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. Current Opinion in Cell Biology 2000, 12:265–275 Contents (chosen by) 265 Cytoskeleton (Desai and Holleran) 266 Cell regulation (Roche, Servant and Weiner) 269 Nucleus and gene expression (Aasland and Weinzierl) 270 Membranes and sorting (Ponnambalam) 271 Membrane permeability (Slesinger) 272 Cell-to-cell contact and extracellular matrix (Pfaff) 273 Cell differentiation (Schuldt and Kudoh) 275 Cell multiplication (Sclafani) • ••

of special interest of outstanding interest

Cytoskeleton Selected by Arshad Desai European Molecular Biology Laboratory, Heidelberg, Germany

An ultrasensitive bacterial motor revealed by monitoring signalling proteins in single cells. Cluzel P, Surette M, Leibler S: Science 2000, 287:1652-1655. • Significance: The first simultaneous measurement correlating the intracellular concentration of the regulator of a biological motor to the output of the motor in single cells. Findings: The authors developed instrumentation that combines fluorescence correlation spectroscopy, an optical technique that allows measurement of the concentration of fluorescent molecules in small volumes, with dark field microscopy. By combining these two techniques, the authors simultaneously correlated the concentration of a green fluorescent protein (GFP)-tagged regulator of the bacterial flagellar motor with the output of the motor in individual bacterial cells. Motor output was visualized by observing the beating of the flagella using dark-field microscopy. By varying the concentration of the GFP-tagged regulator using an inducible promoter, this single cell approach revealed that the bacterial motor has a very steep in vivo response curve to changes in concentration of the regulator. The steepness of this curve was previously not apparent in population-based assays and probably plays an important role in amplifying chemotactic signals during bacterial motility. The single cell approach will be important in quantitative analysis of biochemical networks in vivo. Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization. Lechler T, Shevchenko A, Shevchenko A, Li R: J Cell Biol 2000, 148:363-374. AND

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex. Evangelista M, Klebl BM, Tong AHY, Webb BA, Leeuw T, Leberer E, Whiteway M, Thomas DY, Boone C: J Cell Biol 2000, 148:353-362. • Significance: The first evidence directly implicating type I myosins in the regulation of actin assembly.

Findings: Both groups provide evidence for the physical interaction of a budding yeast type I myosin with the actin-nucleating Arp2/3 complex via a carboxy-terminal acidic domain and the Arp2/3 complex regulator Bee1p — the budding yeast homolog of human Wiskott-Aldrich syndrome protein (WASP) — via a carboxy-terminal SH3 domain. In addition, Lechler et al. show that not only the presence of type I myosins but also their motor activity and their phosphorylation are required for cortical actin polymerization in a permeabilized yeast cell assay. Both groups demonstrate a genetic redundancy between the Arp2/3-complex-binding domains of myosin I and Bee1p. These studies provide the first evidence of a direct connection between type I myosins and the actin nucleation machinery. Centrosome-independent mitotic spindle formation in vertebrates. Khodjakov A, Cole RW, Oakley BR, Rieder CL: Curr Biol 2000, 10:59-67. • Significance: The existence of a robust non-centrosomal pathway for spindle assembly in vertebrate somatic cells is revealed when centrosomes are destroyed in living cells. Findings: The authors use a monkey cell line that constitutively expresses GFP-tagged γ-tubulin, a conserved microtubule-nucleating centrosomal component, to locate centrosomes in cells about to enter mitosis. Once located, they can be destroyed by laser irradiation. Subsequent light and electron microscopic analysis unambiguously revealed that bipolar spindles capable of segregating chromosomes form when either one or both centrosomes are ablated by laser microsurgery. The non-centrosomal pathway for bipolar spindle formation is robust and takes place within the same time frame as normal spindle assembly whether there is either one or no functional centrosomes. These studies reveal that non-centrosomal mechanisms for spindle assembly, previously demonstrated as being prevalent in meiotic cells that lack centrosomes, also exist in centrosome-containing vertebrate somatic cells. Selected by Elizabeth A Holleran University of California, San Francisco, California, USA

Torque generating units of the flagellar motor of Escherichia Coli have a high duty ratio. Ryu WS, Berry RM, Berg HC: Nature 2000, 403:444-447. • Significance: Development of a new motor assay to study the behaviour of a flagellum at higher speeds by tethering the cell body to a glass surface and attaching a bead to the end of a single flagellum. Using different size beads in the optical trap, a variable load could be placed upon the motor. Findings: As in the case of kinesin and myosin, the function of the motor dictates the duty ratio or percentage of time bound during the cycle. Kinesin has a high duty ratio because procession is required more than speed for movement of organells, whereas myosin has a low duty ratio as speed is more important in muscle contraction than procession. For a flagellar motor, a high duty ratio is required to drive viscous loads at high speeds during chemotaxis. Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bidirectional process. Murray JW, Bananis E, Wolkoff AW: Mol Biol Cell 2000, 11:419-433.

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• Significance: This is the first in vitro reconstitution of endosome translocation along microtubules. Exogenous movement does not require cytosol or endogenous motor proteins. Findings: Previous studies of the asialoglycoprotein receptor and ligand system have suggested that endocytic vesicles bind to and move along microtubules under molecular motor directed transport. The ATP-dependent reconstitution of endosomal microtubule-based transport in vitro allows early endocytic vesicle processing and transport to be studied in real time. Endosomes are stretched in this system along microtubules, which suggests that a mechanical basis may underlie endocytic sorting events along microtubule arrays. The yeast kinesin-related protein Smy1p exerts its effects on the class V myosin Myo2p via a physical interaction. Beningo KA, Lillie SH, Brown SS: Mol Biol Cell 2000, 11:691-702. • Significance: The two motor proteins Smy1p, a kinesinrelated protein, and Myo2p a class V myosin, directly interact with one another via their tail domains. Although suppression studies have linked these two motors functionally to one another, this is the first demonstration of a direct physical interaction between these two types of motor proteins. Findings: Two-hybrid analysis reveals that a 69 amino-acid region of the Smy1p tail interacts with the globular part of the Myo2p tail. This interaction is necessary for Smy1p to colocalize with Myo2p. However, overexpression of Smy1p can overcome defects in the head and tail region of Myo2p. The results of this study suggest that Smy1p enhances some aspect of Myo2p function, potentially in the delivery or docking of vesicles at the bud tip. The finding that the behaviour of these two motors is coordinated by a physical interaction adds a new dimension to the field of molecular motors.

Cell regulation Selected by Serge Roche* and Guy Servant† *Centre National de la Recherche Scientifique, Montpellier, France †University of California, San Francisco, California, USA

Function of PI3Kγγ in thymocyte development, T cell activation, and neutrophil migration. Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B, Wakeham A, Itie A, Bouchard D, Kozieradzi I: Science 2000, 287:1040-1046. AND

β2 and-β β3 and PI3Kγγ in chemoattractantRoles of PLC-β mediated signal transduction. Zhong L, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D: Science 2000, 287:1046-1049. AND

Central role for G protein coupled phosphoinositide 3-kinaseγγ in inflammation. Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L, Sozzani S, Mantovani A, Altruda F, Wymann MP: Science 2000, 287:1049-1053. •• Significance: A significant role for phosphoinositide 3-kinaseγ (PI3Kγ) in chemoattractant receptor signaling and inflammation is established from studies in knockout mice. Findings: Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kγ were generated. In neutrophils isolated from these knockout mouse, several chemoattractant (fMLP, C5a, IL-8) receptor-mediated responses, including phosphatidylinositol 3,4,5-triphosphate formation, PKB activation and superoxide production were abolished. Chemoattractantinduced MAP kinase activation was also severely impaired. Neutrophils lacking PI3Kγ showed severe defects in-cell migra-

tion in response to chemoattractants in vitro. Similarly, accumulation of neutrophils at inflammatory sites in vivo was also significantly reduced in PI3Kγ–/– mice. In one of these studies (Sasaki et al.), further data suggest additional roles for PI3Kγ in the regulation of thymocyte survival and proliferation and activation of T cells. Challenge of isolated thymocytes from PI3Kγ–/– mice with adenosine receptor and T cell receptor agonists results in an increased level of apoptosis. Moreover, TCR-induced proliferation is also severely impaired in T cells from PI3Kγ mice. Accordingly, in vivo, PI3Kγ–/– mice exhibit a reduction in thymic cellularity and a reduction in CD8+ cells in the spleen. Other responses such as T-cell receptor-induced cytokine production and T helper cell-dependent responses to hapten antigens are also severely impaired in PI3Kγ–/– mice. These reports confirm a specific function for PI3Kγ in hetrotrimeric G-protein-coupled receptor signalling and highlight a crucial function for PI3Kγ during inflammation. Selected by Serge Roche Centre National de la Recherche Scientifique, Montpellier, France

A subclass of Ras proteins that regulate the degradation of IkappaB. Fenwick C, Na SY, Voll RE, Zhong H, Im SY, Lee JW, Ghosh S: Science 2000, 287:869-873. • Significance: The transcription factor NF-κB plays an important role in a large number of biological responses from cell growth to survival. This report identifies two new members of the small GTP-binding Ras subfamily that control NF-κB activity. Findings: κB-Ras1 and κB-Ras2 were identified in a two-hybrid genetic screen using the IκB cytoplasmic inhibitor of NF-κB as bait. Although κB-Ras 1 and 2 show 30% identity with human Ki-Ras, their primary sequences suggest that they constitutively bind GTP and that they do not attach to the membrane. Moreover, κB-Ras1 and 2 have Drosophila orthologues, indicating that they belong to an evolutionary conserved signalling cascade. κB-Ras binds to the PEST sequence of IκBα and β, which increases their stability. Interestingly, κB-Ras specifically associates with the IκBβ–NF-κB complex in vivo, suggesting that they are specific regulators of IκBβ. AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1. Satoh S, Daigo Y, Furukawa Y, Kato T, Miwa N, Nishiwaki T, Kawasoe T, Ishiguro H, Fujita M, Tokino T et al.: Nat Genet 2000, 24:245-250. • Significance: Auxin is a scaffolding protein that negatively regulates the Wnt–β-catenin signalling pathway. A loss-of-function mutation in auxin is now reported in a subset of human hepatocarcinoma cells that may be involved in β-catenin oncogenic activity and tumour formation. Findings: β-catenin is an important regulator of the Wnt signalling pathway and is essential for development and organogenesis. β-catenin expression is often found to be deregulated in cancers. β-catenin is activated by protein stabilization, and an increase in stability in cancers is achieved by gain-of-function mutations in the corresponding gene. However, β-catenin tends to be active more often than can be accounted for by mutations, suggesting the existence of additional mechanisms for oncogenic activity. The authors now report mutations in auxin — β-catenin ligand that participates in its regulation. These mutations are predicted to prevent β-catenin association and peptidic degradation. The functional importance of such mutations in cancer was measured by the ability of ectopic wild-type auxin to inhibit cancer cell growth

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and induce apoptosis. This report suggests that the auxin gene AXIN1 may be altered in other human cancers. Nuclear translocation and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Hsueh Y-P, Wang T-F, Yang F-C, Sheng M: Nature 2000, 404:298-302. • Significance: Example of a signalling protein that, like βcatenin, is associated with the plasma membrane and cell junctions. It also migrates to the nucleus where it regulates transcription during cell stimulation. Findings: CASK is a cytoplasmic guanylate kinase with multiple protein-binding domains for cellular signalling. CASK is concentrated at neuronal synapses in the brain and associates with various membrane receptors. A two-hybrid screen was performed to search for ligands of the guanlyate-kinase domain (GK) of the protein. The neuronal transcription factor Tbr-1 was identified using this screen and was found to be required for forebrain development. CASK formed a nuclear complex with Trb-1, which increased Trb-1 transcriptional activity. The function of GK in this signalling pathway was, however, not reported. The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases. Luschnig S, Krauss J, Bohmann K, Desjeux I, Nüsslein-Volhard C: Mol Cell 2000, 5:231-241. •• Significance: This report provides important insights into the mechanism by which tyrosine kinase receptors induce cellular responses: in particular it confirms the existence of several independant pathways for signalling and the use of specific intracellular transducers like SHC for cellular responses initiated by a subset of receptors. Findings: dShc loss of function induced a semi-lethal phenotype in Drosophila. In contrast to mammalian Shc, DSHC does not possess a binding site for the Ras activator Grb2, however, it does include a conserved phosphotyrosine-binding domain (PTB) and a Src homology region 2 domain (SH2). A point mutation in the PTB domain mimics the dShc loss of function, implying an important role for the PTB in protein function. DSHC was also found to be required for signalling to the receptor tyrosine kinases (RTK) torso and Drosophila EGF receptor (DER). Although activated DER can mimic the function of the RTK sevenless in the eye, DSHC was dispensible for the function of the later. In addition, epistatic analysis suggested that the transducers DRK (orthologue of the mammalian Grb2) and the adaptors DOS and DSHC act in parallel for torso RTK signalling. This strongly suggests that SHC can function outside of the Ras pathway, as suspected in mammals. Selected by Guy Servant University of California, San Francisco, California, USA

Localization of the G protein βγ complex in living cells during chemotaxis. Jin T, Zhang N, Long Y, Parent CA, Devreotes PN: Science 2000, 287:1034-1036. AND Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW, Bourne HR: Science 2000, 287:1037-1040. • Significance: RhoGTPase activities and an asymmetric distribution of heterotrimeric G proteins may regulate the accumulation of PI3K products at the leading edges of amoebas and neutrophils during chemotaxis.

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Findings: Recent studies performed in Dictyostelium discoideum, a soil amoeba, suggest that PI3K products accumulate preferentially at the leading edge of polarized cells during chemotaxis. Certain pleckstrin homology (PH) domains bind with high affinity and specificity to lipid products of PI3K. The PH domains of the AKT protein kinase was fused with the green fluorescent protein (PHAKT–GFP) and expressed in HL60 cells, a mammalian cell line that can be induced to differentiate into neutrophils. Upon stimulation of these neutrophil-differentiated cells with chemoattractant, PHAKT–GFP translocated from the cytosol selectively to the plasma membrane at the leading edge of polarized cells. This translocation is thought to correspond to the burst of PI3K product formation at the plasma membrane following chemoattractant receptor stimulation. In agreements, a PI3K inhibitor blocked this chemoattractant-induced PHAKT-GFP translocation in HL60 cells. Surprisingly, treatment with Clostridium difficile toxin B, which inactivates RhoGTPases, also prevented chemoattractant-induced translocation of PHAKT-GFP to the plasma membrane of HL60 cells. The β subunit of the D. discoideum G protein was also fused with green fluorescent protein (Gβ–GFP) and expressed in Gβ null cells. Expression of the Gβ–GFP chimera fully restored normal D. discoideum chemoattractant-induced responses, showing that it can compensate for the lack of Gβ. In highly polarized cells, Gβ–GFP was found at the plasma membrane in a shallow anterior–posterior gradient. Stimulation of the polarized cells with a uniformly applied chemoattractant produced binding sites for a PH domain–GFP fusion protein that display a pattern of distribution similar to Gβ–GFP. Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1. Chen C-K, Burns ME, He W, Wensel TG, Baylor DA, Simon MI: Nature, 403:557-560. • Significance: First demonstration that a regulator of G protein signaling (RGS) protein is required for the proper function of a G protein in mammals. Findings: Viable gene-targeted mice lacking RGS9-1 were generated. In single turnover assays performed with rod outer segments (ROS) membranes, GTP was hydrolyzed four times slower with membranes from RGS9–/– mice than with membranes from RGS9+/+ mice. Interestingly, the previously reported phosphodiesterase γ (PDEγ)-induced GTP hydrolysis of Gαt was totally abolished in membranes from RGS9–/–. These observations reveal that PDEγ has no GTPase accelerating activity per se but may recruit and or stabilize the interaction of RGS proteins with activated Gαt. In agreement with these in vitro studies, flash responses from RGS9–/– rod cells showed that the recovery phase, which is dependent on the GTPase activity of Gαt, was significantly prolonged. Moreover, the amplitude of flash responses was not affected in rod cells from RGS9–/– mice suggesting that the principal role of RGS9-1 in vivo is Gαt deactivation. Regulation of carbamoyl phosphate synthetase by MAP kinase. Graves LM, Guy HI, Kozlowski P, Huang M, Lazarowski E, Pope RM, Collins MA, Dahlstrand EN, Earp HS, Evans DR: Nature, 403:328-332. •• Significance: A surprising finding reveals that MAP kinase probably increases cell growth by upregulating the synthesis of pyrimidine nucleotides. Findings: Activated MAP kinase was shown to phosphorylate the multifunctional enzyme CAD in vitro and in vivo. This enzyme catalyzes the synthesis of pyrimidines using three different activities: carbamoyl phosphate synthetase; aspartate transcarbamoylase

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and dihydrooratase. The carbamoyl synthetase activity of CAD is tightly regulated in vivo: it is increased by phosphoribosyl pyrophosphate (PRPP) and inhibited by one of its products, uridine 5′-triphosphate (UTP). Carbamoyl synthetase activity was measured in cell extracts prepared from serum-starved or EGFstimulated rat liver epithelial cells (GN4 cells). In extracts from EGF-stimulated cells UTP did not inhibit the enzyme activity but, instead, increased it. Moreover, PRPP increased carabamoyl sythetase activity to much higher levels in extracts from EGF-stimulated cells compared to those derived from starved cells. Pre-treatment of the cells with a specific MAP kinase inhibitor, PD98059, prevented this increase in carbamoyl synthetase activity. The same observations were made using pure proteins indicating that MAP kinase phosphorylation of CAD is sufficient to modulate its activity. Together, these observations suggest that growth-promoting factors may regulate the production of nucleotides, which is necessary for cell growth, through MAP kinase. In agreement with this conclusion, inhibition of MAP kinase in GN4 cells prevented EGF-induced UTP production. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Raucher D, Stauffer T, Chen W, Shen K, Guo S, York JD, Sheetz MP, Meyer T: Cell 2000, 100:221-228. • Significance: An interesting study showing that agonistinduced modulation of phosphatidylinositol 4,5-bisphosphate (PIP2) levels at the plasma membrane regulates cytoskeleton–plasma-membrane adhesion. Findings: Using optical tweezers, the adhesion force between the plasma membrane and the underlying cytoskeleton was measured in NIH-3T3 mouse fibroblasts. In cells expressing a pleckstrin homology (PH) domain–GFP fusion protein (PHPLCδ–GFP) that binds PIP2 with high affinity, the adhesion force was significantly less than in untransfected cells or in cells transfected with a mutant of PHPLCδ–GFP incapable of interacting with PIP2. Other PH domains with different phospholipid specificity did not affect the adhesion energy. These results suggest that PIP2 may stabilize cytoskeleton–membrane interaction and that modulating the level of PIP2 could modify the adhesion force. Accordingly, decreasing PIP2 levels at the plasma membrane through expression of a plasma-membrane-targeted PIP2-specific 5′phosphatase also reduced the membrane–cytoskeleton adhesion energy. Moreover, agonists acting through G protein coupled receptors and tyrosine kinase receptors that modulate PIP2 levels through activation of phospholipase-C also decreased the adhesion energy between the plasma membrane and the cytoskeleton. PIP2 thus serves as a second messenger that increases interaction between the plasma membrane and the underlying cytoskeleton. Modulating this interaction through the activity of certain receptors that regulate PIP2 levels may play an important role in processes such as membrane protrusion and cell motility. Selected by Orion Weiner University of California, San Francisco, California, USA

Autoinhibition and activation mechanisms of the WiskottAldrich syndrome protein. Kim AS, Kakalis LT, Abdul-Manan N, Liu GA, Rosen MK: Nature 2000, 404:151-158. •• Significance: Crystal structure of the Wiskott-Aldrich syndrome protein (WASP) in its autoinhibited form yields insights into its mechanism of inhibition and activation. Findings: In its GTP-bound form, the Rho GTPase Cdc42 binds to and activates a variety of effectors, such as WASP,

which stimulates de novo polymerization, and PAK, which stimulates serine-threonine phosphorylation and MAP kinase activation. PAK and WASP contain two particularly important domains: a GTPase-binding domain (GBD), which confers binding specificity to Cdc42 and Rac, and a carboxyl terminus that contains the enzymatic domains (activation of the actin nucleating Arp2/3 complex for WASP — known as the VCA domain). PAK and WASP are thought to exist in an autoinhibited form in which the GBD binds to the carboxyl terminus of these proteins, occluding effector function, until the GBD is bound by activated Cdc42, thereby freeing the carboxyl terminus. In support of this hypothesis, it was already known that the carboxyl terminus and GBD of WASP physically interact, and deletion of the GBD results in constitutive activation of WASP and PAK, similar to that seen for activation by Cdc42. To clarify the mechanism of WASP autoinhibition, the authors first used a combination of spectroscopy and affinity-based approaches to specify the minimal domains of the GBD and carboxyl terminus needed for interaction with one another. Then, the crystal structure of the GBD in the autoinhibited form was obtained and compared to those of the GBD alone and the GBD bound to Cdc42. In physiological conditions, the GBD alone is unstructured. When bound to the VCA region of WASP, the GBD becomes structured and masks regions of the carboxyl terminus that are necessary for activation of the Arp2/3 complex. Binding of Cdc42 to the GBD induces a dramatic conformational change in the GBD, resulting in release of the carboxyl terminus. Besides elucidating the structural basis for inhibition and activation of this important signaling molecule, this study provides a fascinating example of an unstructured domain that can be induced to adopt different structures depending on its binding partners. The potential to adopt different structures in different environments may prove to be a more universal property of multifunctional signaling molecules. Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating. Shimada Y, Gulli M-P, Peter M: Nat Cell Biol 2000, 2:117-124. •• Significance: The function of Cdc24, an exchange factor for the Rho GTPase Cdc42, is regulated by its subcellular localization, which is dependent on it via shuttling between the nucleus and cytoplasm. Three proteins involved in the nuclear sequestration and export of Cdc24 are described. Findings: In the budding yeast S. cerevisiae, activation of Cdc42 by its exchange factor Cdc24 is necessary for polarizing the actin cytoskeleton towards an internal bud site during vegetative growth or towards a source of pheromone during mating. Far1 had been previously shown to recruit Cdc24 to Gβγ (a subunit of heteromeric G protein), thereby coupling Cdc42 activation to pheromone receptor activation (Butty AC et al.: Science 1998, 282:1511-1516 and Nern A, Arkowitz RA: J Cell Biol 1999, 144:1187-1202.). Furthermore, Far1 is localized to the nucleus during vegetative growth but accumulates in the cytosol in response to pheromone. In contrast, the spatial and temporal regulation of Cdc24 was not well understood. The authors use a green fluorescent protein (GFP) tagged Cdc24 to assay Cdc24 localization during the cell cycle and during mating in a variety of mutant backgrounds. Cdc24 resides in the nucleus in the G1 phase of the cell cycle and then relocates to the cytoplasm during bud emergence. This export corresponds to expression of the cyclin Cln2, and mutations in the cyclindependent kinase Cdc28–Cln result in permanent nuclear sequestration of Cdc24. Conversely, mutations in Far1 that

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inhibit the nuclear import of Far1 or its ability to interact with Cdc24 result in constitutive cytosolic localization of Cdc24. Finally, a nondegradable Far1 inhibits Cdc24 recruitment to the incipient bud site. These data suggest that Cdc24 is sequestered in the nucleus by Far1 and that it can be released into the cytosol upon degradation of Far1 by Cdc28–Cln. Using similar experiments, the authors also show that pheromoneinduced nuclear export of Cdc24 is accomplished through a different mechanism, involving the export of a Far1–Cdc24 complex from the nucleus by Msn5. Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP. Taunton J, Rowning BA, Coughlin ML, Wu M, Moon RT, Mitchison TJ, Larabell CA: J Cell Biol 2000, 148: 519-530. • Significance: A subset of vesicles in activated or phorbolester treated Xenopus eggs undergo actin-based motility that is correlated with recruitment of the Cdc42 effector protein NWASP. Mammalian endosomes and lysosomes undergo similar actin-based motility, suggesting a novel role for actin assembly in vesicular transport. Findings: In Xenopus eggs activated by fertilization or protein kinase C (PKC)-activating phorbol esters, a subset of vesicles recruited protein kinase C, formed actin-rich comet tails, and moved in a fashion reminiscent of the intracellular bacteria Listeria monocytogenes. A cell-free system consisting of membranes in Xenopus egg extract was used to reconstitute phorbol-ester-induced vesicle motility. This system demonstrated that vesicle motility is dependent on PKC activation and correlates perfectly with recruitment of the Arp2/3 complex activator N-WASP (neuronal isoform of Wiskott-Aldrich syndrome protein). Analysis of the motile vesicles using electron microscopy and fluorescent dyes indicate that they are of lysosomal/endosomal origin. Vesicles from mammalian cells also produced phorbol-ester-mediated motility in Xenopus extracts, and only vesicles from the endosomal/lysosomal compartments, but not other portions of the secretory cascade, were found competent to do so. The next important challenge will be to identify the cis-acting factors that specifically allow lysosomes and endosomes to undergo phorbol-ester-induced Cdc42 activation and N-WASP recruitment.

Nucleus and gene expression Selected by Rein Aasland University of Bergen, Bergen, Norway

The glucocorticoid receptor: Rapid exchange with regulatory sites in living cells. McNally JG, Müller WG, Walker D, Wolford R, Hager GL: Science 2000, 287:1262-1265. • Significance: Transcription factors are often thought to associate tightly with their chromosomal-binding sites and remain bound for prolonged periods. An alternative view is that they interact only transiently with DNA in a ‘hit and run’ mechanism. Support for the latter kind of mechanism is provided here, where it is demonstrated that the glucocorticoid receptor (GR) can cycle rapidly between a chromosomal and a nucleoplasmic localisation. Dynamic behaviour like this could facilitate modification of the receptor, triggered by various signal transduction pathways. Findings: GR was fused to the green fluorescent protein (GFP) and expressed in a cell line that carries some 800–1200 GR-binding sites in a tandem array. The dynamics of association and dissociation of GR–GFP were observed using photobleaching techniques.

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Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Imai S-I, Armstrong CM, Kaeberlein M, Guarente L: Nature 2000, 403:795-800. •• Significance: The yeast chromatin protein Sir2 is required for transcriptional silencing and it has been implicated in regulation of life span. Chromatin silenced by Sir2 displays reduced acetylation of certain lysines at the amino-terminal tails of nucleosomal histones H3 and H4. It was recently shown that Sir2 can catalyse ADP-ribosylation of histone tails using nicotinamide adenine dicucleotide (NAD) as a source of ADP-ribose. An intriguing twist to this story comes with the identification in this work of Sir2 as a NAD-dependent histone deacetylase. Findings: Recombinant Sir2 was found to deacetylate synthetic histone tail oligopeptides in a reaction that was not affected by trichostatin A, a potent inhibitor of other histone deacetylases. The yeast nuclear pore complex: composition, architecture, and transport mechanism. Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT: J Cell Biol 2000, 148:635-651. •• Significance: The nuclear pore complex (NPC) is a large macromolecular assembly responsible for nucleocytoplasmic transport. The NPC has an eight-fold symmetry, and a number of constituents have been identified. In a biochemical tour de force, they have identified most, if not all, components of the yeast NPC. It appears smaller and less complex than previously anticipated with about 30 different polypeptides. Significantly, none of the NPC components appear to be motor proteins, ATPases or GTPases, supporting the view that the NPC acts as a gated pore for facilitated diffusion. Findings: Several hundred peptide fragments derived from a highly enriched NPC preparation were microsequenced and compared with the yeast genome data. 174 different polypeptides were evaluated as NPC components by several criteria, including immunoelectron microscopy. A genetic link between co-suppression and RNA interference in C. elegans. Ketting RF, Plasterk RHA: Nature 2000, 404:296-298. • Significance: Co-suppression takes place when the introduction of a transgene leads to reduced expression of an endogenous homologue as well as of the transgene itself. Here, evidence is provided that co-suppression in Caenorhabditis elegans involves a machinery required for transposon silencing and RNA interference (RNAi; the phenomenon of RNA-guided degradation of mRNAs). Findings: In some cases, the introduction of a transgene leads to reduced expression of an endogenous homolgue as well as of the transgene itself. This phenomenon is called co-suppression. A mutant defective in RNAi but not in transposon silencing, did not exhibit co-suppression. Similarly, another mutant defective in transposon silencing, but not RNAi, was also resistant to co-suppression. Selected by Robert OJ Weinzierl, Imperial College of Science, Technology and Medicine, London, UK

The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo. Deuring R, Fanti L, Armstrong JA, Sarte M, Papoulas O, Prestel M, Daubresse G, Verardo M, Mosely SL, Berloco M et al.: Mol Cell 2000, 5:355-365. • Significance: ATP-dependent remodelling of chromatin structure by large protein complexes makes a major contribution to

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the regulation of gene expression in eukaryotic nuclei. ISWI, a nucleosome-dependent ATPase and a member of the SWI1/SNF2 family, is the central component of several chromatin remodelling complexes in Drosophila (including NURF, CHRAC and ACF). This report describes the effects of null and dominant-negative mutations in the Drosophila ISWI gene on cell viability and gene expression. Findings: Immunofluorescence microscopy studies of labelled anti-ISWI antibodies bound to polytene chromosomes revealed a large number of labelled euchromatic (and some heterochromatic) sites, consistent with a general role for ISWI in transcriptional regulation. The phenotypes of homozygotic mutants show that ISWI plays an essential role in vivo. A detailed analysis revealed abnormalities in the expression of homeotic and segmentation genes and alterations in chromosome morphology. The male X chromosome was shorter and wider than normal, revealing a possible link between histone H4 hyperacetylation and ISWI function. Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones. Galasinski SK, Lively TN, Grebe de Barron A, Goodrich JA: Mol Cell Biol 2000, 20:1923-1930. • Significance: Hyperacetylation of histones is usually tightly coupled to locally increased levels of gene expression. This is achieved by relieving the repressive effects of nucleosomes on transcription factor recruitment and RNA polymerase elongation efficiency. This study shows that the presence of acetyl-Coenzyme A (CoA) has a stimulatory effect, even in the absence of nucleosomes. This points to a direct role for acetyl-CoA in the function of the RNAP II basal transcriptional machinery. Findings: The addition of acetyl-CoA to a highly purified reconsituted in vitro transcription system resulted in substantial stimulation of both basal and activated transcription rates. Further investigations revealed that smaller amounts of TFIID were necessary in the presence of acetyl-CoA to achieve efficient levels of transcription, suggesting that TFIID recruitment was less rate-limiting under these conditions. The results of electrophoretic mobility shift assays and footprinting experiments are compatible with a view that the interaction of acetyl-CoA with TFIID induces a conformational change in the multiprotein complex that increases its affinity for DNA. One of the components of TFIID, TAF II250, contains an acetyltransferase activity and might therefore be involved in this process. β with a novel Structure of the central core domain of TFIIEβ double-stranded DNA-binding surface. Okuda M, Watanabe Y, Okamura H, Hanaoka F, Ohkuma Y, Nishimura Y: EMBO J 2000, 19:1346-1356. • Significance: The basal transcription factor TFIIE is recruited during the final stages of the assembly of RNAPII basal transcriptional machinery on promoter core sequences. TFIIE controls the transition from transcript initiation to elongation. One of the two subunits present in human TFIIE, TFIIEβ, binds to the region of the promoter undergoing melting to form the ‘transcription bubble’. The solution structure presented here sheds new light on the possible function of TFIIE in the transcription process. Findings: The solution structure of the core domain of TFIIEβ reveals a novel double-stranded DNA-binding motif (similar to the ‘winged helix’ domain), which supports the view that TFIIE participates in binding of DNA prior to pro-

moter ‘melting’ (DNA strand separation). This finding is compatible with previous UV photo-crosslinking observations that showed that TFIIE binds between positions –14 and –2 upstream of the transcription start site. In vivo visualization of gene expression using magnetic resonance imaging. Louie AY, Huber MM, Ahrens ET, Rothbacher U, Moats R, Jacobs RE, Fraser SE, Meade TJ: Nat Biotech 2000, 18:321-325. • Significance: The in vivo visualization of gene expression patterns by reporter genes can be carried out in optically transparent organisms using fluorescent proteins, but this approach is not feasible in the majority of organisms because they are optically opaque. This paper describes the development of a new enzymatic substrate that can be specifically visualized by magnetic resonance imaging (MRI). Such an approach offers the exciting possibility of gaining new insights into developmental and physiological processes that occur, for example, in living vertebrates. Findings: β-galactosidase is a commonly used marker gene in reporter constructs to study the regulatory properties of test promoters within intact organisms. The authors describe the synthesis of a compound (‘EgadMe’) consisting of a gadolinium (Gd) chelator and a galactopyranoside residue that blocks the signal from the paramagentic Gd3+ ion. Cleavage of EgadMe by β-galactosidase causes an irreversible transition of this agent to a paramagnetically active state that can be visualized by MRI at cellular resolution (~10 µm). The system was successfully tested by imaging mRNA expression from a reporter gene construct in developing Xenopus embryos.

Membranes and sorting Selected by Vas Ponnambalam University of Dundee, Dundee, UK

Peroxisomes in human fibroblasts have a basic pH. Dansen TB, Wirtz KA, Wanders RJA, Pap EHW: Nat Cell Biol 2000, 2:51-53. •• Significance: The environment of an intracellular compartment is critical for the functions associated with the organelle. The lumen of compartments such as endosomes, lysosomes and the trans-Golgi network have acidic environments essential for enzymatic functions, such as protease cleavage, carbohydrate recognition and processing. Dansen et al. show that human peroxisomes enclose a unique basic environment (pH 8.2 ± 0.3) in contrast to other intracellular organelles. Findings: A membrane-permeable peptide containing a peroxisomal-targeting sequence (SKL) and a pH-sensitive fluorescent moiety (5- and 6-)carboxy-SNAFL-2 was targeted to peroxisomes in primary human fibroblasts. In contrast fibroblasts from patients with Zellweger syndrome have a defect in the PEX6 peroxisomal import protein and cannot incorporate this probe into peroxisomes. The SNAFL-2 probe has a basic form with λ em, max at 625nm (red) and an acidic form with λ em, max at 546nm (green). By determining the ratio of these forms using confocal laser microscopy, the peroxisomes in individual cells were calculated to have a pH ~8.2. In cells from RDCP (rhisomelic form of chrondrodysplasia punctata) type 1 patients with a defect in the PEX7 peroxisomal protein, the peptide probe is still incorporated into peroxisomes, but the pH is approximately 7.2, similar to cytosolic pH. Thus, pEX7 must have an important role in transporting a protein necessary for pH gradient maintenance or ensuring the integrity of the peroxisomal membrane.

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Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Thiele C, Hannah MJ, Fahrenholz F, Huttner WB: Nat Cell Biol 2000, 2:42-49. • Significance: What controls the unique membrane composition of intracellular organelles? Lumenal, transmembrane and cytoplasmic protein sequences in different proteins can mediate targeting to various intracellular compartments such as the ER, Golgi apparatus, lysosomes, peroxisomes and mitochondria. However, analogous to a chicken and egg situation, this still does not explain how key residents are first targeted to a membrane bilayer. One hypothesis (M Bretscher, S Munro, Science 1993, 261:1280-1281.) is that the cholesterol composition of membranes determines bilayer thickness, which in turn specifies localisation of transmembrane proteins along the secretory pathway. Thiele et al. now report that cholesterol can be bound by key synaptic vesicle proteins and cholesterol depletion blocks formation of synaptic-like microvesicles (SLMVs) in a neuroendocrine cell line. Findings: A photoactivatable, radiolabelled cholesterol derivative (photocholesterol) was used to identify 60 kDa (synaptotagmin), 43 kDa (synaptophysin) and 17 kDa (V-ATPase c) cholesterol-binding polypeptides in the PC12 cell line. These cholesterol-labelled polypeptides are absent in a genetic variant of the PC12 line that lacks many secretory granule and SLMV proteins. Immuno-isolated synaptophysin and synaptotagmin from wild-type PC12 cells are shown to have the ability to bind photocholesterol thus indicating that this is an intrinsic property of these SLMV proteins. Depletion of cholesterol using methyl-beta-cyclodextrin causes more than 50% reduction in synaptophysin levels in SLMVs and a dramatic reduction, >90%, in SLMV biogenesis. The authors propose a general mechanism: oligomeric membrane proteins associate with cholesterol to promote the membrane curvature required for vesicle biogenesis. Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP. Taunton J, Rowning BA, Coughlin ML, Wu M, Moon RT, Mitchison TJ, Larabell CA: J Cell Biol 2000, 148:519-530. •• Significance: The movement of membranes along cytoskeletal ‘tracks’ is essential for vesicular targeting and maintenance of organelle structures. Bacteria and viruses can subvert the actin cytokeleton to form actin-rich structures or ‘comet tails’ that power the movement of pathogens through the cell cytoplasm. The authors show that endosomes and lysosomes are also motile and can nucleate the formation of similar actin comet tails. Findings: A cell-free system consisting of membranes, Xenopus egg extract, labelled actin and additional factors or drugs was examined for actin and membrane dynamics using confocal microscopy. Actin comet tail formation was rapidly induced in Xenopus eggs by fertilization or phorbol ester (PMA) treatment; this can be reconstituted in an in vitro cell-free assay. The PMA-stimulated actin comet tails recruit the WiskottAldrich syndrome protein (N-WASP) to the tips of these structures. The actin-binding drug latranculin A inhibits comet tail formation but not N-WASP and actin recruitment to vesicles. However, the Rho GTPase inhibitor toxB completely abolishes both actin and N-WASP assembly and recruitment to large membrane structures resembling endosomes and lysosomes. Using purified membranes from human HeLa cells, the authors show that endosomes and lysosomes, but not ER membranes, are associated with actin comet tails. Electron

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microscopy shows that mutivesicular bodies characteristic of endosomes and coated with N-WASP are propelled at the tips of actin comet tails. These important findings implicate a role for movement of different organelles along the actin cytokeleton that may also be subject to regulation by extrinsic growth factors or stimuli and intracellular signalling. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane interactions. Raucher D, Stauffer T, Chen W, Shen K, Guo S, York JD, Sheetz MP, Meyer T: Cell 2000, 100:221-228. •• Significance: Lipid signals can influence cell function by modulating membrane deformation, vesicle movement and interactions with the cytoskeleton. A key question is what changes in the biological properties of a membrane system take place when the phospholipid composition, is altered but answering this question in vivo is technically difficult. The authors address this question using optical tweezers on single cells to measure plasma- membrane tension and its interaction with the cytoskeleton when phosphatidyl insositol 4,5-biphosphate (PtdIns(4,5)P2) levels are perturbed. Findings: IgG-coated polystyrene beads were used to pull thin membrane tethers from NIH-3T3 fibroblasts using optical tweezers; the tethering force on a bead is approximately proportional to the square root of the plasma membrane–cytoskeleton adhesion energy. The pleckstrin homology (PH) domain of phospholipase Cδ (PLCδ) has a high affinity for PtdIns(4,5)P2 and targets this enzyme to the plasma membrane. Expression of GFP–PH(PLCδ) in transfected cells reduces the plasma membrane–cytoskeleton adhesion energy by more than 50%; in contrast, a mutant protein that is unable to bind PtdIns(4,5)P2 does not affect this property. Expression of a plasma membrane-targeted 5′-lipid phosphatase that hydrolyses PtdIns(4,5)P2 causes a similar reduction in adhesion energy. Both these proteins promote a more rounded cellular appearance and induction of blebs; the adhesion energy between the membrane and cytoskeleton in these blebs is reduced by approximately 90%. Importantly, cell stimulation by growth factors such as PAF, FGF or PDGF causes a significant reduction in adhesion energy, thus linking this to a transient decrease in PtdIns(4,5)P2 levels caused by phosphatidyl insositol 3,4,5trisphosphate (PtdIns(3,4,5)P3) synthesis. These experiments provide an important framework for reconciling membrane dynamics and lipid fluxes caused by growth factor stimulation and intracellular signalling.

Membrane permeability Selected by Paul A Slesinger The Salk Institute, La Jolla, California, USA

Modulation of A-type potassium channels by a family of calcium sensors. Frank An W, Bowlby MR, Betty M, Cao J, Ling H-P, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, Rhodes KJ: Nature 2000, 403:553-556. •• Significance: The rapidly inactivating A-type K+ current influences the membrane excitability of neurones and cardiac cells. Expression of the α subunit from the Kv4 family of inactivating K+ currents that differ slightly from native A-type currents. In this paper, Frank et al. identify an accessory subunit, KChip, which specifically associates with the Kv4 family of K+ channels and restores many of the functional properties of the native A-type currents. Findings: Utilising the yeast two-hybrid system, the amino-terminal domain of Kv4.3 was used as bait to probe a neuronal cDNA library. Three different K+ channel interacting proteins (KChips)

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were identified. Each contained an EF-hand-like motif for binding Ca2+. KChip co-precipitated with Kv4, but not with Kv1 channels, and increased current density, shifted the voltage-dependence of activation, and slowed the rate of inactivation. Mutation of the putative Ca2+-binding domain eliminated the physiological changes but not the association with Kv4. A potassium channel protein encoded by chlorella virus PBCV-1. Plugge B, Gazzarrini S, Nelson M, Cerana R, Van Etten JL, Derst C, DiFrancesco D, Moroni A, Thiel G: Science 2000, 287:1641-1644. •• Significance: Viruses appear to have evolved some of the simplest K+ channels. In this paper, Plugge et al. report that the genome of the plaque-forming chlorella virus (PBCV-1) contains a sequence that encodes a primitive K+ channel. Findings: Sequence analysis of PBCV-1 revealed an open reading frame that encoded a putative K+ channel. The sequence contained two putative transmembrane domains, a pore-loop sequence, a short amino-terminal domain and, surprisingly, no cytoplasmic carboxy-terminal domain. Expression of the cDNA in oocytes yielded constitutively active currents that were K+ selective, weakly voltage-sensitive, inhibited by Ba2+ and the antiviral drug amantadine. Ba2+ and amantadine also inhibited PBCV-1 plague formation, suggesting that the channel activity is required for virus replication. Modulation of N-type calcium channel activity by G-proteins and protein kinase C. Barrett CF, Rittenhouse AR: J Gen Physiol 2000: 115:277-286. • Significance: The mechanism underlying protein kinase C (PKC) potentiation of voltage-gated N-type Ca2+ current is not well understood. In this paper, Barrett and Rittenhouse examine the effect of PKC-mediated phosphorylation on the G-protein-dependent inhibition of voltage-gated N-type Ca2+ channels. Their results suggest that rather than causing a direct increase in channel activity, phosphorylation of the channel simply prevents Gβγ-mediated inhibition. Findings: Ba2+ currents were recorded under a variety of conditions from neonatal rat superior cervical ganglion cells. PKC activation failed to reverse the effects of G-protein-dependent inhibition of Ca2+ channels (+GTPγS), unless preceded by a large depolarizing voltage step to relieve this inhibition. Subsequent phosphorylation then prevented reinhibition by Gproteins. PKC appeared to have no other effects on channel activity, except for preventing G protein-mediated inhibition.

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

Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Vasioukhin V, Bauer C, Yin M, Fuchs E: Cell 2000, 100:209-219. •• Significance: This study describes a novel dynamic mechanism that underlies the formation of cadherin-based epithelial cell to cell contacts. Findings: Primary epidermal keratinocytes in low calcium conditions were induced to develop E-cadherin-mediated adherens junctions (AJ) by the addition of calcium. The adhesion process starts with propulsion of filopodia into neighbouring cells and the clustering of E-cadherin at filopodia tips. This generates a two-rowed adhesion zipper. Experiments using keratinoytes from mice with an ablated α-catenin gene and from transgenic mice expressing a dominant-negative form of the protein VASP showed that α-catenin-dependent recruitment of vinculin, zyxin,

VASP and Mena to the adhesion zipper is required to complete AJ formation. Mice lacking β3 integrins are osteosclerotic because of dysfunctional osteoclasts. McHugh K, Hodivala-Dilke K, Zheng M-H, Namba N, Lam J, Novack D, Feng X, Ross FP, Hynes RO, Teitelbaum SL: J Clin Invest 2000, 105:433-440. • Significance: Blocking experiments with antibodies and peptidomimetics have demonstrated a pivotal role for the integrin αVβ3 in osteoclast function and inititated the development of integrin aVβ3 inhibitors as potential antiosteoporosis drugs. Evaluation of mice lacking β3 integrins now buttresses this strategy and reveals the mechanism used by the knockout mice to compensate for dysfunctional osteoclasts. Findings: β3-integrin-deficient mice do not develop obvious skeletal abnormalities before the age of four months, when significant increases in bone mass and density become apparent. Compared to wild type, mutant mice contain over three-fold more osteoclasts, which display cell-morphological defects and significantly reduced bone resorptive capacities. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Jiménez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N: Nat Med 2000, 6:41-48. • Significance: This report shows for the first time that thrombospondin-1 (TSP-1), a large extracellular protein secreted by many cell types, inhibits neovascularization in vivo by a mechanism that requires the transmembrane receptor CD36 and the src-kinase fyn. Moveover this mechanism triggers endothelial cell apoptosis. Findings: Mice deficient for CD36 or fyn are insensitive to the potent anti-angiogenic activity of TSP-1 but not to that of angiostatin. TSP-1 induces the recruitment of fyn to complexes containing CD36. It activates p38 mitogen-activated protein kinases in a manner dependent on fyn and caspase-3like proteases and induces endothelial cell apoptosis in vitro and in vivo. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Ashkar S, Weber GF, Panoutsakopoulou V, Sanchirico ME, Jansson M, Zawaideh S, Rittling SR, Denhardt DT, Glimcher MJ, Cantor H: Science 2000, 287:860-864. •• Significance: Previous studies indicated that the extracellular matrix protein osteopontin is produced by T cells during the early stages of bacterial infections, where it induces inflammatory responses by interaction with macrophages. This study now shows that it governs cellular immunity against viral and bacterial infections by regulation of interleukin (IL)-12 and IL-10 production of macrophages. Findings: Osteopontin-deficient mice had severely impaired immunity to viral and bacterial infection accompanied by diminished IL-12 and increased IL-10 levels in affected lymph nodes. Purified osteopontin induced IL-12 production of macrophages via its receptor integrin αVβ3 and diminished IL-10 production via an interaction with CD44. Cell surface-localized matrix metalloproteinase-9 proteβ and promotes tumor invasion and olytically activates TGF-β angiogenesis. Yu Q, Stamenkovic I: Genes Dev 2000, 14:163-176. • Significance: A novel functional relationship is discovered between the hyaluronan receptor CD44, the matrix metalloproteinase-9 (MMP-9) and the cytokine TGF-β.

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Findings: In a recent study (Q Yu, I Stamenkovic, Genes Dev 1999, 13:35-48.), these authors demonstrated that metalloproteinase-9 (MMP-9) is activated by its coclustering with CD44 on tumor cells. Using a chimeric protein consisting of MMP-9 linked to the transmembrane and cytoplasmic domains of CD44, they now show that CD44-anchored MMP-9 efficiently promotes tumor invasion. Moreover, it proteolytically cleaves and activates latent TGF-β in tumor cell cultures and solid tumors, as well as in keratinocyte cultures from wild-type, but not from CD44-deficient, mice. CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor. Ergün S, Kilic N, Ziegeler G, Hansen A, Nollau P, Götze J, Wurmbach J-H, Horst A, Weil J, Fernando M, Wagener C: Mol Cell 2000, 5:311-320. • Significance: Novel angiogenic functions are ascribed to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) (also known as biliary glycoprotein or C-CAM), a member of the immunoglobulin family of cell-adhesion receptors. Findings: Both recombinant and native CEACAM1 induce proliferation and a chemotactic response when applied to endothelial cell cultures. Furthermore, these proteins have potent angiogenic activities in in vitro and in vivo assays either alone or in synergy with vascular endothelial growth factor (VEGF). VEGF induced CEACAM1 expression in endothelial cells. In situ CEACAM1 was detected in endothelial cells and in basement membranes of small angiogenic blood vessels. Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin. Akimov S, Krylow D, Fleischman LF, Belkin AM: J Cell Biol 2000, 148:825-838. • Significance: This study establishes a novel function for cellsurface-associated tissue transglutaminase (tTG) as an integrin-associated coreceptor for fibronectin (FN). Findings: Transfection studies indicate that tTG expression promotes cell adhesion and cell spreading on the 42 kDa gelatin-binding FN fragment independently of tTG enzymatic activity and integrin receptors. tTG stably associates with β1 and β3, but not β2 integrins, in coimmunoprecipitation experiments. It also aids the formation of ternary complexes containing tTG, integrin and the 42 kDa FN fragment. Moreover, tTG colocalizes with β1 integrins and amplifies tyrosine phosphorylation of focal adhesion kinase in cells that adhere to FN or its 42 kDa fragment. Disruption of the β-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E. Durbeej M, Cohn RD, Hrstka RF, Moore SA, Allamand V, Davidson BL, Williamson RA, Campbell KP: Mol Cell 2000, 5:141-151. • Significance: Sarcoglycans (SG) and dystroglycans (DG) collaborate to form the dystrophin–glycoprotein complex (DGC), which protects muscle cells from contraction-induced damage by conferring a link between the extracellular matrix and the actin–cytoskeleton. Mice deficient in β-SG now provide a novel insight into the function of the DGC and the pathogenesis of limb girdle muscular dystrophy type 2E. Findings: β-SG-null mice were viable and fertile but developed a severe muscular dystrophy, cardiomyopathy and vascular smooth muscle irregularities. Biochemically, this was attributable to the disruption of SG–DG complexes in membranes of skeletal, cardiac and smooth muscle cells.

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Cell differentiation Selected by Alison Schuldt Wellcome/CRC Institute, Cambridge, UK

The Neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation. McCartney BM, Kulikauskas RM, LaJeunesse DR, Fehon RG: Development 2000, 127:1315-1324. •• Significance: The authors identify a Drosophila homologue of the tumor suppressor gene, Merlin. They show that this gene functions as a heterodimer with another gene, expanded from the same protein superfamily. Together, they regulate both cell proliferation and differentiation. Specifically, a role for regulating differentiation through decapentaplegic (dpp)-mediated pathways is highlighted. Findings: Loss of Merlin expression results in hyperplasia of the affected tissue, but does not prevent differentiation. Merlin colocalises with expanded in Drosophila tissues and cell culture. The amino-terminal region of Merlin interacts with the carboxy-terminal region of Expanded in vitro. Loss of both Merlin and expanded also disrupts dpp expression and thus differentiation of the developing eye and leg. Mammalian hepatocyte differentiation requires the tranα. Li J, Ning G, Duncan SA: Genes Dev scription factor HNF-4α 2000, 14:464-474. • Significance: HNF-4α, a transcription factor of the nuclear hormone receptor family, is expressed during mammalian liver development. Targeted disruption of the HNF-4α gene has previously shown that HNF-4α is essential for embryos to complete gastrulation (Chen WS et al. Genes Dev 1994, 8:2466-2477.) Here, the authors use a complementation technique to generate embryos that rescues the function of HNF-4s during gastrulation and highlight its later function during hepatocyte differentiation. Findings: HNF-4α is not required for hepatocyte specification, but is essential for normal differentiation of this lineage. Loss of HNF-4α disrupts expression of several genes required to mediate hepatocyte differentiation, including HNF-1α and PXR. mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex differentiation in Caenorhabditis elegans. Li W, Boswell R, Wood WB: Dev Biol 2000, 218:172-182. • Significance: mag-1 is a functional homologue of Drosophila mago nashi. Here, the authors investigate the function of Mag-1 during C. elegans development and demonstrate that mag-1 is essential for maintaining the oogenesis pathway during sex determination. Findings: Disruption of mag-1 function using the RNA interference technique results in a masculinisation of the germ line. Epistasis analysis shows that mag-1 acts upstream of a number of genes important for promoting spermatogenesis. In addition, Mag-1 is required during embryogenesis for organisation of the hypodermis. Selected by Tetsuhiro Kudoh National Institute of Child Health and Human Development, Bethesda, Maryland, USA

The LIM homeobox gene Lhx9 is essential for mouse gonad formation. Birk OS, Casiano DE, Wassif CA, Cogliati T, Zhao L, Zhao Y, Grinberg A, Huang S, Kreidberg JA, Parker KL, et al.: Nature 2000, 403:909-913. • Significance: The authors discovered that LIM homeobox gene Lhx9 knockout mice specifically failed to form gonads,

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thus identifying a new player in gonad development and sexual differentiation. In addition, Lhx9 provides a new candidate for the cause of human gonadal agenesis. Findings: Lhx9 is expressed in the urogenital ridge that gives rise to the gonad. The authors generated Lhx9 homozygous null mice (–/–) and observed a defect specifically in the gonad, which caused phenotypical feminity. Although this gene is also expressed in the pancreas, limbs and central nervous system, the phenotype is restricted to the gonad, possibly because of redundancy with other LIM homeobox genes. In Lhx9–/– mice, at embryonic day E11.5, somatic cells of the genital ridge fail to proliferate and a discrete gonad fail to form. In variance with other gonad mutants including Sf1–/– and Wt1–/– mice, Lhx9–/– mice do not display massive apoptosis. Finally, Sf1 is suppressed in the genital ridge in Lhx9–/– mice, whereas Lhx9 is not effected in Sf1–/– mice, suggesting Lhx9 functions upstream of Sf1. β signalling pathways Interaction between Wnt and TGF-β during formation of Spemann’s organizer. Nishita M, Hashimoto MK, Ogata S, Laurent MN, Ueno N, Shibuya H, Cho KW: Nature 2000, 403:781-785. • Significance: The signaling cascades of the Wnt and TGF-β superfamilies are involved in various cell differentiation and cell proliferation processes. In vertebrates, these signals are required for the formation of the dorsal organizing center. In this paper, the authors found that the Wnt downstream components — β-catenin and Lef1/TCF — formed a complex with Smad4, which is the essential mediator of TGF-β signaling. These data suggest, for the first time, possible cross talk between the Wnt and TGF-β signaling cascades. Findings: In animal cap reporter assays, the promotor of the early organiser gene, twin is activated by Xwnt8 overexpression. This activation requires the Lef1 recognition motif in the promotor and an upstream region where putative Smad4 recognition sites are located. The authors found that dominantnegative Smad4 suppressed the Xwnt8-mediated activation of the twin promotor and Smad4 bound to this region in footprint analysis. In addition, the Smad4 protein also form a complex with Lef1 and β-catenin. In a luciferase reporter assay, the synergistic effect of Smad4 with Lef1 was observed and, to some extent, this effect was independent from Smad4–DNA interaction. It was, however, dependent upon protein–protein interactions between Smad4 and Lef1/β-catenin. naked cuticle encodes an inducible antagonist of Wnt signalling. Zeng W, Wharton KA Jr, Mack JA, Wang K, Gadbaw M, Suyama K, Klein PS, Scott MP: Nature 2000, 403:789-795. • Significance: A new Wnt antagonist was discovered to be the gene responsible for Drosophila mutant naked cuticle (nkd). Overexpression of nkd antagonized Wnt signaling in Drosophila as well as in Xenopus. Though the mechanism for nkd antagonization of Wnt signaling is not clear, nkd seems to a new type of general regulator of Wnt signaling. Findings: The Drosophila mutant nkd shows multiple segmentation defects, which are also seen when the Wnt molecule wingless (Wg) are over expressed as well as in the mutants of negative regulator in the Wg pathway. The nkd gene was identified from P-element insertion and found to be a high-affinity calcium-binding EF hand protein, similar to the recoverin family of myristoil switch proteins. The expression of nkd overlaps with Wg in many tissues and seemed to be upregulated by Wg. Misexpression of nkd causes several abnormalities that are indistinguishable from many Wg loss-of-function phenotypes.

The antagonistic effects of nkd were also tested in Xenopus embryos. Activin-induced animal cap extention, which depends on Wnt signaling, was inhibited by nkd overexpression. Secondary axis formation caused by Xwnt8 mRNA injection was also antagonized by co-expression of nkd mRNA. Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif. Germain S, Howell M, Esslemont GM, Hill CS: Genes Dev 2000,14:435-451. • Significance: An activin-like signal regulates mesoendoderm formation in vertebrates. As the downstream event the Smad2–Smad4 complex is known to interact with the wingedhelix transcription factor Fast1 to activate activin responsive element. The authors analyzed an activin-inducible distal element (DE) in the Xenopus gooscoid (gsc) promoter, and found that Smad2 also interacts with the paired-like homeobox genes mixer and milk to bind the DE element. This finding generalises the function of Smads, which can interact with different types of transcription factors to mediate activin-like signal in mesoendodermal induction. Findings: As the DE in the gsc promoter possesses a paired-like homeobox transcription factor recognition sequence, the authors analyzed mix family homeodomain proteins for candidates for the DE-binding protein (DEBP). Although mix1, mixer and milk bound to the DE, only mixer and milk bound to Smad2 protein as well, which is another component of DEBP complex. In the mixer and milk proteins, a short carboxy-terminal stretch of amino acids is found conserved within the Smad-interacting motif (SIM), which resides at Fast1’s carboxy terminus. In cultured cells, mixer and milk formed a complex with Smad2 and activated the DE promoter in reporter assays. These findings suggest that mixer and milk are good DEBP candidates and are essential for activin-dependent gsc promotor activation. Transient depletion of xDnmt1 leads to premature gene activation in Xenopus embryos. Stancheva I, Meehan RR: Genes Dev 2000, 14:313-327. • Significance: Transcriptional silencing in early cleavage stage embryos is a widely conserved, but the molecular mechanism has remained unclear. In Xenopus, zygotic transcription begins at the midblastula transition (MBT). In this report, the authors found that methylation may be the major regulator of preMBT silencing of transcription. Findings: Maternal DNA methyltransferase (xDnmt1) mRNA was depleted by antisense injection, which caused a decrease in methylation of genomic DNA and morphological abnormalities, including gastrulation defects, axis truncation and cell death. These abnormalities were rescued by coinjection of the Dnmt1 protein. Strikingly, the authors observed incorporation of radiolabeled nucleotides in xDnmt1-depleted embryos approximately two cell cycles earlier than the start of normal MBT. This suggests that in these embryos, zygotic transcription starts before MBT. Cerberus, Xbra and otx2, which are early mesoendodermal markers expressed after the MBT period, were examined and, in fact, found to be transcribed before normal MBT in depleted embryos. Later stage-specific genes such as muscle actin and neural β-tubulin were not induced. The Xbra promoter was analyzed for methylation levels and found to be undermethylated in the depleted embryos. These findings suggest that methylation has a general role for silencing of early MBT genes.

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

Kinetic analysis of a molecular model of the budding yeast cell cycle. Chen KC, Csikasz-Nagy A, Gyorffy B, Val J, Novak B, Tyson JJ: Mol Biol Cell 2000, 11:369-391. •• Significance: This study provides a unique view of the cell cycle, based on mathematical modeling, similar to the studies on biochemical kinetics. This model accounts for many of the details of physiology, biochemistry and genetics of cell-cycle control in budding yeast. Findings: A set of differential equations is used to describe the time course of the major classes of CDK (cyclin-dependent kinase) activities in the cell cycle and to build a mathematical model of the cell cycle. These equations are based on the plethora of data that had been published on the S. cerevisiae cell cycle. The model is then used to simulate the effect of different conditions and mutations on the cell cycle with reasonable accuracy. Activating the DNA damage checkpoint in a developmental context. Su TT, Walker J, Stumpff J: Curr Biol 2000, 10:119-126. • Significance: Different mechanisms may be used at different points during development to block cell division in response to the DNA-damage checkpoint. In pre-gastrula cleavage divisions, which lack a G2 phase, anaphase is blocked. In post-blastoderm embryos, there is a G2 phase and mitotic entry is blocked. Findings: X-ray irradiation of post-blastoderm Drosophila embryos delays mitosis by inhibitory phosphorylation of CDK1 and to a minor extent, by exclusion of mitotic cyclins from the nucleus. The delay of cell division has little effect on gastrulation, which implies that development dictates cell proliferation and not vice versa. Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7. Nguyen VQ, Co C, Irie K, Li JJ: Curr Biol 2000, 10:195-205. • Significance: The putative DNA helicase Mcm (mini-chromosome maintenance) protein complex is required for DNA replication in eukaryotes. In G1 phase of the cell cycle, the Mcm complex is bound to chromatin at the origin. As the Mcm is displaced from chromatin during DNA replication, CDK is shown to export the complex from the nucleus. In this manner, re-replication of origins is prevented in the same S phase. Findings: The Mcm protein complex is detected in live cells through the use of GFP (green fluorescent protein) fusions produced by recombinant methods in S. cerevisiae. The major S/M phase form of CDK — Cdc28/Clb — is shown to be necessary and sufficient for Mcm nuclear export. If the Mcm complex is bound to chromatin in G1 phase, it is refractory to CDK action. DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H, Liu D, Elledge SJ, Mak TW: Science 2000, 287:1824-1827.

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•• Significance: This study emphasizes the importance of the Chk2/Rad53 kinase on the DNA-damage checkpoint, which is dependent on p53. Chk2/Rad53 kinase stabilizes p53 after irradiation by gamma rays but not by UV-light. Thus, p53 may become compromised in cancer cells by the loss of Chk2/Rad53 kinase, which acts as a tumor suppressor. Findings: Recombinant mouse cells lacking Chk2/Rad53 kinase were generated by knockout mouse technology. These cells are more resistant to gamma rays because they fail to maintain p53-dependent G2 arrest and do not undergo apoptosis. The mutant cells do not stabilize p53 because of the loss of Chk2/Rad53 kinase phosphorylation of p53. This phosphorylation of p53 is at the Mdm2-binding site and thus may prevent Mdm2 from inhibiting p53. Arrest of G(1)-S progression by the p53-inducible gene PC3 is Rb dependent and relies on the inhibition of cyclin D1 transcription. Guardavaccaro D, Corrente G, Covone F, Micheli L, D’Agnano I, Starace G, Caruso M, Tirone F: Mol Cell Biol 2000, 20:1797-1815. • Significance: This study emphasizes the importance of Rb (retinoblastoma) protein in cellular differentiation. The PC3 gene present in differentiated cells, such as neurons, is antiproliferative and is regulated by p53. The PC3 protein is shown in this paper to act as a transcriptional regulator of cyclin D1. Thus, reduction of cyclin D1 would reduce CDK4 levels and produce cell cycle arrest due to Rb protein dephosphorylation and activation. Findings: The PC3 gene was cloned previously as a p53inducible gene. Expression of PC3 produces downregulation of cyclin D1 mRNA levels and Rb protein hypophosphorylation. Overexpression of cyclin D1 makes cells resistant to the effect of PC3. Rb is downstream of PC3 as Rb cells are resistant to the effect of PC3. CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis. Noton E, Diffley JF: Mol Cell 2000, 5:85-95. •• Significance: It is known that cells must complete mitosis before the origins of DNA replication can be reset for the next S phase. In G1 phase, a pre-replication complex is then formed at these origins. This pre-replication complex is converted to a post-replication complex in S phase. This study shows that CDK inactivation by the APC/C (anaphase promoting complex or cyclosome) is the only essential step for origin resetting. Findings: In yeast (S. cerevisiae), Sic1, a CDK inhibitor, is used to inhibit Cdc28/Clb, which is the S/M phase CDK of yeast. CDK inhibition is shown to cause pre-replication complex formation even in cells blocked in S phase or in mitosis. Re-replication can occur at these origins if the cells are allowed to proceed in the cell cycle by removal of the CDK inhibition. If CDK is inhibited in this fashion, but the APC is also inactivated, origin resetting still occurs.