bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell nucleus and DNA fragmentation are not required for apoptosis.
Authors: Schulze-Osthoff K , Walczak H , Dröge W , Krammer P H
Published: May 2004

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.
Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons.
Authors: Takeda S , Okabe S , Funakoshi T , Hirokawa N
Published: May 2004

Neurofilaments (NFs) are composed of triplet proteins, NF-H, NF-M, and NF-L. To understand the dynamics of NFs in vivo, we studied the dynamics of NF-H and compared them to those of NF-L, using the combination of microinjection technique and fluorescence recovery after photobleaching. In the case of NF-L protein, the bleached zone gradually restored its fluorescence intensity with a recovery half time of approximately 35 min. On the other hand, recovery of the bleached zone of NF-H was considerably faster, taking place in approximately 19 min. However, in both cases the bleached zone was stationary. Thus, it was suggested that NF-H is the dynamic component of the NF array and is interchangeable, but that it assembles with the other neurofilament triplet proteins in a more exchangeable way, implying that the location of NF-H is in the periphery of the core NF array mainly composed of NF-L subunits. Immunoelectron microscopy investigations of the incorporation sites of NF-H labeled with biotin compounds also revealed the lateral insertion of NF-H subunits into the preexisting NF array, taking after the pattern seen in the case of NF-L. In summary, our results demonstrate that the dynamics of the L and H subunit proteins in situ are quite different from each other, suggesting different and separated mechanisms or structural specialization underlying the behavior of the two proteins.
A novel microtubule-based motor protein (KIF4) for organelle transports, whose expression is regulated developmentally.
Authors: Sekine Y , Okada Y , Noda Y , Kondo S , Aizawa H , Takemura R , Hirokawa N
Published: May 2004

To understand the mechanisms of transport for organelles in the axon, we isolated and sequenced the cDNA encoding KIF4 from murine brain, and characterized the molecule biochemically and immunocytochemically. Complete amino acid sequence analysis of KIF4 and ultrastructural studies of KIF4 molecules expressed in Sf9 cells revealed that the protein contains 1,231 amino acid residues (M(r) 139,550) and that the molecule (116-nm rod with globular heads and tail) consists of three domains: an NH2-terminal globular motor domain, a central alpha-helical stalk domain and a COOH-terminal tail domain. KIF4 protein has the property of nucleotide-dependent binding to microtubules, microtubule-activated ATPase activity, and microtubule plus-end-directed motility. Northern blot analysis and in situ hybridization demonstrated that KIF4 is strongly expressed in juvenile tissues including differentiated young neurons, while its expression is decreased considerably in adult mice except in spleen. Immunocytochemical studies revealed that KIF4 colocalized with membranous organelles both in growth cones of differentiated neurons and in the cytoplasm of cultured fibroblasts. During mitotic phase of cell cycle, KIF4 appears to colocalize with membranous organelles in the mitotic spindle. Hence we conclude that KIF4 is a novel microtubule-associated anterograde motor protein for membranous organelles, the expression of which is regulated developmentally.
Regulation of bFGF gene expression and subcellular distribution of bFGF protein in adrenal medullary cells.
Authors: Stachowiak M K , Moffett J , Joy A , Puchacz E , Florkiewicz R , Stachowiak E K
Published: May 2004

Basic fibroblast growth factor (bFGF), a potent mitogenic/neurotrophic factor, controls the development and plasticity of many types of neural cells. In adrenal chromaffin cells, the appearance of bFGF protein coincided with the establishment of functional innervation, suggesting induction by trans-synaptic signals. In cultured bovine adrenal medullary cells Western blot analysis revealed 18-, 23-, and 24-kD bFGF isoforms in the cytosolic and nuclear fractions. Stimulation of acetylcholine nicotinic receptors or hormonal angiotensin II receptors or the direct stimulation of adenylate cyclase with forskolin or protein kinase C (PKC) with PMA increased the content of all bFGF isoforms. Increases in the levels of intracellular bFGF did not result in detectable presence of bFGF proteins in culture medium. Instead, bFGF proteins accumulated in the cytoplasm or the nucleus depending on whether PKC or cAMP pathways were activated. The long-term nuclear forskolin-induced accumulation of bFGF was prevented by cycloheximide or by antisense bFGF oligonucleotide and was also accompanied by an increase in bFGF mRNA. We used luciferase reporter plasmids containing the human bFGF promoter to show that the induction of bFGF resulted from transcriptional activation of the bFGF gene and was mediated by regulatory sequences located upstream from its transcription start site. Stimulation of bFGF gene expression by forskolin and PMA was synergistic and was mediated through different promoter regions. The results suggest that stimulation by cAMP and PKC is mediated through novel cis elements. The regulation of bFGF protein content also involves posttranscriptional mechanisms since changes in the levels of individual bFGF isoforms were different depending on whether cells were treated with carbachol or angiotensin II, forskolin, or PMA. The present study indicates that bFGF is an intracrine cytoplasmic-nuclear factor, whose expression is regulated by trans-synaptic and hormonal stimuli and which may act as a direct mediator of genomic responses to afferent stimulation.
Retrieval of HDEL proteins is required for growth of yeast cells.
Authors: Townsley F M , Frigerio G , Pelham H R
Published: May 2004

The ERD2 gene of Saccharomyces cerevisiae encodes the receptor which retrieves HDEL-containing containing ER proteins from the Golgi apparatus. Viable erd2 mutants have been isolated that show no obvious HDEL-dependent retention of the luminal ER protein BiP, suggesting that retrieval of HDEL proteins is not essential for growth. However, cells that lack Erd2p completely have a defective Golgi apparatus and cannot grow. This observation led to the suggestion that the receptor had a second function, possibly related to its ability to recycle from Golgi to ER. In this paper we investigate the requirements for Erd2p to support growth. We show that mutations that block its recycling also prevent growth. In addition, we show that all mutant receptors that can support growth have a residual ability to retrieve BiP, which is detectable when they are overexpressed. Mere recycling of an inactive form of the receptor, mediated by a cytoplasmic KKXX sequence, is not sufficient for growth. Furthermore, saturation of the receptor by expression of an HDEL-tagged version of pro-alpha factor inhibits growth, even of strains that do not show obvious BiP retention. We conclude that growth requires the HDEL-dependent retrieval of one or more proteins, and that these proteins can be recognized even under conditions where BiP is secreted. Genetic screens have failed to identify any one protein whose loss could account for the Erd2p requirement. Therefore, a growth may require the retention of multiple HDEL proteins in the ER, or alternatively the removal of such proteins from the Golgi apparatus.
The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-alpha catenin fusion molecules.
Authors: Nagafuchi A , Ishihara S , Tsukita S
Published: May 2004

The carboxyl terminus-truncated cadherin (nonfunctional cadherin) has no cell adhesion activity probably because of its failure to associate with cytoplasmic proteins called alpha and beta catenin. To rescue this nonfunctional cadherin as adhesion molecules, we constructed three cDNAs for fusion proteins between nonfunctional E-cadherin and alpha catenin, nE alpha, nE alpha N, and nE alpha C, where the intact, amino-terminal and carboxy-terminal half of alpha catenin, respectively, were directly linked to the nonfunctional E-cadherin, and introduced them into mouse L cells. The subcellular distribution and cell adhesion activity of nE alpha and nE alpha C molecules was similar to those of intact E-cadherin transfectants: they bound to cytoskeletons, were concentrated at cell-cell adhesion sites and showed strong cell adhesion activity. nE alpha N molecules, which also bound to cytoskeletons, showed very poor cell adhesion activity. Taken together, we conclude that in the formation of the cadherin-catenin complex, the mechanical association of alpha catenin, especially its carboxy-terminal half, with E-cadherin is a key step for the cadherin-mediated cell adhesion. Close comparison revealed that the behavior of nE alpha molecules during cytokinesis was quite different from that of intact E-cadherin, and that the intercellular motility, i.e., the cell movement in a confluent sheet, was significantly suppressed in nE alpha transfectants although it was facilitated in E-cadherin transfectants. Considering that nE alpha was not associated with endogenous beta catenin in transfectants, the difference in the nature of cell adhesion between nE alpha and intact E-cadherin transfectants may be explained by the function of beta catenin. The possible functions of beta catenin are discussed with a special reference to its role as a negative regulator for the cadherin-mediated cell adhesion system.
Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line.
Authors: Watabe M , Nagafuchi A , Tsukita S , Takeichi M
Published: May 2004

PC9 lung carcinoma cells cannot tightly associate with one another, and therefore grow singly, despite their expression of E-cadherin, because of their lack of alpha-catenin, a cadherin-associated protein. However, when the E-cadherin is activated by transfection with alpha-catenin cDNA, they form spherical aggregates, each consisting of an enclosed monolayer cell sheet. In the present work, we examined whether the alpha-catenin-transfected cell layers expressed epithelial phenotypes, by determining the distribution of various cell adhesion molecules on their surfaces, including E-cadherin, ZO-1, desmoplakin, integrins, and laminin. In untransfected PC9 cells, all these molecules were randomly distributed on their cell surface. In the transfected cells, however, each of them was redistributed into a characteristic polarized pattern without a change in the amount of expression. Electron microscopic study demonstrated that the alpha-catenin-transfected cell layers acquired apical-basal polarity typical of simple epithelia; they formed microvilli only on the outer surface of the aggregates, and a junctional complex composed of tight junction adherens junction, and desmosome arranged in this order. These results indicate that the activation of E-cadherin triggered the formation of the junctional complex and the polarized distribution of cell surface proteins and structures. We also found that, in untransfected PC9 cells, ZO-1 formed condensed clusters and colocalized with E-cadherin, but that other adhesion molecules rarely showed such colocalization with E-cadherin, suggesting that there is some specific interaction between ZO-1 and E-cadherin even in the absence of cell-cell contacts. In addition, we found that the activation of E-cadherin caused a retardation of PC9 cell growth. Thus, we concluded that the E-cadherin-catenin adhesion system is essential not only for structural organization of epithelial cells but also for the control of their growth.
HVEM tomography of the trans-Golgi network: structural insights and identification of a lace-like vesicle coat.
Authors: Ladinsky M S , Kremer J R , Furcinitti P S , McIntosh J R , Howell K E
Published: May 2004

High voltage electron microscopy and computer axial tomography have been used to study the 3-D structure of trans-Golgi cisternae and trans-Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the techique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279). Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy and subsequent tomographic reconstruction. Resolution of the reconstructions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were made from regions with pronounced TGN. Most regions analyzed contained multiple (2-4) Golgi cisternae that stain with ceramide. These "peel off" from the closely stacked cisternae and are continuous at their ends with tubules that contribute to the TGN. Most vesicular profiles visualized in the TGN are connected to TGN tubules. The budding of vesicles appears to occur synchronously along the length of a TGN tubule. Two distinct coats were visualized on budding vesicles: clathrin cages and a novel, lace-like structure. Individual TGN tubules produce vesicles of only one coat type. These observations lead to the following predictions: (a) sorting of molecules must occur prior to the formation of TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lace-like coats form an exocytic vesicles.
Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane.
Authors: Leno G H , Munshi R
Published: May 2004

We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact-inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact-inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha-32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.
The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme.
Authors: DeTomaso A W , Blanco G , Mercer R W
Published: May 2004

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K-ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.
The Caenorhabditis elegans muscle-affecting gene unc-87 encodes a novel thin filament-associated protein.
Authors: Goetinck S , Waterston R H
Published: May 2004

Mutations in the unc-87 gene of Caenorhabditis elegans affect the structure and function of bodywall muscle, resulting in variable paralysis. We cloned the unc-87 gene by taking advantage of a transposon-induced allele of unc-87 and the correspondence of the genetic and physical maps in C. elegans. A genomic clone was isolated that alleviates the mutant phenotype when introduced into unc-87 mutants. Sequence analysis of a corresponding cDNA clone predicts a 357-amino acid, 40-kD protein that is similar to portions of the vertebrate smooth muscle proteins calponin and SM22 alpha, the Drosophila muscle protein mp20, the deduced product of the C. elegans cDNA cm7g3, and the rat neuronal protein np25. Analysis of the genomic sequence and of various transcripts represented in a cDNA library suggest that unc-87 mRNAs are subject to alternative splicing. Immunohistochemistry of wildtype and mutant animals with antibodies to an unc-87 fusion protein indicates that the gene product is localized to the I-band of bodywall muscle. Studies of the UNC-87 protein in other muscle mutants suggest that the unc-87 gene product associates with thin filaments, in a manner that does not depend on the presence of the thin filament protein tropomyosin.
Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast.
Authors: Scherthan H , Bähler J , Kohli J
Published: May 2004

Interactions between homologous chromosomes (pairing, recombination) are of central importance for meiosis. We studied entire chromosomes and defined chromosomal subregions in synchronous meiotic cultures of Schizosaccharomyces pombe by fluorescence in situ hybridization. Probes of different complexity were applied to spread nuclei, to delineate whole chromosomes, to visualize repeated sequences of centromeres, telomeres, and ribosomal DNA, and to study unique sequences of different chromosomal regions. In diploid nuclei, homologous chromosomes share a joint territory even before entry into meiosis. The centromeres of all chromosomes are clustered in vegetative and meiotic prophase cells, whereas the telomeres cluster near the nucleolus early in meiosis and maintain this configuration throughout meiotic prophase. Telomeres and centromeres appear to play crucial roles for chromosome organization and pairing, both in vegetative cells and during meiosis. Homologous pairing of unique sequences shows regional differences and is most frequent near centromeres and telomeres. Multiple homologous interactions are formed independently of each other. Pairing increases during meiosis, but not all chromosomal regions become closely paired in every meiosis. There is no detectable axial compaction of chromosomes in meiotic prophase. S. pombe does not form mature synaptonemal complexes, but axial element-like structures (linear elements), which were analyzed in parallel. Their appearance coincides with pairing of interstitial chromosomal regions. Axial elements may define minimal structures required for efficient pairing and recombination of meiotic chromosomes.
Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure.
Authors: Belmont A S , Bruce K
Published: May 2004

We have used light microscopy and serial thin-section electron microscopy to visualize intermediates of chromosome decondensation during G1 progression in synchronized CHO cells. In early G1, tightly coiled 100-130-nm "chromonema" fibers are visualized within partially decondensed chromatin masses. Progression from early to middle G1 is accompanied by a progressive uncoiling and straightening of these chromonema fibers. Further decondensation in later G1 and early S phase results in predominantly 60-80-nm chromonema fibers that can be traced up to 2-3 microns in length as discrete fibers. Abrupt transitions in diameter from 100-130 to 60-80 nm along individual fibers are suggestive of coiling of the 60-80-nm chromonema fibers to form the thicker 100-130-nm chromonema fiber. Local unfolding of these chromonema fibers, corresponding to DNA regions tens to hundreds of kilobases in length, reveal more loosely folded and extended 30-nm chromatin fibers. Kinks and supercoils appear as prominent features at all observed levels of folding. These results are inconsistent with prevailing models of chromosome structure and, instead, suggest a folded chromonema model of chromosome structure.
Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins.
Authors: Falk M M , Kumar N M , Gilula N B
Published: May 2004

Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation-competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the connexins. Variable amounts of similar connexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell.
Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains.
Authors: Banfield D K , Lewis M J , Rabouille C , Warren G , Pelham H R
Published: May 2004

The yeast Sed5 protein, which is required for vesicular transport between ER and Golgi complex, is a membrane protein of the syntaxin family. These proteins are thought to provide the specific targets that are recognized by transport vesicles. We have investigated the mechanism by which Sed5 protein is itself localized. Expression of epitope-tagged versions of the yeast, Drosophila and rat Sed5 homologues in COS cells results in a perinuclear distribution; immuno-EM reveals that the majority of the protein is in a tubulo-vesicular compartment on the cis side of the Golgi apparatus. A similar distribution was obtained with a chimeric molecule consisting of a plasma membrane syntaxin with the Drosophila Sed5 transmembrane domain. This indicates that the membrane-spanning domain contains targeting information, as is the case with resident Golgi enzymes. However, alterations to the transmembrane domain of Drosophila Sed5 itself did not result in its mistargeting, implying that an additional targeting mechanism exists which involves only the cytoplasmic part of the protein. This was confirmed by modifying the transmembrane domain of the yeast Sed5 protein: substitution with the corresponding region from the Sso1 protein (a plasma membrane syntaxin homologue) did not affect yeast Sed5 function in vivo.
Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.
Authors: Bacon R A , Cohen C J , Lewin D A , Mellman I
Published: May 2004

We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning.
Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype.
Authors: Kislauskis E H , Zhu X , Singer R H
Published: May 2004

We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.
Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase).
Authors: Tassan J P , Schultz S J , Bartek J , Nigg E A
Published: May 2004

The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).
Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils.
Authors: Gilbert S H , Perry K , Fay F S
Published: May 2004

During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]i increased rapidly from < or = 100 nM to > or = 400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of approximately 200 nM. The initial Ca2+ spike was insensitive acutely to removal of extracellular Ca2+ but was abolished by treatments expected to deplete internal Ca2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lamellipod activity and suppressed the Ca2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca2+ spike on stimulation. Although cell flattening and lamellipod activity were induced by chemoattractant when the normal Ca2+ response was blocked, cells failed to polarize and crawl, indicating that Ca2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.
Differential perturbations in the morphogenesis of anterior structures induced by overexpression of truncated XB- and N-cadherins in Xenopus embryos.
Authors: Dufour S , Saint-Jeannet J P , Broders F , Wedlich D , Thiery J P
Published: May 2004

Cadherins, a family of Ca-dependent adhesion molecules, have been proposed to act as regulators of morphogenetic processes and to be major effectors in the maintenance of tissue integrity. In this study, we have compared the effects of the expression of two truncated cadherins during early neurogenesis in Xenopus laevis. mRNA encoding deleted forms of XB- and N-cadherin lacking most of the extracellular domain were injected into the four animal dorsal blastomeres of 32-cell stage Xenopus embryos. These truncated cadherins altered the cohesion of cells derived from the injected blastomeres and induced morphogenetic defects in the anterior neural tissue to which they chiefly contributed. Truncated XB-cadherin was more efficient than N-cadherin in inducing these perturbations. Moreover, the coexpression of both truncated cadherins had additive perturbation effects on neural development. The two truncated cadherins can interact with the three known catenins, but with distinct affinities. These results suggest that the adhesive signal mediated by cadherins can be perturbed by overexpressing their cytoplasmic domains by competing with different affinity with catenins and/or a common anchor structure. Therefore, the correct regulation of cadherin function through the cytoplasmic domain appears to be a crucial step in the formation of the neural tissue.
Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells.
Authors: Re F , Zanetti A , Sironi M , Polentarutti N , Lanfrancone L , Dejana E , Colotta F
Published: May 2004

When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a half-life of approximately 10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-Arg-Gly-Asp-Ser-coated microbeads (approximately 10 microbeads/cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene-transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis.
Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.
Authors: Balzac F , Retta S F , Albini A , Melchiorri A , Koteliansky V E , Geuna M , Silengo L , Tarone G
Published: May 2004

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.
Characterization of the yeast (1-->6)-beta-glucan biosynthetic components, Kre6p and Skn1p, and genetic interactions between the PKC1 pathway and extracellular matrix assembly.
Authors: Roemer T , Paravicini G , Payton M A , Bussey H
Published: May 2004

A characterization of the S. cerevisiae KRE6 and SKN1 gene products extends previous genetic studies on their role in (1-->6)-beta-glucan biosynthesis (Roemer, T., and H. Bussey. 1991. Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl. Acad. Sci. USA. 88:11295-11299; Roemer, T., S. Delaney, and H. Bussey. 1993. SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048). KRE6 and SKN1 are predicted to encode homologous proteins that participate in assembly of the cell wall polymer (1-->6)-beta-glucan. KRE6 and SKN1 encode phosphorylated integral-membrane glycoproteins, with Kre6p likely localized within a Golgi subcompartment. Deletion of both these genes is shown to result in a dramatic disorganization of cell wall ultrastructure. Consistent with their direct role in the assembly of this polymer, both Kre6p and Skn1p possess COOH-terminal domains with significant sequence similarity to two recently identified glucan-binding proteins. Deletion of the yeast protein kinase C homolog, PKC1, leads to a lysis defect (Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J. Cell Biol. 116:1221-1229). Kre6p when even mildly overproduced, can suppress this pkc1 lysis defect. When mutated, several KRE pathway genes and members of the PKC1-mediated MAP kinase pathway have synthetic lethal interactions as double mutants. These suppression and synthetic lethal interactions, as well as reduced beta-glucan and mannan levels in the pkc1 null wall, support a role for the PKC1 pathway functioning in cell wall assembly. PKC1 potentially participates in cell wall assembly by regulating the synthesis of cell wall components, including (1-->6)-beta-glucan.
Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere.
Authors: Sullivan K F , Hechenberger M , Masri K
Published: May 2004

Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2-terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.
A novel FK506- and rapamycin-binding protein (FPR3 gene product) in the yeast Saccharomyces cerevisiae is a proline rotamase localized to the nucleolus.
Authors: Benton B M , Zang J H , Thorner J
Published: May 2004

The gene (FPR3) encoding a novel type of peptidylpropyl-cis-trans-isomerase (PPIase) was isolated during a search for previously unidentified nuclear proteins in Saccharomyces cerevisiae. PPIases are thought to act in conjunction with protein chaperones because they accelerate the rate of conformational interconversions around proline residues in polypeptides. The FPR3 gene product (Fpr3) is 413 amino acids long. The 111 COOH-terminal residues of Fpr3 share greater than 40% amino acid identity with a particular class of PPIases, termed FK506-binding proteins (FKBPs) because they are the intracellular receptors for two immunosuppressive compounds, rapamycin and FK506. When expressed in and purified from Escherichia coli, both full-length Fpr3 and its isolated COOH-terminal domain exhibit readily detectable PPIase activity. Both fpr3 delta null mutants and cells expressing FPR3 from its own promoter on a multicopy plasmid have no discernible growth phenotype and do not display any alteration in sensitivity to the growth-inhibitory effects of either FK506 or rapamycin. In S. cerevisiae, the gene for a 112-residue cytosolic FKBP (FPR1) and the gene for a 135-residue ER-associated FKBP (FPR2) have been described before. Even fpr1 fpr2 fpr3 triple mutants are viable. However, in cells carrying an fpr1 delta mutation (which confers resistance to rapamycin), overexpression from the GAL1 promoter of the C-terminal domain of Fpr3, but not full-length Fpr3, restored sensitivity to rapamycin. Conversely, overproduction from the GAL1 promoter of full-length Fpr3, but not its COOH-terminal domain, is growth inhibitory in both normal cells and fpr1 delta mutants. In fpr1 delta cells, the toxic effect of Fpr3 overproduction can be reversed by rapamycin. Overproduction of the NH2-terminal domain of Fpr3 is also growth inhibitory in normal cells and fpr1 delta mutants, but this toxicity is not ameliorated in fpr1 delta cells by rapamycin. The NH2-terminal domain of Fpr3 contains long stretches of acidic residues alternating with blocks of basic residues, a structure that resembles sequences found in nucleolar proteins, including S. cerevisiae NSR1 and mammalian nucleolin. Indirect immunofluorescence with polyclonal antibodies raised against either the NH2- or the COOH-terminal segments of Fpr3 expressed in E. coli demonstrated that Fpr3 is located exclusively in the nucleolus.
Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast.
Authors: Gaynor E C , te Heesen S , Graham T R , Aebi M , Emr S D
Published: May 2004

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.