular stimuli including growth factors. Although its gene expression is thought to be regulated by a Ras/mitogen-activated protein kinase (MAP kinase) cascade, both papers suggest an additional involvement of a PI 3-K/Akt signaling pathway, which may regulate protein synthesis (Muise-Helmericks et al.), stability and nuclear localization (Diehl et al.). Findings: Quiescent cells stimulated with serum increased their translation of cyclin D1 mRNA (Muise-Helmerick et al.). This effect was not related to the MAP kinase cascade but was rather dependent on a PI 3-K/Akt signaling pathway, that was blocked by herbimycin A. Diehl et al. showed that the glycogen synthase kinase 3-β (GSK3-β) phosphorylates cyclin D1 on Thr286 inducing its proteasomal degradation. GSK3-β is known to be inactivated by both PI 3-K and Akt in vivo. An activated form of Ras that no longer binds PI 3-K could not stabilize cyclin D1, in favour of the involvement of PI 3-K in this process. GSK3-β may also regulate cyclin D1 localization as overexpression of the kinase caused cyclin D1 redistribution to the cytoplasm and a nonphosphorylatable form of cyclin D1 showed protein stability and a nuclear localization. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4/MAPKKK. Takeda M, Saito H. Cell 1998, 95:521-530. •• Significance: The growth arrest and DNA damageinducible gene 45 (GADD45) was originally identified as a gene that is quickly induced upon DNA damage. This report now strongly suggests that GADD45 and two new GADD-like molecules activate the mitogen activated protein kinase kinase kinase 4 (MTK/MEKK4) pathway, and provides a molecular mechanism by which environmental stress induces the p38 and Jun amino-terminal kinase (JNK) mitogen activated protein kinase (MAP kinase) pathways. Findings: GADD45 (called GADD45α) and two new GADD45-like proteins (GADD45β and γ) were pulled out in a two-hybrid approach using MTK as a bait in the search for interactors of the p38/JNK kinases activator MTK/MEKK4. All three GADD proteins bound to MTK and increased its kinase activity. GADD45-like genes were induced upon an environmental cellular stress. When overexpressed, they increased p38 and JNK kinase activities and induced apoptosis. Calcium promotes cell survival through CaM-K kinase activation of the protein-kinase-B pathway. Yano S, Tokumitsu H, Soderling TR: Nature 1998, 396:584-587. • Significance: This paper unravels the signalling pathway by which intracellular Ca2+ induces cell survival in neurons. Findings: A modest increase in intracellular Ca2+ can promote cell survival, however, this effect does not involve a phosphatidylinositol 3 kinase (PI 3-K) or mitogen-activated protein kinase (MAP kinase) pathway. Protein kinase B (PKB)/Akt is known to phosphorylate and inhibit the activity of the pro-apoptotic Bcl-2 family member BAD. Yamo et al. show that PKB/Akt

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is a substrate of the Ca2+/calmodulin kinase kinase (CaM-K kinase) both in vitro and in vivo by using a transient transfection assay. Serum withdrawal induced apoptosis in neuroblastoma cells that was prevented by expression of a constitutive active form of CaM-K kinase. Conversely, protection from apoptosis by increasing intracellular concentration of Ca2+ with the neurotransmitter N-methyl-D-aspartate (NMDA) was blocked by expressing dominant-negative forms of CaM-K kinase and PKB/Akt. Selected by Giulio Superti-Furga European Molecular Biology Laboratory, Heidelberg, Germany

Regulation of cell death protease caspase-9 by phosphorylation. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC: Science 1998, 282:1318-1321. •• Significance: Several cases of caspases affecting protein kinase activity are known but this is the first case showing regulation of caspase activity by phosphorylation. Findings: Extracts from cells expressing the activated Ras oncoprotein are refractory to caspase activation by cytochrome C release from the mitochondria. Treatment of extracts with phosphatase restores sensitivity suggesting that phosphorylation is involved. Investigating candidate kinases in the Ras pathway, the authors identify the Akt/PKB kinase as the kinase that in vivo and in vitro phosphorylates caspase 9 at Ser196 and thereby inhibits the activity of the caspase. It is possible, that phosphorylation of caspase 9 represents a novel mechanism by which Akt inhibits apoptosis.

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CNK, a RAF-binding multidomain protein required for RAS signaling. Therrien M, Wong AM, Rubin GM: Cell 1998, 95:343-353. •• Significance: A novel, tyrosine phosphorylated protein, localizing at cell junctions and functioning upstream or in parallel to Raf in Ras/mitogen-activated protein kinase (MAP kinase) signaling pathways is identified. Finding: The kinase suppressor of Ras (KSR) had previously been identified genetically by the authors as a downstream effector of Ras and has since been shown to be a regulator of the Ras/MAP kinase signalling module. This paper describes a screen for modifiers of a dominant-negative KSR. As an aside, the authors identify Src42A, one of the two known Src genes of Drosophila, as a gene negatively affecting the Ras signalling pathway. The paper focuses on the connector enhancer of KSR (CNK), an enhancer of the dominant-negative KSR phenotype. CNK contains several protein–protein interaction domains typical of proteins involved in signalling, such as a SAM domain, a PDZ domain, a pleckstrin homology (PH) domain and two proline-rich regions with SH3-domain-binding potential. A novel, functionally-relevant domain (termed CRIC [conserved region in CNK]), is also identified by comparing the fly CNK with human and worm homologs found in database searches. The authors prove genetically that CNK is required for different Ras-dependent pathways. CNK is shown to localize to the cytoplasm and to cell membranes, to interact physically with Raf and to become tyrosine phosphorylated after Sevenless receptor tyrosine kinase activation in transfected Schneider cells and after epidermal growth factor stimulation in transfected COS cells.

Nucleus and gene expression Disruption of the p70s6k/p85s6k gene reveals a small mouse phenotype and a new functional S6 kinase. Shima H, Pende M, Chen Y, Fumagalli S, Thomas G, Kozma SC: EMBO J 1998, 17:6649-6659. •• Significance: The p70S6 kinase knock-out mouse confirms a role for this kinase in growth control and leads to the discovery of a second, related, compensating kinase. Finding: The p70 S6 kinase knockout mice are smaller than the wild type. Surprisingly, ribosomal S6 phosphorylation occurs normally in stimulated cells recovered from these knockout mice but this process is still sensitive to rapamycin suggesting the unexpected existence of a second S6 kinase. The authors indeed identify a cDNA highly related to p70 S6 kinase, S6K2, and show that the message for this new S6 kinase is upregulated in the p70S6 kinase knockout mice. Differentiation of CD4+ T cells to Th1 cells requires MAP kinase JNK2. Yang DD, Conze D, Whitmarsh AJ, Barrey T, Davis RJ, Rincon M, Flavell RA: Immunity 1998, 9:575-585. •• Significance: The Jun amino-terminal kinase (JNK)2 mitogenactivated protein kinase is crucial to the differentiation of certain effector T cells and no JNK2 leads to an increase, rather than decrease of AP-1 transcription factor activity in the affected cells. Finding: Mice deficient for the JNK2 gene (all four isoforms) show no obviously abnormal phenotype; however, Th1 effector T cells fail to differentiate properly due to a defect in the IFNγ secretion, whereas Th2 cells produce IL-4 normally, suggesting different requirement and regulation of JNK activity in distinct Tcell populations. By crossing the JNK2-/- mice with AP-1 luciferase reporter mice, the authors show that in Th1 cells, lack of JNK2 leads to an increase in AP-1 activity, suggesting that JNK2 may actually inhibit AP-1 activity.

Selected by Elisa Izaurralde University of Geneva, Geneva, Switzerland

A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Pellizzoni L, Kataoka N, Charroux B, Dreyfuss G: Cell 1998, 95:615-624. •• Significance: The survival of motor neurons (SMN) protein, is the product of the spinal muscular atrophy (SMA) disease gene. SMN has previously been shown to play a role in snRNP assembly in the cytoplasm. This manuscript provides evidence for a novel function for SMN in pre-mRNA splicing. Findings: A dominant-negative mutation of SMN is described. This causes a dramatic reorganisation of nuclear substructures: coiled bodies, gems and speckles merge into several large accumulations in the nucleus. This mutation also inhibits premRNA splicing in vitro, whereas the wild-type protein stimulates splicing. Similarly, mutated SMN found in SMA patients cannot stimulate splicing. These findings provide a link between the function of SMN in splicing and the SMA disease. Localization of ASH1 mRNA particles in living yeast. Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM: Mol Cell 1998, 2:437-445. •• Significance: Messenger RNA localisation provides an important mechanism to generate cell asymmetry but methods to visualise RNA movement in real time are not available. This manuscript describes a novel and elegant approach to visualise RNA movement in real time in living cells. Findings: ASH1 mRNA asymmetrically localises to the bud tip in Saccharomyces cerevisiae. To visualise its localisation, a reporter RNA consisting of ASH1 mRNA sequences and six binding sites for the RNA binding protein MS2 was expressed in yeast cells. In these cells the MS2 protein was produced as

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a fusion with the green fluorescent protein (GFP). Because the MS2–GFP protein avidly binds to the reporter RNA, formation and localisation of GFP-containing ribonucleoprotein particles could be followed by video microscopy in living yeast. NTF2 mediates nuclear import of Ran. Ribbeck K, Lipowsky G, Kent HM, Stewart M, Görlich D: EMBO J 1998, 17:6587-6598. • Significance: The nuclear transport factor 2 (NTF2) was originally identified as an activity that stimulates nuclear import in vitro. Although NTF2 is highly conserved and is essential in yeast, its precise function remained obscure. This manuscript shows that the role of NTF2 is to mediate nuclear import of the small GTPase Ran, a key regulator of nuclear transport processes. Findings: NTF2 binds to Ran GDP, the cytoplasmic form of Ran, and facilitates its translocation through the nuclear pore complex. The release of Ran from NTF2 into the nucleus requires nucleotide exchange to generate Ran GTP, for which NTF2 has no affinity. The subsequent nuclear accumulation of Ran also requires nuclear binding sites, which are provided by the importin β family of transport receptors Selected by Rein Aasland Department of Molecular Biology, University of Bergen, Norway

Localization of bacterial DNA polymerase: evidence for a factory model of replication. Lemon KP, Grossman AD: Science 1998, 282:1516-1519. •• Significance: Two models for DNA replication have been proposed; either the DNA polymerase moves along DNA or the DNA is pulled through an immobilised polymerase. In this paper the authors provide evidence for the latter model since they could show that DNA polymerase is largely stationary in bacterial cells. Findings: A fusion protein between PolC, the catalytic subunit of the replicative DNA polymerase and green fluorescent protein (GFP) was expressed in Bacillus subtilis. The PolC–GFP protein was observed in living cells by fluorescence microscopy. In slowly-growing cells, PolC–GFP was shown to localise to one or two discrete foci close to midcell. dMi-2, a Hunchback-interacting protein that functions in Polycomb repression. Kehle J, Beuchle D, Treuheit S, Christen B, Kennison JA, Bienz M, Müller J: Science 1998, 282:1897-1900. • Significance: The Polycomb-group (Pc-G) of proteins is required for maintaining stable patterns of repression of the homeotic genes during embryogenesis. Repressors, like Hunchback (Hb), are thought to recruit Pc-G proteins to Polycomb response elements. Inactive chromatin may subsequently spread from these elements. The Mi-2 protein contains a helicase-type ATPase and two domains also found in other Pc-G proteins. Mammalian Mi-2 is a component of a histone deacetylase, an activity associated with repression. In this work dMi-2 is identified as a possible link between Hb and Pc-G function. Findings: dMi-2 was found to interact with Hb in a two-hybrid screen and mutant dMi-2 alleles were shown to act as enhancers of Polycomb. The Heterochromatin Protein 1 prevents telomere fusions in Drosophila. Fanti L, Giovinazzo G, Berloco M, Pimpinelli S: Mol Cell 1998, 2:527-538. • Significance: Heterochromatin Protein 1 (HP1) is associated with heterochromatin in polytene chromosomes as well as in mitotic chromosomes. The protein is also localised to certain euchromatic regions and to telomeres. HP1 is encoded by a suppressor

of position-effect variegation and it has been shown to be required for proper chromosome segregation. Furthermore, a population of HP1 is associated with the origin of replication complex (ORC). In this work, evidence for a new function for HP1 is provided; the protein appears to prevent formation of telomere fusions. Findings: The authors characterised in detail the association of HP1 with telomeres and showed that it is independent of two classes of telomeric repeats. In most cells carrying different combinations of mutant HP1-alleles, accumulation of telomere fusions and other chromosomal abberations was observed. Requirement of RSF and FACT for transcription of chromatin templates in vitro. LeRoy G, Orphanides G, Lane WS, Reinberg D: Science 1998, 282:1900-1904. • Significance: The basic transcription machinery, RNA polymerase II and the general transcription factors, is not capable of transcribing chromatin templates in vitro. Several chromatin remodeling factors have been identified and some of these, including RSF (remodeling and spacing factor) described in this work, can facilitate transcription initiation in chromatin. Similarly, a factor called FACT (facilitates chromatin transcription) can aid the polymerase in transcription elongation on chromatin templates. In the present work, RSF and FACT were shown to be sufficient for aiding RNA polymerase and the general transcription factors in activator-dependent transcription from chromatin templates in an in vitro reconstituted system. Findings: Reconstituted transcription was observed in a system containing recombinant or affinity purified proteins. The chromatin template, however, was assembled with the aid of a cell extract and subsequently purified by gel filtration. Selected by Peter Verrijzer Imperial Cancer Research Fund, London, UK

Tankyrase, a Poly(ADP-Ribose) polymerase at human telomeres. Smith S, Giriat I, Schmitt A, de Lange T: Science 1998, 282:1484-1487. •• Significance: The length of human telomeres is a key determinant of cellular ageing. This paper describes the identification of a novel potential regulator of telomere length. Findings: The DNA sequences at the ends of human chromosomes, the telomeres, are specifically bound by TRF1. This protein is believed to negatively regulate telomere length by blocking telomerase function at the telomere ends. TRF1 is found to be specifically bound by tankyrase, an ankyrin repeat protein that is homologous to poly(ADP-ribose) polymerase (PARP). Tankyrase has PARP activity, with itself and TRF1 as substrates. Interestingly, ADP-ribosylation of TRF1 inhibits its binding to telomeric DNA in vitro, suggesting a novel mechanism for the regulation of telomere length. Dissecting the regulatory circuitry of a eukaryotic genome Holstege FCP. Jennings EG, Wyrick JJ, Lee TI, Hengartner CJ, Green MR, Golub TR, Lander ES, Young RA: Cell 1998, 95:717-726. •• Significance: The role of specific components of the yeast RNA polymerase II transcription machinery in gene expression is examined by genome-wide expression analysis. This study reveals several unanticipated intricacies of the mechanisms by which genes are regulated. Findings: High-density oligonucleotide array technology is effectively used to probe the contributions of various components of the transcription machinery to the control of gene

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expression at the level of a complete genome. The results shed new light on various aspects of the functioning of the basal transcription machinery, co-activators, signalling pathways and histone acetyltransferases.

Membranes and sorting Selected by Karl Matter University of Geneva, Geneva, Switzerland

A novel direct interaction of endoplasmic reticulum with microtubules. Klopfenstein DRC, Kappeler F, Hauri HP: EMBO J 1998, 17:6168-6177. •• Significance: This paper provides the first example of a transmembrane protein that directly links a membrane-bound organelle to microtubules. Findings: The endoplasmic reticulum specific transmembrane protein p63 is shown to bind microtubules in vivo and in vitro and its overexpression level is demonstrated to influence the morphology of both the endoplasmic reticulum and the microtubule network. A structural explanation for the recognition of tyrosinebased endocytotic signals. Owen DJ, Evans PR: Science 1998, 282:1327-1332. •• Significance: The crystal structure of an adaptor subunit complexed to endocytosis determinants of the ‘YXX hydrophobic residue’ type provides a structural explanation for the biochemical characteristics of this type of endocytosis determinant. Findings: The crystal structure of the signal-binding domain of µ2 containing peptides representing either the internalization determinant of the epidermal growth factor receptor or the one of TGN38 (trans-Golgi network protein of 38 kDa) was solved to 2.7 Å resolution. Both peptides were found to be in an elongated position with the tyrosine residue engaged in hydrophobic interactions and in a network of hydrogen bonds with µ2 residues. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Caron E, Hall A: Science 1998, 282:1717-1721. •• Significance: Two different types of macrophage receptors are shown to require different GTPases for phagocytosis, reflecting the different biological consequences of their activation(e.g. in the stimulation of an inflammatory response). Findings: By expressing wild-type and mutant GTPases in fibroblasts and macrophages, Fc receptor-mediated phagocytosis is shown to require Cdc42, Rac, and Rho. Phagocytosis mediated by the complement receptor is shown to depend on Rho only.

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New COP1-binding motifs involved in ER retrieval. Cosson P, Lefkir Y, Démollière C, Letourneur F: EMBO J 1998, 17:6863-6870. •• Significance: A novel determinant is described that mediates coatomer protein 1 (COPI)-dependent localization to the endoplasmic reticulum. Findings: The δ subunit of COPI is shown to interact with a sequence that contains a critical aromatic residue. If connected to the cytoplasmic domain of a reporter protein, this sequence mediates retention in the endoplasmic reticulum in yeast and mammalian cells. Similarly, the previously identified tyrosinedependent endoplasmic reticulum retention signal of the CD3ε subunit also interacts with COPI. Fab1p phosphatidylinositol-3-phosphate 5-kinase function essential for protein sorting in the multivesicular body. Odorizzi G, Babst M, Emr SD: Cell 1998, 95:847-858. •• Significance: This paper demonstrates that phosphatidylinositol-3-phosphate 5-kinase activity is important for the sorting of membrane proteins to internal membranes in endocytic multivesicular bodies. Findings: Ste2p and carboxypeptidase S are shown to be transported to the yeast vacuole lumen in a manner that depends on VPS vacuolar protein sorting gene products that are important for normal endosome function. Luminal sorting appears to occur at the level of endosomes and requires the enzymatic activity of the phosphatidylinositol-3-phosphate 5-kinase Fab1p. Defining the functions of trans-SNARE pairs. Ungermann C, Sato K, Wickner W: Nature 1998, 396:543-548. AND

Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion. Peters C, Mayer A: Nature 1998, 396:575-580. •• Significance: Fusion of primed cellular membranes is suggested to be a three step process: a GTPase-dependent tethering step, a SNARE-dependent docking step, and a Ca2+/calmodulin-dependent fusion reaction. Findings: The homotypic fusion of yeast vacuoles is shown to be initated by a Ypt-7-dependent tethering step followed by the formation of trans-SNARE complexes that stabilize the docking complex. The SNARE complexes can then be dissociated without reducing the fusion efficiency. Upon completion of the docking step, Ca2+ is released from vacuoles which activates a calmodulin-dependent fusion reaction.

Membrane permeability Integral membrane protein sorting to vacuoles in plant cells: evidence for two pathways. Jiang L, Rogers JC: J Cell Biol 143:1183-1199. •• Significance: Transport of transmembrane proteins to the two types of plant vacuoles involves two different mechanisms: one involves transport to the Golgi and recognizes specific transmembrane domains; the other one bypasses the Golgi and recognizes specific cytosolic domains. Findings: In tobacco protoplasts, a chimeric reporter protein consisting of a glycosylated luminal domain and the transmembrane domain of BP-80, the proposed vacuolar sorting receptor, is transported to lytic vacuoles via the Golgi complex. The cytoplasmic domain of a membrane protein of protein storage vacuoles, but not the one of a membrane protein of lytic vacuoles, prevents transport to the Golgi and mediates incorporation into protein storage vacuoles.

Selected by Paul A Slesinger The Salk Institute, La Jolla, CA

Membrane phospholipid control of nucleotide sensitivity of KATP channels. Shyng S-L, Nichols CG: Science 1998, 282:1138-1141. AND

PIP2 and PIP as determinants for ATP inhibition of KATP channels. Baukrowitz T, Schulte U, Oliver D, Herlitze S, Krauter T, Tucker SJ, Ruppersberg JP, Fakler B: Science 1998, 282:1141-1060. •• Significance: KATP channels comprise a class of inwardly rectifying K+ channels that are closed by intracellular ATP. One puzzle concerning KATP channels has been that normal levels of intracellular ATP are sufficient to keep these channels closed, raising the question of what their function is in cells. These two papers demonstrate for the first time that phospholipids, such as phos-

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phatidylinositol-4,5-bisphosphate (PIP2), dramatically decrease the sensitivity of KATP channels to closure by intracellular ATP. Thus, under certain conditions, KATP channels may open even in the presence of physiological concentrations of ATP. Findings: KATP channels (composed of SUR1 and Kir6.2 subunits) recorded in excised patches became less sensitive to inhibition by ATP after exogenous application of PIP2. Both papers implicated the carboxy-terminal domain of Kir6.2 in the regulation by ATP and by PIP2, suggesting a similar mechanism of action. Baukrowitz et al. also demonstrated that manipulating intracellular levels of PIP2 by activating a metabotropic receptor (which couples to phospholipase C and depletes PIP2) inhibits KATP channel activity. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH: Nature 1998, 396:269-272. •• Significance: Much research has focused on the identification of the endothelium-derived hyperpolarizing factor (EDHF), an elusive factor which induces vasorelaxation. In this paper, Edwards et al. provide evidence that implicates K+ ions as an EDHF. K+ released from the endothelial cells hyperpolarizes smooth muscle via activation of inwardly rectifying K+ channels and Na+/K+ ATPase. Findings: Acetylcholine-induced and K+-induced hyperpolarization in smooth-muscle were blocked by a combination of barium (an inhibitor of inwardly rectifying K + channels) and ouabain (an inhibitor of Na+/K+ ATPase). Barium and ouabain also inhibited muscle contractions induced by acetylcholine or elevated extracellular K+. Whether K+ is a universal EDHF remains to be determined. Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme. Feng G, Tintrup H, Kirsch J, Nichol MC, Kuhse J, Betz H, Sanes JR: Science 1998, 282:1321-1324. •• Significance: The cytoplasmic protein gephyrin is hypothesised to be involved in the clustering of postsynaptic ionotropic glycine receptors. In this paper, Feng et al. confirm that gephyrin is important for clustering of glycine receptors but also provide evidence that gephyrin is essential for the activity of molybdoenzymes. Gephyrin has homology with other proteins implicated in molybdenum cofactor metabolism. The disorganisation of glycine receptors may be important for humans with molybdenum cofactor deficiency. Findings: Feng et al. constructed a mutant mouse with a null mutation in the gephyrin gene (geph) . Homozygote geph-/mice lacked gephyrin protein, developed normally but were hyper-responsive to tactile stimulation and died within one day of birth. The behavioural defects were consistent with a loss of glycine-mediated inhibition. In addition, two molybdenum-containing enzymes were undetectable in geph-/- mice. Patch-clamp and amperometric recordings from norepinephrine transporters: channel activity and voltage-dependent uptake. Galli A, Blakely RD, DeFelice LJ: Proc Natl Acad Sci USA 1998, 95:13260-13265. •• Significance: In the carrier model of gating, neurotransmitter transporters mediate the uptake of neurotransmitters by slowly alternating between two open states. Neurotransmitter transporters have also been shown to possess an open channel state where both gates are open (similar to ion channels) and ions can freely flow through the transporter. Now, Galli et al.

provide evidence that norepinephrine (NE) also fluxes through the transporter while in the open channel state. Findings: Galli et al. combined high-resolution patch-clamp recordings of ionic currents through norepinephrine transporters (NET) with microamperometric recordings (measured by oxidation of NE) of NE transport in real-time. The amplitude of NE oxidative currents suggested that NET sustain rates of 30,000 molecules of NE per second. Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake. Levy LM, Warr O, Attwell D: J Neurosci 1998, 18:9620-9628. • Significance: The glial glutamate transporter (GLT-1) plays an important role in removing extracellular glutamate following synaptic activity. There is disagreement over the stoichiometry of the glutamate transporters. In this paper, Levy et al. provide evidence that GLT-1 cotransports one glutamate with three Na+ and one H+, and countertransports one K+. Findings: Levy et al. examined the stoichiometry of GLT-1 by creating a mammalian cell line (Chinese hamster ovary) in which GLT-1 is expressed under the control of an inducible promoter. The stoichiometry was estimated using an analogue of glutamate that is not transported (dihydrokainate) and measuring the reversal potential of the ionic current in different solutions.

Cell-to-cell contact and extracellular matrix Selected by Martin Pfaff Ecole Normale Supérieure, Lyon, France

Pyk2 and src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK– cell migration. Sieg D, Ilic D, Jones KC, Damsky CH, Hunter T, Schlaepfer DD: EMBO J 1998, 17:5933-5947. • Significance: This study provides an important functional comparison of the two related cell-adhesion dependent tyrosine kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2). Findings: Compared to wild-type cells, FAK-deficient fibroblasts showed increased levels of Pyk2 tyrosine phosphorylation and of Pyk2-associated in vitro kinase activity after adhesion to fibronectin. Overexpression of Pyk2 and FAK variants, as well as of the negative regulator of src-kinases, p50csk, indicated that Pyk2 and src kinases together compensate for FAK in adhesiondependent signals to the mitogen-activated protein kinase pathway, but cannot replace FAK to reconstitute cell migration. α induction of CD44-mediated leukocyte adhesion by TNF-α sulfation. Maiti A, Maki G, Johnson P: Science 1998, 282:941-943. • Significance: Sulfation of CD44 is newly identified as a posttranslational modification regulating its hyaluronan binding activity in leukocytes. Findings: Tumor necrosis factor (TNF)α treatment of the human leukemic cell line SR91 caused a slight increase in CD44 expression and enhanced binding to hyaluronan or to an endothelial cell layer. These interactions were shown to depend on both hyaluronan and CD44. In addition, a fivefold elevation in the sulfate content of CD44 was noted following the cytokine treatment. Abrogation of sulfation with the sulfo-transferase inhibitor, sodium chlorate, abolished CD44 binding activities, but not the TNF-α-induced increase in expression.

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Importance of the basement protein SPARC for viability and fertility in Caenorabditis elegans. Fitzgerald M, Schwarzbauer, JE: Curr Biol 1998, 8:1285-1288. • Significance: The gene disruption of SPARC (secreted protein acidic and rich in cysteine also known as BM-40/osteonectin) resulted in mostly normal mice with defects in lens cell differentiation. In contrast, loss-of-function SPARC mutations in Caenorhabditis elegans caused a much more severe phenotype impressively prooving its unique biological importance. Findings: A fusion protein between SPARC and green fluorescent protein could be used to study SPARC expression in vivo. Then loss-of-function mutants were created by the techniques of RNA interference. This resulted in lethality, sterility or reduced fecundity of significant proportions of the progeny, which appeared reduced in size and transparent due to the lack of gut granules. An essential role for ectodomain shedding in mammalian development. Peschon J, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell WE, Castner BJ, Johnson RS, Fitzner JN et al.: Science 1998, 282:1281-1284. •• Significance: The functional knockout of the membraneanchored protease, tumor necrosis factor (TNF)-α converting enzyme (TACE), a member of the ADAM (a disintegrin and metalloprotease domain) family, reveals its pivotal role in the proteolytic release of biologically active ectodomains probably from many different receptor proteins. Findings: Mice homozygous for TACE lacking metalloprotease activity, displayed a much more severe phenotype than mice lacking TNF or TNF receptors that rather ressembled the phenotype found in mice lacking transforming growth factor (TGF)-α. Experiments performed with wild-type and TACE-deficient cells demonstrated TACE-mediated ectodomain shedding of TGF-α, TNF receptor and L-selectin. Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Miranti C, Leng L, Maschberger P, Brugge JS, Shattil SJ: Curr Biol 1998, 8:1289-1299. •• Significance: This paper characterizes a novel integrin-triggered signaling pathway that is independent of actin polymerization and involves the nonreceptor tyrosine kinase Syk, specific for hematopoietic cells. Findings: A Chinese Hamster Ovary cell line, stably transfected with integrin αIIbβ3, the platelet fibrinogen receptor, was used to reconstitute an integrin signaling pathway by additional transient transfection with the kinase Syk, the guanine nucleotide exchange factor vav1 and other signaling molecules. Combined results showed that ligand binding to integrin αIIbβ3 initiated signals to Syk and vav1, which could not be inhibited by cytochalasin D and subsequently triggered a series of independent downstream reactions. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins. Hiraoka N, Allen E, Apel IJ, Gyetko MR, Weiss SJ: Cell 1998, 95:365-377. •• Significance: This study shows that invasion and neovascularization of fibrin gels by endothelial cells are unexpectedly independent of plasminogen activation, but require metalloproteinases (MMPs). Findings: Tissue explants embedded in fibrin gels were used as an ex vivo model of neovessel formation. Explants from mice deficient in the activation of plasminogen showed unaffected

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neovascularization. On the other hand, inhibitors of MMPs (and not of serine proteases) completely blocked neovessel formation in this model. Among MMPs expressed by endothelial cells, the membrane-type 1 MMP displayed the highest fibrinolytic activity, and its expression in MT1-MMP-deficient cells conferred the ability to invade fibrin gels. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all αV integrins. Bader B, Rayburn H, Crowley D, Hynes RO: Cell 1998, 95:507-519. •• Significance: Results from the targeted disruption of the gene for the integrin αV chain necessitate reevaluation of the primacy of αV integrins in many biological functions. Findings: αV-null embryos died in two waves: 80% between embryonic day 10 and 12, and 20% perinatally. Histological analyses indicated normal development until day 9.5 of gestation. Lethality at day 10–12 was caused by pericardial edema and placental defects. The αV-neonates died of severe intracranial and intestinal hemorrhages after birth, however, most histological aspects of implantation, vascular development, and organogenesis proceeded almost normally in complete absence of the integrin αV chain. The polysialic acid units of the neural cell adhesion molecule N-CAM form filament bundle networks. Toikka J, Aalto J, Häyrinen J, Pelliniemi LJ, Finne J: J Biol Chem 1998, 273:28557-28559. •• Significance: Atomic force microscopy reveals an unexpected capacity of sialic acid polymers to assemble into filament bundle networks. This indicates a novel molecular mechanism potentially contributing to cell–cell interactions. Findings: Oligomers of 12 or more α2-8-linked N-acetylneuraminic acid residues, and purified polysialylated glycopeptides of N-CAM formed branched filament bundle networks, when dried onto cleaved mica surfaces in the presence of calcium ions. Treatment with sialidase inhibited the filament assembly and glycopeptides without polysialic acid did not form these structures. The nonreceptor protein tyrosine phosphatase PTP1B binds to the cytoplasmic domain of N-cadherin and regulates the cadherin-actin linkage. Balsamo J, Arregui C, Leung T, Lilien J: J Cell Biol 1998, 143:523-532. AND

Impaired integrin-mediated adhesion and signaling in fibroblasts expressing a dominant-negative mutant PTP1B. Arregui C, Balsamo J, Lilien J: J Cell Biol 1998, 143:861-873. • Significance: These two cell transfection studies identify novel roles for the tyrosine phosphatase PTP1B in N-cadherin based cell–cell adhesion and in integrin-mediated cellmatrix adhesion. Findings: A protein tyrosine phosphatase associated with N-cadherin in embryonic chick retina cells was identified as chicken PTP1B. Binding to N-cadherin was confirmed in mouse L cells transfected with wild-type or catalytically inactive enzyme. Mutant PTP1B downregulated cadherin-mediated adhesion as well as adhesion and spreading on fibronectin. Compromised dephosphorylation of β catenin in the first case and of src-kinase in the latter case were identified as potential causes of the defects. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. Ilic D, Almeida EAC, Schlaepfer DD, Dazin P, Aizawa S, Damsky CH: J Cell Biol 1998, 143:547-560.

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• Significance: An apoptotic pathway, which becomes active when cell adhesion signals from the focal adhesion kinase (FAK) are missing, is characterized for the first time as a process monitored by p53. Findings: Embryoid bodies and endothelial cells from focal adhesion kinase (FAK)-deficient mice showed increased apoptosis, when cultured in suspension in the absence of serum. This could be suppressed by inactivation of p53. Interference with FAK function by transfection of fibroblasts with dominant negative FAK constructs induced their apoptosis in serum-free medium. This pathway was dependent on p53, on the atypical protein kinase C isoform PKC λ/ι and on cytosolic phospholipase A2. Integrins induce activation of EGF receptor: role in MAP kinase induction and adhesion-dependent cell survival. Moro L, Venturino M, Bozzo C, Silengo L, Altruda F, Beguinot L, Tarone G, Defilippi P: EMBO J 1998, 17:6622-6632. •• Significance: This report unravels a novel signaling mechanism in which integrins induce the activation of a growth factor receptor in the absence of growth factor ligands. Findings: In serum-starved primary fibroblasts, in an endothelial cell line and in NIH-3T3 cells transfected with epidermal growth factor receptor (EGFR), adhesion to matrix or to antiintegrin antibodies triggered a transient tyrosine phosphorylation of EGFR. EGFR activation was not inhibited by cytochalasin D and depended on intrinsic EGFR kinase activity. Requirements of this pathway, for example for adhesioninduced cell survival, were only evident in cells expressing EGFR above a critical threshold level.

Cell differentiation Selected by Eric A Miska Wellcome/Cancer Research Campaign Institute, Cambridge, UK

Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression. Lin JH, Saito T, Anderson DJ, Lance-Jones C, Jessell TM, Arber S: Cell 1998, 95:393-407. •• Significance: Correlation of ETS gene expression with synapse formation suggests a novel role for ETS genes in organising the sensory-motor circuit in the chick spinal cord. Findings: ETS proteins are known to be involved in cell-type specification of various lineages. In this study the authors demonstrate that ETS gene expression can define motor neuron pools and subsets of muscle sensory afferents in the sensory-motor circuit. In particular, two highly similar ETS genes, PEA3 and ER81, were specifically expressed in a subset of motor neurons in the limb of chick spinal cords. PEA3 and ER81 were also found to specify a subgroup of sensory afferent neurons at the time of monosynaptic connection formation. Interestingly, ER81 and PEA3 expression in motor and sensory neurons correlated with circuit formation involving a common muscle target. Thus, the connection of neurons with similar expression patterns of ETS genes is suggestive of an involvement of these genes in establishing a homophilic interaction between sensory and motor neurons. Neuronal patterning by BMPs: a requirement for GDF7 in the generation of a discrete class of commissural interneurons in the mouse spinal cord. Lee Kj, Mendelsohn M, Jessell TM: Genes Dev 1998, 12:3394-3407. •• Significance: Evidence for an essential role of bone morphogenic protein (BMP) signalling from the roof-plate in the developing central nervous system is demonstrated.

Findings: The authors examined the effect of GDF7, a transforming growth factor (TGF)β superfamily member (BMP subclass), on the specification of neuronal cell types in the developing mouse nervous system. GDF7 was found to be expressed selectively by roof plate cells. GDF7 promoted the differentiation of two dorsal sensory interneuron classes, D1A and D1B. Gdf7 null mice were generated using gene-targeting. Homozygous mice developed a severe hydrocephalus phenotype. Mutant embryos specifically lacked D1A neurons whereas D1B neurons were unaffected. Thus, GDF7 is established as a roof-plate derived signal for neuron cell fate determination. Development of neuroendocrine lineages requires the bHLH-PAS transcription factor SIM1. Michaud JL, Rosenquist T, May NR, Fan CM: Genes Dev 1998, 12:3264-3275. • Significance: In Drosophila the transcription factor sim is essential for midline development, acting upstream of drifter, a POU-domain transcription factor. This knockout study establishes mouse Sim1 as being required for differentiation of neuroendocrine lineages acting upstream of another POU-domain transcription factor, Brn2, suggesting a conserved pathway between flies and mammals. Findings: The SIM1 transcription factor, a member of the bHLH-PAS family, is expressed during the development of the hypothalamic-pituitary axis in the hypothalamic nuclei in mice. The authors created Sim1 null mice using gene targeting. Homozygous mice died shortly after birth. The paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of these mice were found to be hypocellular. Secretory neurons in the nuclei were examined by analysing marker expression. At least five types of secretory neurons were found to be lacking from PVN, SON and the anterior periventricular nucleus. In the mutant mice, Brn2, a POU domain transcription factor required for the terminal differentiation of a subset of secretory neurons, was not expressed in PVN/SON.

Cell multiplication Selected by Robert A Sclafani University of Colorado Health Sciences Center, Denver, Colorado, USA

Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ: Nature 1998, 396:84-88. •• Significance: This paper demonstrates that the molecular changes required for immortalization of cells is different in different cell types. It also may provide a simple method for cellular immortalization of normal human cells, which requires only a small number of genetic changes in contrast to previous methods. Findings: Human primary keratinocytes and mammary epithelial cells can be immortalized by a combination of telomerase and human papilloma virus E7 protein expression. Previous results by Bodnar et al. showed that telomerase expression alone was sufficient to immortalize fibroblasts. As E7 inhibits the retinoblastoma/p16 pathway, immortalization can also be accomplished by downregulation of the cyclin-dependent kinase inhibitor p16 and telomerase expression. In contrast, elimination of p53 or the p53 regulator p19ARF is not needed for immortalization. Phosphorylation and activation of 13S condensin by cdc2 in vitro. Kimura K, Hirano M, Kobayashi R, Hirano T: Science 1998, 282:487-490.

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•• Significance: For many years, it has been known that phosphorylation of important substrates by Cdc2 (or cyclin dependent kinase 1 [CDK1]) is needed for mitosis. These substrates presumably would produce the events that occur in mitosis, such as nuclear envelope breakdown, chromosome condensation and segregation. Yet these substrates have not been identified. This paper demonstrates that condensin, which is a multisubunit protein complex need for chromosome condensation, is a bona fide mitotic substrate of Cdc2. Findings: The activity of frog condensin was measured in vitro by the introduction of positive supercoils into plasmid DNA. Condensin from mitotic extracts is phosphorylated and active, whereas interphase condensin is inactive and unphosphorylated. This correlates well with Cdc2 being active during mitosis. Cdc2–cyclin B increased condensin activity in vitro. Depletion of Cdc2 from mitotic extracts inactivates condensin, whereas addition of Cdc2–cyclin B to the depleted extract can restore condensin activity. Phospho-specific antibodes were used to identify three condensin residues, Thr1314, 1348 and 1353 in the carboxy-terminus, as sites of phosphorylation by Cdc2. Control of cyclin D1, p27(Kip1), and cell cycle progression in human capillary endothelial cells by cell shape and cytoskeletal tension. Huang S, Chen CS, Ingber DE: Mol Biol Cell 1998, 9:3179-3193. •• Significance: It is well known that regulation of the cell cycle occurs at the restriction point in G1 phase. Activation of the cyclin dependent kinase (CDK) machinery occurs at this point by growth factors or hormones and causes production of cyclin D1, which activates CDK4 and CDK6. This paper demonstrates that cell shape and cytoskeletal tension can also activate this CDK machinery in endothelial cells and are required during G1 phase in addition to growth factors for cellular proliferation. Findings: Human capillary endothelial cells that are not attached to the extracellular matrix (ECM) fail to enter the cell cycle even in the presence of growth factors. This ‘shapedependent block’ is ineffective in cells that have past the restriction point and are in S phase. During this block, the level of cyclin D1 protein is low, whereas the CDK inhibitor (CDKI) p27 level is high. Consequently, retinoblastoma protein is active, hypophosphorylated and inhibits cell cycle progression. Cell shape and cytoskeletal tension are needed to cause retinoblastoma phosphorylation and produce cell proliferation. Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase plx1. Qian YW, Erikson E, Maller JL: Science 1998, 282:1701-1704. •• Significance: Polo-like kinases are found in many eukaryotes and are important for mitosis, yet it is not known how they are activated. Polo-like kinases activate Cdc25C, which is a phosphatase that activates cyclin dependent kinase 1 (CDK1)

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during the G2 to M transition, and are needed for spindle assembly and cyclin B degradation. This paper demonstrates that a protein kinase cascade is needed to activate Plx1, the polo-like kinase of Xenopus laevis. Findings: Xenopus Polo-like kinase kinase 1 (xPlkk1), a protein kinase that activates Plx1, was identified and purified to homogeneity. The xPlkk1 gene was cloned and recombinant protein was expressed in insect cells. The recombinant protein had biochemical properties similar to the native protein. Injection of recombinant xPlkk1 into oocytes accelerated both Plx1 activation and the G2 to M transition. Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase cdk. Mimura S, Takisawa H: EMBO J 1998, 17:5699-5707. • Significance: The role of cyclin dependent kinases (CDKs) in DNA replication is not known. Previous work by Zou and Stillman suggested that Cdc45, a protein needed for the initiation of DNA replication, is loaded onto the origin by CDK phosphorylation. This paper demonstrates a similar finding in vitro by using frog egg extracts, but more importantly, shows that Cd45’s function may be the loading of DNA polymerase a. Findings: Sperm chromatin is added to frog egg extracts and DNA replication is observed. Depletion of XCdc45, the frog homologue of yeast Cdc45, or inhibition of CDK activity by the CDK inhibitor p21 blocks both DNA replication and the binding of polymerase to chromatin. Depletion of CDK by suc1-beads also inhibited these processes. In contrast, binding of minichromosome maintenance (Mcm) proteins is not affected. This shows that loading of polymerase and loading of the Mcm complex may be independent reactions. XCdc45 is shown to physically interact with polymerase α. Xcdc45 co-localizes with the polymerase on chromatin during S phase in nuclei. Localization of bacterial DNA polymerase: evidence for a factory model of replication. Lemon KP, Grossman AD: Science 1998, 282:1516-1519. • Significance: A fundamental question in chromosomal replication is whether the DNA polymerase machine moves along the DNA or does the DNA move through the fixed polymerase. This paper supports the latter hypothesis by showing that DNA polymerase C (polC) of the bacterium Bacillus subtilis is localized to a discrete intracellular mid-cellular position. Findings: A green fluorescent protein tagged polC is expressed in Bacillus subtilis. Fluorescent images of live cells were captured with a digital camera. By using different physiological conditions and genetic alterations the number of replication forks was varied. The number of fluorescent foci increased with the increasing number of forks. In all cases, the polC–GFP protein remained fixed near midcell, rather than being distributed randomly.