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BBA - Molecular Cell Research (v.1744, #2)

Editorial Board (pp. ii).

Live cell imaging reveals distinct roles in cell cycle regulation for Nek2A and Nek2B by Lynda Fletcher; George J. Cerniglia; Tim J. Yen; Ruth J. Muschel (pp. 89-92).

Two splice variants of Nek2 kinase, a member of the NIMA-related family, have been identified as Nek2A and Nek2B. Nek2A regulates centrosome disjunction, spindle formation checkpoint signaling, and faithful chromosome segregation. A specific role for Nek2B has not yet been identified. Here, we have examined the distinct roles of Nek2A and Nek2B using timelapse video microscopy to follow the fate of cells progressing through the cell cycle in the absence of either Nek2A or Nek2B. We show that the down-regulation of Nek2B leads to a mitotic delay in the majority of cells. Upon exiting mitosis, cells exhibit mitotic defects such as the formation of multinucleated cells. Such phenotypes are not observed in cells that exit mitosis in the absence of Nek2A. These observations suggest that Nek2B may be required for the execution of mitotic exit.

Keywords: Centrosome; Mitotic delay; Multinucleation; Nek2

The plant Golgi apparatus—Going with the flow by Chris Hawes; B�atrice Satiat-Jeunemaitre (pp. 93-107).

The plant Golgi apparatus is composed of many separate stacks of cisternae which are often associated with the endoplasmic reticulum and which in many cell types are motile. In this review, we discuss the latest data on the molecular regulation of Golgi function. The concept of the Golgi as a distinct organelle is challenged and the possibility of a continuum between the endoplasmic reticulum and Golgi is proposed.

Keywords: Plant Golgi apparatus; Membrane flow; Protein trafficking

Golgi reassembly after mitosis: The AAA family meets the ubiquitin family by Hemmo H. Meyer (pp. 108-119).

The Golgi apparatus in animal cells breaks down at the onset of mitosis and is later rebuilt in the two daughter cells. Two AAA ATPases, NSF and p97/VCP, have been implicated in regulating membrane fusion steps that lead to regrowth of Golgi cisternae from mitotic fragments. NSF dissociates complexes of SNARE proteins, thereby reactivating them to mediate membrane fusion. However, NSF has a second function in regulating SNARE pairing together with the ubiquitin-like protein GATE-16. p97/VCP, on the other hand, is involved in a cycle of ubiquitination and deubiquitination of an unknown target that governs Golgi membrane dynamics. Here, these findings are reviewed and discussed in the context of the increasingly evident role of ubiquitin in membrane traffic processes.

Keywords: Membrane fusion; Ubiquitin; CDC48; Valosin-containing protein; VCIP135; Deubiquitinating enzyme

SNAREs and traffic by Wanjin Hong (pp. 120-144).

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are now generally accepted to be the major players in the final stage of the docking and the subsequent fusion of diverse vesicle-mediated transport events. The SNARE-mediated process is conserved evolutionally from yeast to human, as well as mechanistically and structurally across different transport events in eukaryotic cells. In the post-genomic era, a fairly complete list of “all�? SNAREs in several organisms (including human) can now be made. This review aims to summarize the key properties and the mechanism of action of SNAREs in mammalian cells.

Keywords: Soluble; N; -ethylmaleimide-sensitive factor attachment protein receptor; Vesicle fusion; Membrane traffic; Synaptic transmission; Exocytosis; Endocytosis

MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes by Maria D. Mavrouli; Sotiris Tsakas; Georgios L. Theodorou; Maria Lampropoulou; Vassilis J. Marmaras (pp. 145-156).

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.

Keywords: Prophenoloxidase; Mitogen activating protein kinase; Signalling; Phagocytosis; Prophenoloxidase-activating proteinase; Insect immunity

N-benzyloxycarbonyl-l-proline: An in vitro and in vivo inhibitor of prolidase by Anna Lupi; Antonio Rossi; Patrizia Vaghi; Angelo Gallanti; Giuseppe Cetta; Antonella Forlino (pp. 157-163).

Prolidase deficiency (PD) is a recessive disorder of the connective tissue caused by mutations in the prolidase, a specific peptidase, cleaving the dipeptides with a C-terminal prolyl and hydroxyprolyl residue. PD is a complex syndrome characterized mainly by intractable skin lesions, recurrent respiratory infections and mental retardation. The relation between prolidase biological functions and the disease is still largely unknown. We studied the effect of a prolidase inhibitor, N-benzyloxycarbonyl-l-proline (Cbz-Pro), in vitro on prolidase from human fibroblasts and in vivo on murine erythrocytes prolidase. A 90% inhibition was detected incubating cellular extracts at 1:1 ratio of Gly-Pro substrate: Cbz-Pro inhibitor. Pulse experiments performed incubating human fibroblasts with 6 mM Cbz-Pro revealed that the inhibitor uptake was completed in about 1 min. The Cbz-Pro uptake was saturable and pH dependent. Long-term incubation of fibroblasts with Cbz-Pro caused mitochondria depolarization and increased cellular death as reported for long-term culture of fibroblasts from PD patients. An inhibitory effect of Cbz-Pro has also been shown in vivo. Our results demonstrated that Cbz-Pro is a potent inhibitor of prolidase in cultured fibroblasts and it can be used in vivo to better characterize the prolidase enzyme and further investigate PD physiopathology.

Keywords: N; -benzyloxycarbonyl-; l; -proline; Prolidase; Prolidase deficiency; Prolidase inhibition; Capillary electrophoresis

Early signaling interactions between the insulin and leptin pathways in bovine myogenic cells by A. Lulu Strat; Theresa A. Kokta; Michael V. Dodson; Arieh Gertler; Zida Wu; Rodney A. Hill (pp. 164-175).

Cross-talk between hormone signaling pathways provides mechanisms to facilitate flexibility in the cellular response to extracellular conditions. One function of insulin is to signal high extracellular glucose, while leptin may signal the abundance of extracellular lipid, both energy sources being readily utilized by muscle. The present study reports early signaling events in the insulin and leptin cascades in primary bovine myogenic cells (BMC). BMC were treated with insulin, or leptin for 1, 10, 30 and 120 min, or pretreated with leptin for 10 min followed by insulin for 1, 10, 30 and 120 min. BMC were insulin resistant, showing a significant inhibition of IRS-1 association with the insulin receptor (IR) following insulin stimulation, a corresponding increase in PI 3-kinase association with the IR, and a slow and modest increase in GLUT4 recruitment to the plasma membrane. Pretreatment of BMC for 10 min leptin, followed by insulin time-course, caused IRS-1 recruitment to be unresponsive, but evoked a rapid, phasic response of PI 3-kinase recruitment to the IR and abrogated the response of GLUT4 translocation to the plasma membrane evoked by insulin alone. The lack of insulin response was independent of IR abundance or affinity. JAK-2 association with the ObR and JAK-2 tyrosine phosphorylation were responsive to all three treatments. Insulin alone down-regulated the leptin signaling pathway, JAK-2 association with ObR decreased at all time-points, and JAK-2 phosphorylation decreased similarly. Leptin alone also appeared to down-regulate JAK-2 association with the ObR, but stimulated the down-regulated pathway to signal, JAK-2 tyrosine phosphorylation being increased at later time-points. Pretreatment with leptin followed by insulin time-course showed marked up-regulation of the early leptin signaling pathway, JAK-2 association with the ObR being increased by insulin while JAK-2 tyrosine phosphorylation was also increased. The contrasting responses of BMC to insulin alone, leptin alone and the sequential leptin–insulin treatment may point to the ability of these cells to respond to energy substrate availability, as bovine muscle has evolved to utilize lipids and fatty acids in response to a metabolism which provides only limited glucose. This cross-talk between insulin and leptin signaling pathways points to a better understanding of the mechanisms driving energy substrate utilization in ruminant muscle and may provide a useful model for greater understanding of the molecular mechanisms underlying the development of insulin resistance and Type 2 diabetes in man.

Keywords: Abbreviations; IR; insulin receptor kinase; IRS-1; insulin receptor substrate-1; PI; phosphatidylinositol; GLUT4; glucose transporter 4; ObR; leptin receptor; JAK-2; Janus kinase-2; BMC; bovine myogenic cellsInsulin resistance; Signal transduction; Insulin receptor; IR; IRS-1; PI 3-kinase; GLUT4; Leptin; Leptin receptor; ObR; JAK-2; Bovine

Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements by László Bene; Szollosi János Szöllősi; Gergely Szentesi; László Damjanovich; Rezso Rezső Gáspár Jr.; Thomas A. Waldmann; Sándor Damjanovich (pp. 176-198).

Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Rα, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence.

Keywords: Abbreviations; mAb; monoclonal antibody; Fab; Fab fragment; FRET; fluorescence resonance energy transfer; MHCI and MHCII; major hystocompatibility complex class I and class II; IL-2Rα; interleukin-2 receptor α subunit; TrfR; transferrin receptorFluorescence anisotropy; Receptor clustering; MHCI and MHCII glycoprotein; IL-2Rα; Transferrin receptor; CD45

NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum by Bernard Lardy; Mireille Bof; Laurence Aubry; Marie H�l�ne Paclet; Fran�oise Morel; Michel Satre; G�rard Klein (pp. 199-212).

Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91^phox) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22^phox) and of the cytosolic factor p67^phox. Null-mutants of either noxA, noxB, noxC or p22^phox show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA_myc2 in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22^phox null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22^phox null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.

Keywords: NADPH oxidase; Dictyostelium; Cell-type differentiation; EF-hand

The role of reactive oxygen species in TNFα-dependent expression of the receptor for advanced glycation end products in human umbilical vein endothelial cells by Tapan K. Mukherjee; Srirupa Mukhopadhyay; John R. Hoidal (pp. 213-223).

Engagement of the receptor for advanced glycation end products (RAGE) by its signal transduction ligands is implicated in the development and progression of atherosclerosis. TNFα, a proinflammatory cytokine, is a potent inducer of RAGE expression in endothelial cells. In the present study, we demonstrate that reactive oxygen species (ROS) generated by TNFα stimulated human umbilical vein endothelial cells (HUVECs) induce RAGE expression. The complex III of mitochondrial respiratory chain appears to be the primary source of ROS. The gp91phox subunit of NADPH oxidase appears to be the source of ROS that induces TNFα-dependent mitochondrial ROS generation and subsequent RAGE expression. We also demonstrate that the ROS-mediated RAGE induction occurs via activation of NF-κB, a proinflammatory transcription factor. Thus, stimulation of HUVECs by TNFα evokes the following sequence of events: stimulation of NADPH oxidase → generation of ROS → activation of the mitochondrial respiratory chain → stimulation of NF-κB activity → induction of RAGE expression.

Keywords: TNFα; ROS; NADPH oxidase; Mitochondria; RAGE; HUVEC

Vasoactive intestinal peptide (VIP) induces c- fos expression in LNCaP prostate cancer cells through a mechanism that involves Ca²⁺ signalling. Implications in angiogenesis and neuroendocrine differentiation by Beatriz Collado; Mar�a G. S�nchez; In�s D�az-Laviada; Juan C. Prieto; Mar�a J. Carmena (pp. 224-233).

The effect of vasoactive intestinal peptide (VIP) on intracellular Ca²⁺ levels and its relationship with the expression of c- fos and vascular endothelial growth factor (VEGF) as well as with neuroendocrine (NE) differentiation were investigated in human prostate LNCaP cells. VIP induced the expression of c- fos mRNA as studied by reverse transcription polymerase chain reaction (RT-PCR). It was accompanied by VIP stimulation of c- fos protein synthesis, as measured by Western blot analysis. VIP enhanced intracellular Ca²⁺ levels as evaluated using the calcium probe fura-2. VIP regulation of c- fos expression depended on [Ca²⁺]_i concentration since the intracellular calcium chelator BAPTA/AM decreased c- fos expression (both mRNA and protein) to basal levels. As shown by means of real-time RT-PCR, VIP stimulated VEGF mRNA expression: the effect was inhibited by 40% in the presence of curcumin (an inhibitor of AP-1 binding), and it was dependent on Ca²⁺ since BAPTA/AM inhibited this VIP action by 43%. Similar observations were made on the effects of BAPTA/AM and curcumin on VIP stimulation of VEGF protein expression. Simultaneous treatment of cells with the protein kinase A inhibitor H89 and BAPTA/AM completely blocked this VIP effect, whereas each agent alone led only to a partial inhibition. In addition, the calcium chelator blocked by 37% the ability of VIP to induce NE cell differentiation as estimated by the observation of neurite development. These features support a VIP signalling pathway that could be mediated through both cAMP and [Ca²⁺]_i increase in prostate LNCaP cancer cells. Moreover, our data suggest the implication of c-Fos on the induction of the main angiogenic factor VEGF since the promoter region of the VEGF gene possesses AP-1 (i.e., c-Fos/c-Jun heterodimer) response elements. This feature represents a link between the nuclear oncogene c- fos, angiogenesis and NE differentiation by means of an initiating signal upon VIP receptors.

Keywords: VIP receptor; Prostate cancer; c-; fos; Ca; ²⁺; cAMP; VEGF

Functional properties of a recombinant bacterial DING protein: Comparison with a homologous human protein by Ken Scott; Liyun Wu (pp. 234-244).

DING proteins are highly-conserved proteins with poorly-defined cell-signalling roles in mammals. Conserved homologues are also commonplace in plants, though not as yet functionally characterized. Poor availability of the proteins, and a lack of genetic structure, hamper progress in elucidating the roles of these eukaryotic DING proteins, but highly-homologous hypothetical DING proteins have recently been identified in Pseudomonas genomes. We have cloned and expressed a DING protein from P. fluorescens SWB25 in Escherichia coli. The recombinant protein, and its natural human homologue, act as phosphate-binding proteins, as predicted by structural homologies with other bacterial proteins. The recombinant protein also displays other functional similarities with mammalian DING proteins, in that, like the human version, it acts as a mitogen for cultured human cells, and can bind cotinine, known to be a binding ligand for a rat neuronal DING protein.

Keywords: DING protein; Cell-signaling; Pseudomonas fluorescens; Human; Mitogen; Phosphate-binding

Paclitaxel resistance in cells with reduced β-tubulin by Yaqing Wang; Fernando Cabral (pp. 245-255).

We previously described the isolation of colcemid resistant Chinese hamster ovary cell lines containing α- and β-tubulin mutations that increase microtubule assembly and stability. By analyzing colcemid sensitive revertants from one of the β-tubulin mutants, we now find that loss or inactivation of the mutant allele represents the most common mechanism of reversion. Consistent with this loss, the revertants have 35% less tubulin at steady state, no evidence for the presence of a mutant polypeptide, and a normal extent of tubulin polymerization. In addition to the loss of colcemid resistance, the revertant cells exhibit increased resistance to paclitaxel relative to wild-type cells. This paclitaxel resistance can be suppressed by transfecting the revertant cells with a cDNA for wild-type β-tubulin, indicating that the reduction in tubulin in the revertant cells is responsible for the resistance phenotype. We propose that reducing tubulin levels may represent a novel mechanism of paclitaxel resistance.

Keywords: Abbreviations; 2D; two-dimensional; CHO; Chinese hamster ovary; GST; glutathione-; S; -transferase; HA; hemagglutinin; MAP; microtubule associated protein; MDR; multidrug resistance; MTB; microtubule buffer; OD; optical density; RT-PCR; reverse transcription-polymerase chain reaction; TTA; tetracycline transactivator; UTR; untranslated regionColcemid resistance; Gene inactivation; Microtubule assembly; Drug resistance; Tetracycline regulated expression

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BBA - Molecular Cell Research (v.1744, #2)