bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Plasma Membrane Anchoring of Newly Synthesized p59  fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3
Authors: Hof Wouter van 't , Resh Marilyn D.
Published: July 2002

The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [35S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20– 60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1–2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH2-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Gαo-, Gαs-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10–20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.
ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells
Authors: Faúndez Victor , Horng Jim-Tong , Kelly Regis B.
Published: July 2002

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041–1049). A peptide spanning the effector domain of human ARF1 (2–17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.
Fibroblast Growth Factor 4 Directs Gap Junction Expression in the Mesenchyme of the Vertebrate Limb Bud
Authors: Makarenkova H. , Becker D.L. , Tickle C. , Warner A.E.
Published: July 2002

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell–cell communication and polarizing signaling are intimately connected.
Accumulation of Muscle Ankyrin Repeat Protein Transcript Reveals Local Activation of Primary Myotube Endcompartments during Muscle Morphogenesis
Authors: Baumeister Anja , Arber Silvia , Caroni Pico
Published: July 2002

The characteristic shapes and positions of each individual body muscle are established during the process of muscle morphogenesis in response to patterning information from the surrounding mesenchyme. Throughout muscle morphogenesis, primary myotubes are arranged in small parallel bundles, each myotube spanning the forming muscles from end to end. This unique arrangement potentially assigns a crucial role to primary myotube end regions for muscle morphogenesis.

We have cloned muscle ankyrin repeat protein (MARP) as a gene induced in adult rat skeletal muscle by denervation. MARP is the rodent homologue of human C-193 (Chu, W., D.K. Burns, R.A. Swerick, and D.H. Presky. 1995. J. Biol. Chem. 270:10236–10245) and is identical to rat cardiac ankyrin repeat protein. (Zou, Y., S. Evans, J. Chen, H.-C. Kuo, R.P. Harvey, and K.R. Chien. 1997. Development. 124:793–804). In denervated muscle fibers, MARP transcript accumulated in a unique perisynaptic pattern. MARP was also expressed in large blood vessels and in cardiac muscle, where it was further induced by cardiac hypertrophy. During embryonic development, MARP was expressed in forming skeletal muscle. In situ hybridization analysis in mouse embryos revealed that MARP transcript exclusively accumulates at the end regions of primary myotubes during muscle morphogenesis. This closely coincided with the expression of thrombospondin-4 in adjacent prospective tendon mesenchyme, suggesting that these two compartments may constitute a functional unit involved in muscle morphogenesis. Transfection experiments established that MARP protein accumulates in the nucleus and that the levels of both MARP mRNA and protein are controlled by rapid degradation mechanisms characteristic of regulatory early response genes. The results establish the existence of novel regulatory muscle fiber subcompartments associated with muscle morphogenesis and denervation and suggest that MARP may be a crucial nuclear cofactor in local signaling pathways from prospective tendon mesenchyme to forming muscle and from activated muscle interstitial cells to denervated muscle fibers.
Embryonic Expression of the Putative γ Subunit of the Sodium Pump Is Required for Acquisition of Fluid Transport Capacity during Mouse Blastocyst Development
Authors: Jones D. Holstead , Davies Tyler C. , Kidder Gerald M.
Published: July 2002

The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic α subunit and a noncatalytic, glycosylated β subunit. Recently, a putative third subunit, the γ subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the α and β subunits and is closely associated with the ouabain binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the γ subunit in preimplantation mouse development, where Na+,K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the γ subunit mRNA accumulates continuously from the eight-cell stage onward and that it cosediments with polyribosomes from its time of first appearance. Confocal immunofluorescence microscopy revealed that the γ subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage. In contrast with the α and β subunits, the γ subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos were treated with antisense oligodeoxynucleotide complementary to the γ subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+,K+-ATPase enzymatic activity was unaffected as determined by a direct phosphorylation assay (“back door” phosphorylation) applied to plasma membrane preparations. These results indicate that the γ subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression is essential for blastocoel formation in the mouse.
Caveolin Transfection Results in Caveolae Formation but Not Apical Sorting of Glycosylphosphatidylinositol (GPI)-anchored Proteins in Epithelial Cells
Authors: Lipardi Concetta , Mora Rosalia , Colomer Veronica , Paladino Simona , Nitsch Lucio , Rodriguez-Boulan Enrique , Zurzolo Chiara
Published: July 2002

Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The “raft” hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI- anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI- anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42–53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI- anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.
Nopp140 Functions as a Molecular Link Between the Nucleolus and the Coiled Bodies
Authors: Isaac Cynthia , Yang Yunfeng , Thomas Meier U.
Published: July 2002

Coiled bodies are small nuclear organelles that are highly enriched in small nuclear RNAs, and that have long been thought to be associated with the nucleolus. Here we use mutational analysis, transient transfections, and the yeast two-hybrid system to show that the nucleolar phosphoprotein Nopp140 functions as a molecular link between the two prominent nuclear organelles. Exogenous Nopp140 accumulated in the nucleolus rapidly, but only after a lag phase in coiled bodies, suggesting a pathway between the two organelles. The expression of partial Nopp140 constructs exerted dominant negative effects on the endogenous Nopp140 by chasing it and other antigens that were common to both organelles out of the nucleolus. The alternating positively and negatively charged repeat domain of Nopp140 was required for targeting to both organelles. In addition, partial Nopp140 constructs caused formation of novel structures in the nucleoplasm and, in the case of the conserved carboxy terminus, led to the dispersal of coiled bodies. As a final link, we identified the coiled body–specific protein p80 coilin in a yeast two-hybrid screen with Nopp140. The interaction of the two proteins was confirmed by coimmunoprecipitation. Taken together, Nopp140 appeared to shuttle between the nucleolus and the coiled bodies, and to chaperone the transport of other molecules.
Functional Characteristics of ES Cell–derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein
Authors: Kolossov E. , Fleischmann B.K. , Liu Q. , Bloch W. , Viatchenko-Karpinski S. , Manzke O. , Ji G.J. , Bohlen H. , Addicks K. , Hescheler J.
Published: July 2002

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions.

In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac α-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1–2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs.

Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.
Neurotrophin Regulation of β-Actin mRNA and Protein Localization within Growth Cones
Authors: Zhang H.L. , Singer R.H. , Bassell G.J.
Published: July 2002

Neurotrophins play an essential role in the regulation of actin-dependent changes in growth cone shape and motility. We have studied whether neurotrophin signaling can promote the localization of β-actin mRNA and protein within growth cones. The regulated localization of specific mRNAs within neuronal processes and growth cones could provide a mechanism to modulate cytoskeletal composition and growth cone dynamics during neuronal development. We have previously shown that β-actin mRNA is localized in granules that were distributed throughout processes and growth cones of cultured neurons. In this study, we demonstrate that the localization of β-actin mRNA and protein to growth cones of forebrain neurons is stimulated by neurotrophin-3 (NT-3). A similar response was observed when neurons were exposed to forskolin or db-cAMP, suggesting an involvement of a cAMP signaling pathway. NT-3 treatment resulted in a rapid and transient stimulation of PKA activity that preceded the localization of β-actin mRNA. Localization of β-actin mRNA was blocked by prior treatment of cells with Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A. Depolymerization of microtubules, but not microfilaments, inhibited the NT-3–induced localization of β-actin mRNA. These results suggest that NT-3 activates a cAMP-dependent signaling mechanism to promote the microtubule-dependent localization of β-actin mRNA within growth cones.
Comparative Analysis of P73 and P53 Regulation and Effector Functions
Authors: Fang Li , Lee Sam W. , Aaronson Stuart A.
Published: July 2002

p53 is mutated in ∼50% of human cancers, whereas mutations of the related p73 gene are rare. p73 can activate p53-responsive promoters and induce apoptosis when overexpressed in certain p53-deficient tumor cells. We show that p73 isoforms, p73α and p73β, can each induce permanent growth arrest with markers of replicative senescence when overexpressed in a tetracycline-regulatable manner in human cancer cells lacking functional p53. Human homologue of mouse double minute 2 gene product (hMDM2), but not an NH2-terminal deletion mutant, coimmunoprecipated with p73α or p73β, and inhibited p73 transcriptional activity as with p53. In contrast to p53, ectopically expressed hemagglutinin (HA)-tagged p73 proteins were not stabilized by treatment with several DNA damaging agents. Furthermore, unlike normal p53, which increases in response to DNA damage due to enhanced protein stability in MCF7 cells, endogenous p73 protein levels were not increased in these cells under the same conditions. Thus, although p73 has an ability, comparable to that of p53, to suppress tumor cell growth in p53-deficient cells, p73 induction is regulated differently from p53. These findings suggest that the selective pressures for p53 rather than p73 inactivation in tumors may reflect their differential responses to stresses such as DNA damage, rather than their capacities to induce permanent growth arrest or apoptosis programs.
Alternative Splicing Regulates the Subcellular Localization of a-Kinase Anchoring Protein 18 Isoforms
Authors: Trotter Kevin W. , Fraser Iain D.C. , Scott Gregory K. , Stutts M. Jackson , Scott John D. , Milgram Sharon L.
Published: July 2002

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca2+ channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18β and AKAP18γ, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18α) and AKAP18β target the plasma membrane when expressed in HEK-293 cells, while AKAP18γ is cytosolic. When expressed in epithelial cells, AKAP18α is targeted to lateral membranes, whereas AKAP18β is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18β with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.
Fusion of Small Peroxisomal Vesicles in Vitro Reconstructs an Early Step in the in Vivo Multistep Peroxisome Assembly Pathway of Yarrowia lipolytica
Authors: Titorenko Vladimir I. , Chan Honey , Rachubinski Richard A.
Published: July 2002

We have identified and purified six subforms of peroxisomes, designated P1 to P6, from the yeast, Yarrowia lipolytica. An analysis of trafficking of peroxisomal proteins in vivo suggests the existence of a multistep peroxisome assembly pathway in Y. lipolytica. This pathway operates by conversion of peroxisomal subforms in the direction P1, P2→P3→P4→P5→P6 and involves the import of various peroxisomal proteins into distinct vesicular intermediates. We have also reconstituted in vitro the fusion of the earliest intermediates in the pathway, small peroxisomal vesicles P1 and P2. Their fusion leads to the formation of a larger and more dense peroxisomal vesicle, P3. Fusion of P1 and P2 in vitro requires cytosol and ATP hydrolysis and is inhibited by antibodies to two membrane-associated ATPases of the AAA family, Pex1p and Pex6p. We provide evidence that the fusion in vitro of P1 and P2 peroxisomes reconstructs an actual early step in the peroxisome assembly pathway operating in vivo in Y. lipolytica.
Regulated Expression ofnulloIs Required for the Formation of Distinct Apical and Basal Adherens Junctions in theDrosophilaBlastoderm
Authors: Hunter Christine , Wieschaus Eric
Published: July 2002

During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila β-catenin homologue Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. In this paper, we show that the basal accumulation of Arm colocalizes with DE-cadherin and Dα-catenin, and corresponds to a region of tight membrane association, which we refer to as the basal junction. Although the two junctions are similar in components and function, they differ in their response to the novel cellularization protein Nullo. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation.
Localization and Activity of Myosin Light Chain Kinase Isoforms during the Cell Cycle
Authors: Poperechnaya Angela , Varlamova Olga , Lin Pei-ju , Stull James T. , Bresnick Anne R.
Published: July 2002

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH2-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH2-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735–744) act cooperatively to regulate the level of Ser 19–phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.
Mutagenesis of the Phosphatidylinositol 4,5-Bisphosphate (Pip2) Binding Site in the Nh2-Terminal Domain of Ezrin Correlates with Its Altered Cellular Distribution
Authors: Barret Cécile , Roy Christian , Montcourrier Philippe , Mangeat Paul , Niggli Verena
Published: July 2002

The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidyl-inositol 4,5-bisphosphate (PIP2)-containing liposomes via its NH2-terminal domain. Using internal deletions and COOH-terminal truncations, determinants of PIP2 binding were located to amino acids 12–115 and 233–310. Both regions contain a KK(X)nK/RK motif conserved in the ezrin/radixin/moesin family. K/N mutations of residues 253 and 254 or 262 and 263 did not affect cosedimentation of ezrin 1-333 with PIP2-containing liposomes, but their combination almost completely abolished the capacity for interaction. Similarly, double mutation of Lys 63, 64 to Asn only partially reduced lipid interaction, but combined with the double mutation K253N, K254N, the interaction of PIP2 with ezrin 1-333 was strongly inhibited. Similar data were obtained with full-length ezrin. When residues 253, 254, 262, and 263 were mutated in full-length ezrin, the in vitro interaction with the cytoplasmic tail of CD44 was not impaired but was no longer PIP2 dependent. This construct was also expressed in COS1 and A431 cells. Unlike wild-type ezrin, it was not any more localized to dorsal actin-rich structures, but redistributed to the cytoplasm without strongly affecting the actin-rich structures. We have thus identified determinants of the PIP2 binding site in ezrin whose mutagenesis correlates with an altered cellular localization.
RhoA is required for monocyte tail retraction during transendothelial migration
Authors: Worthylake Rebecca A. , Lemoine Sean , Watson Joanna M. , Burridge Keith
Published: July 2002

Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of β2 integrin in the unretracted tails.
Rapid nitric oxide–induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme
Authors: Mullershausen Florian , Russwurm Michael , Thompson W. Joseph , Liu Li , Koesling Doris , Friebe Andreas
Published: July 2002

Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.
Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells
Authors: Ohta Toshiro , Essner Russell , Ryu Jung-Hwa , Palazzo Robert E. , Uetake Yumi , Kuriyama Ryoko
Published: July 2002

By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.
Huntingtin toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1
Authors: Meriin Anatoli B. , Zhang Xiaoqian , He Xiangwei , Newnam Gary P. , Chernoff Yury O. , Sherman Michael Y.
Published: July 2002

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1–151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1–85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.
Impaired Host Defense, Hematopoiesis, Granulomatous Inflammation and Type 1–Type 2 Cytokine Balance in Mice Lacking CC Chemokine Receptor 1
Authors: Gao Ji-Liang , Wynn Thomas A. , Chang Yun , Lee Eric J. , Broxmeyer Hal E. , Cooper Scott , Tiffany H. Lee , Westphal Heiner , Kwon-Chung June , Murphy Philip M.
Published: July 2002

CC chemokine receptor 1 (CCR1) is expressed in neutrophils, monocytes, lymphocytes, and eosinophils, and binds the leukocyte chemoattractant and hematopoiesis regulator macrophage inflammatory protein (MIP)-1α, as well as several related CC chemokines. Four other CCR subtypes are known; their leukocyte and chemokine specificities overlap with, but are not identical to, CCR1, suggesting that CCR1 has both redundant and specific biologic roles. To test this, we have developed CCR1-deficient mice (−/−) by targeted gene disruption. Although the distribution of mature leukocytes was normal, steady state and induced trafficking and proliferation of myeloid progenitor cells were disordered in −/− mice. Moreover, mature neutrophils from −/− mice failed to chemotax in vitro and failed to mobilize into peripheral blood in vivo in response to MIP-1α. Consistent with this, −/− mice had accelerated mortality when challenged with Aspergillus fumigatus, a fungus controlled principally by neutrophils. To test the role of CCR1 in granuloma formation, we injected Schistosoma mansoni eggs intravenously, and observed a 40% reduction in the size of lung granulomas in −/− mice compared to +/+ littermates. This was associated with increased interferon-γ and decreased interleukin-4 production in −/− versus +/+ lung lymph node cells stimulated with egg-specific antigen, suggesting that CCR1 influences the inflammatory response not only through direct effects on leukocyte chemotaxis, but also through effects on the type 1–type 2 cytokine balance. Thus CCR1 has nonredundant functions in hematopoiesis, host defense, and inflammation.
The Direct Binding of a p58 Killer Cell Inhibitory Receptor to Human Histocompatibility Leukocyte Antigen (HLA)-Cw4 Exhibits Peptide Selectivity
Authors: Rajagopalan Sumati , Long Eric O.
Published: July 2002

Natural killer (NK) cells in mice and humans express a number of structurally diverse receptors that inhibit target cell lysis upon recognition of major histocompatibility complex (MHC) class I molecules expressed on targets. The contribution of peptide to the structural features of class I required for NK cell inhibition appears to vary depending on the type of receptor engaged. Thus, while there is no peptide specificity in NK inhibition mediated by Ly-49A in the mouse, human histocompatibility antigen (HLA)-B*2705–specific NK clones displayed selectivity for peptides. In this report, we examine the role of peptide in the recognition of HLA-C by the defined killer cell inhibitory receptor (KIR) cl42 with established specificity for HLA-Cw4. Binding of soluble KIR cl42 molecules to HLA-Cw4 expressed on transporter associated with antigen presentation (TAP)-deficient RMA-S cells occurred only upon exogenous peptide loading. Moreover, there was peptide selectivity in that certain substitutions at positions 7 and 8 of the nonamer peptide QYDDAVYKL abolished Cw4 interaction with KIR cl42 despite similar surface expression of HLA-C. The specificity of this direct interaction between peptideloaded HLA-Cw4 on RMA-S cells and soluble KIR cl42 correlated with recognition by NK clones in that they were inhibited only by HLA-Cw4 loaded with the appropriate peptides.
Identification of a Gal/GalNAc Lectin in the Protozoan Hartmannella vermiformis as a Potential Receptor for Attachment and Invasion by the Legionnaires' Disease Bacterium
Authors: Venkataraman Chandrasekar , Haack Bradley J. , Bondada Subbarao , Kwaik Yousef Abu
Published: July 2002

The Legionnaire's disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen which invades and replicates within two evolutionarily distant hosts, free-living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaire's disease. Although attachment and invasion of human macrophages by L. pneumophila is mediated in part by the complement receptors CR1 and CR3, the protozoan receptor involved in bacterial attachment and invasion has not been identified. To define the molecular events involved in invasion of protozoa by L. pneumophila, we examined the role of protein tyrosine phosphorylation of the protozoan host Hartmannella vermiformis upon attachment and invasion by L. pneumophila. Bacterial attachment and invasion were associated with a time-dependent tyrosine dephosphorylation of multiple host cell proteins. This host cell response was highly specific for live L. pneumophila, required contact with viable bacteria, and was completely reversible following washing off the bacteria from the host cell surface. Tyrosine dephosphorylation of host proteins was blocked by a tyrosine phosphatase inhibitor but not by tyrosine kinase inhibitors. One of the tyrosine dephosphorylated proteins was identified as the 170-kD galactose/N-acetylgalactosamine–inhibitable lectin (Gal/GalNAc) using immunoprecipitation and immunoblotting by antibodies generated against the Gal/GalNAc lectin of the protozoan Entamoeba histolytica. This Gal/GalNAc–inhibitable lectin has been shown previously to mediate adherence of E. histolytica to mammalian epithelial cells. Uptake of L. pneumophila by H. vermiformis was specifically inhibited by two monovalent sugars, Gal and GalNAc, and by mABs generated against the 170-kD lectin of E. histolytica. Interestingly, inhibition of invasion by Gal and GalNAc was associated with inhibition of bacterial-induced tyrosine dephosphorylation of H. vermiformis proteins. High stringency DNA hybridization confirmed the presence of the 170-kD lectin gene in H. vermiformis. We conclude that attachment of L. pneumophila to the H. vermiformis 170-kD lectin is required for invasion and is associated with tyrosine dephosphorylation of the Gal lectin and other host proteins. This is the first demonstration of a potential receptor used by L. pneumophila to invade protozoa.
The Lymphocyte Function–associated Antigen 1 I Domain Is a Transient Binding Module for Intercellular Adhesion Molecule (ICAM)-1 and ICAM-3 in Hydrodynamic Flow
Authors: Knorr Ruth , Dustin Michael L.
Published: July 2002

The I domain of lymphocyte function–associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1–containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.
Circulating Activated Platelets Reconstitute Lymphocyte Homing and Immunity in L-selectin–Deficient Mice
Authors: Diacovo Thomas G. , Catalina Michelle D. , Siegelman Mark H. , Andrian Ulrich H. von
Published: July 2002

Peripheral lymph nodes (PLN) are critical for immunologic memory formation in response to antigens that penetrate the skin. Blood-borne lymphocytes first encounter such antigens after they home to PLN through a multi-step adhesion process that is normally initiated by L-selectin (CD62L) in high endothelial venules (HEV). Since naive T cells can not enter PLN normally in L-selectin–deficient mice, a delayed type hypersensitivity response to cutaneously applied antigen cannot be mounted. In this study, we report that the administration of activated platelets into the systemic circulation of L-selectin knockout mice restores lymphocyte trafficking to PLN, and reconstitutes T cell–mediated immunity in response to a cutaneous antigen. These effects required platelet-expressed P-selectin that allows activated platelets to transiently form a bridge between lymphocytes and HEV, thereby enabling lymphocytes to undergo subsequent β2 integrin-dependent firm adhesion. These profound effects of platelet-mediated cell–cell interactions on lymphocyte trafficking and formation of immunologic memory may impact on a variety of autoimmune and inflammatory conditions.
A Gene Encoding Antigenic Peptides of Human Squamous Cell Carcinoma Recognized by Cytotoxic T Lymphocytes
Authors: Shichijo Shigeki , Nakao Masanobu , Imai Yasuhisa , Takasu Hideo , Kawamoto Mayumi , Niiya Fumihiko , Yang Damu , Toh Yuji , Yamana Hideaki , Itoh Kyogo
Published: July 2002

Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients.
Role of Nerve Growth Factor in Cutaneous Wound Healing: Accelerating Effects in Normal and Healing-impaired Diabetic Mice
Authors: Matsuda Hiroshi , Koyama Hiromi , Sato Hiroaki , Sawada Junko , Itakura Atsuko , Tanaka Akane , Matsumoto Masahiro , Konno Katsuhiko , Ushio Hiroko , Matsuda Kuniko
Published: July 2002

Four full-thickness skin wounds made in normal mice led to the significant increase in levels of nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA and protein of NGF were detected in newly formed epithelial cells at the edge of the wound and fibroblasts consistent with the granulation tissue produced in the wound space, suggests that NGF was also produced at the wounded skin site. Topical application of NGF into the wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing linear wounds in normal and diabetic mice. These findings suggested that NGF immediately and constitutively released in response to cutaneous injury may contribute to wound healing through broader biological activities, and NGF improved the diabetic impaired response of wound healing.
Caspase-mediated Cleavage of Focal Adhesion Kinase pp125FAK and Disassembly of Focal Adhesions in Human Endothelial Cell Apoptosis
Authors: Levkau Bodo , Herren Barbara , Koyama Hidenori , Ross Russell , Raines Elaine W.
Published: July 2002

Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation–induced apoptosis in confluent monolayers of human umbilical vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase (pp125FAK), an important component of the focal adhesion complex, and identify pp125FAK as a novel substrate for caspase-3 and caspase-3–like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125FAK and paxillin from focal adhesion sites and their redistribution into the characteristic membrane blebs of apoptotically dying cells. Cleavage of pp125FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130Cas (Cas, Crk-associated substrate) and vinculin, to the COOH terminally truncated pp125FAK is abolished. Therefore, caspase-mediated cleavage of pp125FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.
bcl-x Prevents Apoptotic Cell Death of Both Primitive and Definitive Erythrocytes at the End of Maturation
Authors: Motoyama Noboru , Kimura Tohru , Takahashi Tomomi , Watanabe Takeshi , Nakano Toru
Published: July 2002

bcl-x is a member of the bcl-2 gene family, which regulates apoptotic cell death in various cell lineages. There is circumstantial evidence suggesting that bcl-x might play a role in the apoptosis of erythroid lineage cells, although there is no direct evidence. In this study, we used Bcl-X null mouse embryonic stem (ES) cells, and showed that Bcl-X is indispensable for the production of both embryonic primitive erythrocytes (EryP) and adult definitive erythrocytes (EryD) at the end of their maturation. In vivo, bcl-x−/− ES cells did not contribute to circulating EryD in adult chimeric mice that were produced by blastocyst microinjection of the bcl-x−/− ES cells. bcl-x−/− EryP and EryD were produced by in vitro differentiation induction of ES cells on macrophage colony-stimulating factor–deficient stromal cell line OP9, and further analysis was carried out. The emergence of immature EryP and EryD from bcl-x−/− ES cells was similar to that from bcl-x+/+ ES cells. However, prominent cell death of bcl-x−/− EryP and EryD occurred when the cells matured. The data show that the antiapoptotic function of bcl-x acts at the very end of erythroid maturation.