bluuebunny/crossref_metadata_2025 huggingface dataset:
gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation.
Authors: Spang A , Geissler S , Grein K , Schiebel E
Published: May 2004

Tub4p is a novel tubulin in Saccharomyces cerevisiae that most closely resembles gamma-tubulin. We report in this manuscript that the essential Tub4p is associated with the inner and outer plaques of the yeast microtubule organizing center, the spindle pole body (SPB). These SPB substructures are involved in the attachment of the nuclear and cytoplasmic microtubules, respectively (Byers, B., and L. Goetsch. 1975. J. Bacteriol. 124:511-523). Study of a temperature sensitive tub4-1 allele revealed that TUB4 has essential functions in microtubule organization. Remarkably, SPB duplication and separation are not impaired in tub4-1 cells incubated at the nonpermissive temperature. However, SPBs from such cells contain less or misdirected nuclear microtubules. Further analysis revealed that tub4-1 cells are able to assemble a short bipolar spindle, suggesting that the defect in microtubule organization occurs after spindle formation. A role of Tub4p in microtubule organization is further suggested by an increase in chromosome loss in tub4-1 cells. In addition, cell cycle arrest and survival of tub4-1 cells is dependent on the mitotic checkpoint control gene BUB2 (Hoyt, M.A., L. Totis, B.T. Roberts. 1991. Cell. 66:507-517), one of the cell's monitors of spindle integrity.
Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.
Authors: Marschall L G , Jeng R L , Mulholland J , Stearns T
Published: May 2004

gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.
Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.
Authors: Matthies H J , McDonald H B , Goldstein L S , Theurkauf W E
Published: May 2004

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.
ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway.
Authors: Tansey M G , Chu G C , Merlie J P
Published: May 2004

AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Since some RTKs act through Ras and phosphatidylinositol 3-kinase (PI3K), we investigated the role of these pathways in ARIA signaling. Expression of activated Ras or Raf mimicked ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocked the effect of ARIA. ARIA rapidly activated erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulated association of PI3K with erbB3, expression of an activated PI3K led to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolished the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways.
Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation.
Authors: Albers K M , Perrone T N , Goodness T P , Jones M E , Green M A , Davis B M
Published: May 2004

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT-3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross-sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.
eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling.
Authors: Starich T A , Lee R Y , Panzarella C , Avery L , Shaw J E
Published: May 2004

The Drosophila melanogaster genes Passover and l(1)ogre and the Caenorhabditis elegans gene unc-7 define a gene family whose function is not known. We have isolated and characterized the C. elegans gene eat-5, which is required for synchronized pharyngeal muscle contractions, and find that it is a new member of this family. Simultaneous electrical and video recordings reveal that in eat-5 mutants, action potentials of muscles in the anterior and posterior pharynx are unsynchronized. Injection of carboxyfluorescein into muscles of the posterior pharynx demonstrates that all pharyngeal muscles are dye-coupled in wild-type animals; in eat-5 mutants, however, muscles of the anterior pharynx are no longer dye-coupled to posterior pharyngeal muscles. We show that a gene fusion of eat-5 to the green fluorescent protein is expressed in pharyngeal muscles. unc-7 and eat-5 are two of at least sixteen members of this family in C. elegans as determined by database searches and PCR-based screens. The amino acid sequences of five of these members in C. elegans have been deduced from cDNA sequences. Polypeptides of the family are predicted to have four transmembrane domains with cytoplasmic amino and carboxyl termini. We have constructed fusions of one of these polypeptides with beta-galactosidase and with green fluorescent protein. The fusion proteins appear to be localized in a punctate pattern at or near plasma membranes. We speculate that this gene family is required for the formation of gap junctions.
The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA.
Authors: Schilke B , Forster J , Davis J , James P , Walter W , Laloraya S , Johnson J , Miao B , Craig E
Published: May 2004

SSH1, a newly identified member of the heat shock protein (hsp70) multigene family of the budding yeast Saccharomyces cerevisiae, encodes a protein localized to the mitochondrial matrix. Deletion of the SSH1 gene results in extremely slow growth at 23 degrees C or 30 degrees C, but nearly wild-type growth at 37 degrees C. The matrix of the mitochondria contains another hsp70, Ssc1, which is essential for growth and required for translocation of proteins into mitochondria. Unlike SSC1 mutants, an SSH1 mutant showed no detectable defects in import of several proteins from the cytosol to the matrix compared to wild type. Increased expression of Ssc1 partially suppressed the cold-sensitive growth defect of the SSH1 mutant, suggesting that when present in increased amounts, Ssc1 can at least partially carry out the normal functions of Ssh1. Spontaneous suppressors of the cold-sensitive phenotype of an SSH1 null mutant were obtained at a high frequency at 23 degrees C, and were all found to be respiration deficient. 15 of 16 suppressors that were analyzed lacked mitochondrial DNA, while the 16th had reduced amounts. We suggest that Ssh1 is required for normal mitochondrial DNA replication, and that disruption of this process in ssh1 cells results in a defect in mitochondrial function at low temperatures.
ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization.
Authors: Arnoult C , Zeng Y , Florman H M
Published: May 2004

The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization.
Lipid-modified, cysteinyl-containing peptides of diverse structures are efficiently S-acylated at the plasma membrane of mammalian cells.
Authors: Schroeder H , Leventis R , Shahinian S , Walton P A , Silvius J R
Published: May 2004

A variety of cysteine-containing, lipid-modified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid-modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S-acylation of plasma membrane-associated proteins in various characteristics, including inhibition by tunicamycin. The substrate range of the S-acylation reaction encompasses, but is not limited to, lipopeptides incorporating the motifs myristoylGC- and -CXC(farnesyl)-OCH3, which are reversibly S-acylated in various intracellular proteins. Mass-spectrometric analysis indicates that palmitoyl residues constitute the predominant but not the only type of S-acyl group coupled to a lipopeptide carrying the myristoylGC- motif, with smaller amounts of S-stearoyl and S-oleoyl substituents also detectable. Fluorescence microscopy using NBD-labeled cysteinyl lipopeptides reveals that the products of lipopeptide S-acylation, which cannot diffuse between membranes, are in almost all cases localized preferentially to the plasma membrane. This preferential localization is found even at reduced temperatures where vesicular transport from the Golgi complex to the plasma membrane is suppressed, strongly suggesting that the plasma membrane itself is the preferred site of S-acylation of these species. Uniquely among the lipopeptides studied, species incorporating an unphysiological N-myristoylcysteinyl- motif also show substantial formation of S-acylated products in a second, intracellular compartment identified as the Golgi complex by its labeling with a fluorescent ceramide. Our results suggest that distinct S-acyltransferases exist in the Golgi complex and plasma membrane compartments and that S-acylation of motifs such as myristoylGC- occurs specifically at the plasma membrane, affording efficient targeting of cellular proteins bearing such motifs to this membrane compartment.
Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities.
Authors: Kelley C A , Sellers J R , Gard D L , Bui D , Adelstein R S , Baines I C
Published: May 2004

There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. We compared the enzymatic activities as well as the subcellular localizations of these isoforms in Xenopus cells. MHC-A and MHC-B were purified from cells by immunoprecipitation with isoform-specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A was 3.3-fold faster than that by MHC-B. Likewise, the Vmax of the actin-activated Mg(2+)-ATPase activity of MHC-A was 2.6-fold greater than that of MHC-B. Immunofluorescence microscopy demonstrated distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium was dramatically enriched for MHC-B suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A was absent from the cell periphery and was arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also had distinct localizations throughout mitosis. During prophase, the MHC-B redistributed to the nuclear membrane, and then resumed its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localized to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalized to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions.
emo-1, a Caenorhabditis elegans Sec61p gamma homologue, is required for oocyte development and ovulation.
Authors: Iwasaki K , McCarter J , Francis R , Schedl T
Published: May 2004

emo-1(oz1) is a member of a class of hermaphrodite sterile mutations in Caenorhabditis elegans that produce endomitotic oocytes in the gonad arm. Oocytes in emo-1(oz1) mutants exhibit multiple defects during oogenesis. After meiotic maturation, ovulation fails, trapping oocytes in the gonad arm where they become endomitotic. emo-1 encodes a homologue of the Sec61p gamma subunit, a protein necessary for translocation of secretory and transmembrane proteins into the endoplasmic reticulum of yeast and mammalian cells. A putative emo-1 null mutation, oz151, displays embryonic lethality. The oz1 sterile mutation is a transposable element insertion into the emo-1 3' untranslated region that almost completely eliminates germline mRNA accumulation. Genetic mosaic analysis using the oz1 allele indicates that emo-1(+) expression in germ cells is required for fertility. The J67 monoclonal antibody, which recognizes an oocyte surface antigen (Strome, S. 1986. In Gametogenesis and the Early Embryo. J.G. Gall, editor. Alan R. Liss, Inc., New York. 77-95.), does not stain oz1 oocytes, a finding consistent with defective protein transport in the mutant. We propose that the emo-1 gene product acts in the transport of secreted and transmembrane proteins in C. elegans oocytes, and is necessary for both oogenesis and the coupling of ovulation with meiotic maturation.
Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles.
Authors: Haass N K , Kartenbeck M A , Leube R E
Published: May 2004

Certain properties of the highly specialized synaptic transmitter vesicles are shared by constitutively occurring vesicles. We and others have thus identified a cDNA in various nonneuroendocrine cell types of rat and human that is related to synaptophysin, one of the major synaptic vesicle membrane proteins, which we termed pantophysin. Here we characterize the gene structure, mRNA and protein expression, and intracellular distribution of pantophysin. Its mRNA is detected in murine cell types of nonneuroendocrine as well as of neuroendocrine origin. The intron/exon structure of the murine pantophysin gene is identical to that of synaptophysin except for the last intron that is absent in pantophysin. The encoded polypeptide of calculated mol wt 28,926 shares many sequence features with synaptophysin, most notably the four hydrophobic putative transmembrane domains, although the cytoplasmic end domains are completely different. Using antibodies against the unique carboxy terminus pantophysin can be detected by immunofluorescence microscopy in both exocrine and endocrine cells of human pancreas, and in cultured cells, colocalizing with constitutive secretory and endocytotic vesicle markers in nonneuroendocrine cells and with synaptophysin in cDNA-transfected epithelial cells. By immunoelectron microscopy, the majority of pantophysin reactivity is detected at vesicles with a diameter of < 100 nm that have a smooth surface and an electron-translucent interior. Using cell fractionation in combination with immunoisolation, these vesicles are enriched in a light fraction and shown to contain the cellular vSNARE cellubrevin and the ubiquitous SCAMPs in epithelial cells and synaptophysin in neuroendocrine or cDNA-transfected nonneuroendocrine cells and neuroendocrine tissues. Pantophysin is therefore a broadly distributed marker of small cytoplasmic transport vesicles independent of their content.
Symplekin, a novel type of tight junction plaque protein.
Authors: Keon B H , Schäfer S , Kuhn C , Grund C , Franke W W
Published: May 2004

Using a monoclonal antibody we have identified and cDNA-cloned a novel type of protein localized, by light and electron microscopy, to the plaque associated with the cytoplasmic face of the tight junction-containing zone (zonula occludens) of polar epithelial cells and of Sertoli cells of testis, but absent from the junctions of vascular endothelia. The approximately 3.7-kb mRNA encodes a polypeptide of 1142 amino acids (calculated molecular weight 126.5 kD, pI 6.25), for which the name "symplekin" (from Greek sigma upsilon mu pi lambda epsilon kappa epsilon iota, nu, to tie together, to weave, to be intertwined) is proposed. However, both the mRNA and the protein can also be detected in a wide range of cell types that do not form tight junctions or are even completely devoid of any stable cell contacts. Careful analyses have revealed that the protein occurs in all these diverse cells in the nucleoplasm, and only in those cells forming tight junctions is it recruited, partly but specifically, to the plaque structure of the zonula occludens. We discuss symplekin as a representative of a group of dual residence proteins which occur and probably function in the nucleus as well as in the plaques exclusive for either tight junctions, adherens junctions, or desmosomes.
Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells.
Authors: Li H , Liu T F , Lazrak A , Peracchia C , Goldberg G S , Lampe P D , Johnson R G
Published: May 2004

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7-hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.
The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition.
Authors: Ishov A M , Maul G G
Published: May 2004

After DNA viruses enter the nucleus, they initiate a transcriptional cascade which is followed by replication. We investigated whether these processes take place at specific nuclear sites or, as suggested by the mode of entry, randomly throughout the nucleus. Three distinct nuclear domains, nuclear factor-1 sites, coiled bodies, and nuclear domain 10 (ND10), were used as markers to investigate the relative position of DNA virus replication sites. We found that all three nuclear domains had a very high spatial correlation with each other in uninfected cells. After adenoviral infection, nuclear factor 1 and coiled bodies were found associated with some viral replication domains. Simian virus 40 begins replication adjacent to ND10 but adenovirus 5 and herpes simplex type 1 modified ND10s before replication. Adenovirus E4orf 3 gene deletion mutants retain ND10 and begin replication at the peripheries of ND10. The same was found for the herpes simplex virus type 1 immediate early gene 1 mutants. That the deposition and replication of adenovirus 5 and herpesvirus type 1 at ND10 was not a mutant phenotype was confirmed by finding the input wild-type virus juxtaposed to ND10. The transport of viral genomes to ND10 does not require viral gene expression. Thus, the peripheries of ND10 represent preferred sites where early steps of transcription and replication of at least three DNA virus families take place, suggesting a new set of functional properties for this large nuclear domain.
Regulation of RAR beta 2 mRNA expression: evidence for an inhibitory peptide encoded in the 5'-untranslated region.
Authors: Reynolds K , Zimmer A M , Zimmer A
Published: May 2004

Regulation of mRNA translation and stability plays an important role in the control of gene expression during embryonic development. We have recently shown that the tissue-specific expression of the RAR beta 2 gene in mouse embryos is regulated at the translational level by short upstream open reading frames (uORFs) In the 5'-untranslated region (Zimmer, A., A.M. Zimmer, and K. Reynolds. 1994. J. Cell Biol. 127:1111-1119). To gain insight into the molecular mechanism, we have performed a systematic mutational analysis of the uORFs. Two series of constructs were tested: in one series, each uORF was individually inactivated by introducing a point mutation in its start codon; in the second series, all but one ORF were inactivated. Our results indicate that individual uORFs may have different functions. uORF4 seems to inhibit translation of the major ORF in heart and brain, while uORFs 2 and 5 appear to be important for efficient translation in all tissues. To determine whether the polypeptide encoded by uORF4 or the act of translating it, is the significant event, we introduced point mutations to create silent mutations or amino acid substitutions in uORF4. Our results indicate that the uORF4 amino acid coding sequence is important for the inhibitory effect on translation of the downstream major ORF.
Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus.
Authors: Kielian M , Klimjack M R , Ghosh S , Duffus W A
Published: May 2004

Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell-cell and virus-liposome fusion assays. During the low pH- induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.
unc-68 encodes a ryanodine receptor involved in regulating C. elegans body-wall muscle contraction.
Authors: Maryon E B , Coronado R , Anderson P
Published: May 2004

Striated muscle contraction is elicited by the release of stored calcium ions through ryanodine receptor channels in the sarcoplasmic reticulum. ryr-1 is a C. elegans ryanodine receptor homologue that is expressed in body-wall muscle cells used for locomotion. Using genetic methods, we show that ryr-1 is the previously identified locus unc-68. First, transposon-induced deletions within ryr-1 are alleles of unc-68. Second, transformation of unc-68 mutants with ryr-1 genomic DNA results in rescue of the Unc phenotype. unc-68 mutants move poorly, exhibiting an incomplete flaccid paralysis, yet have normal muscle ultrastructure. The mutants are insensitive to the paralytic effects of ryanodine, and lack detectable ryanodine-binding activity. The Unc-68 phenotype suggests that ryanodine receptors are not essential for excitation-contraction coupling in nematodes, but act to amplify a (calcium) signal that is sufficient for contraction.
Glycine 699 is pivotal for the motor activity of skeletal muscle myosin.
Authors: Kinose F , Wang S X , Kidambi U S , Moncman C L , Winkelmann D A
Published: May 2004

Myosin couples ATP hydrolysis to the translocation of actin filaments to power many forms of cellular motility. A striking feature of the structure of the muscle myosin head domain is a 9-nm long "lever arm" that has been postulated to produce a 5-10-nm power stroke. This motion must be coupled to conformational changes around the actin and nucleotide binding sites. The linkage of these sites to the lever arm has been analyzed by site-directed mutagenesis of a conserved glycine residue (G699) found in a bend joining two helices containing the highly reactive and mobile cysteine residues, SH1 and SH2. Alanine mutagenesis of this glycine (G699A) dramatically alters the motor activity of skeletal muscle myosin, inhibiting the velocity of actin filament movement by > 100-fold. Analysis of the defect in the G699A mutant myosin is consistent with a marked slowing of the transition within the motor domain from a strong binding to a weak binding interaction with actin. This result is interpreted in terms of the role of this residue (G699) as a pivot point for motion of the lever arm. The recombinant myosin used in these experiments has been produced in a unique expression system. A shuttle vector containing a regulated muscle-specific promoter has been developed for the stable expression of recombinant myosin in C2C12 cells. The vector uses the promoter/enhancer region, the first two and the last five exons of an embryonic rat myosin gene, to regulate the expression of an embryonic chicken muscle myosin cDNA. Stable cell lines transfected with this vector express the unique genetically engineered myosin after differentiation into myotubes. The myosin assembles into myofibrils, copurifies with the endogenous myosin, and contains a complement of muscle-specific myosin light chains. The functional activity of the recombinant myosin is readily analyzed with an in vitro motility assay using a species-specific anti-S2 mAb to selectively assay the recombinant protein. This expression system has facilitated manipulation and analysis of the skeletal muscle myosin motor domain and is also amenable to a wide range of structure-function experiments addressing questions unique to the muscle-specific cytoarchitecture and myosin isoforms.
Chemoattractant-mediated increases in cGMP induce changes in Dictyostelium myosin II heavy chain-specific protein kinase C activities.
Authors: Dembinsky A , Rubin H , Ravid S
Published: May 2004

Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP (Abu-Elneel et al. 1996. J. Biol. Chem. 271:977- 984). Recent studies have indicated that cAMP-induced cGMP accumulation plays a role in the regulation of myosin II phosphorylation and localization (Liu, G., and P. Newell. 1991. J. Cell. Sci. 98: 483-490). This report describes the roles of cAMP and cGMP in the regulation of MHC-PKC membrane association, phosphorylation, and activity (hereafter termed MHC-PKC activities). cAMP stimulation of Dictyostelium cells resulted in translocation of MHC-PKC from the cytosol to the membrane fraction, as well as increasing in MHC-PKC phosphorylation and in its kinase activity. We present evidence that MHC is phosphorylated by MHC-PKC in the cell cortex which leads to myosin II dissociation from the cytoskeleton. Use of Dictyostelium mutants that exhibit aberrant cAMP-induced increases in cGMP accumulation revealed that MHC-PKC activities are regulated by cGMP. Dictyostelium streamer F mutant (stmF), which produces a prolonged peak of cGMP accumulation upon cAMP stimulation, exhibits prolonged increases in MHC-PKC activities. In contrast, Dictyostelium KI-10 mutant that lacks the normal cAMP-induced cGMP response, or KI-4 mutant that shows nearly normal cAMP-induced cGMP response but has aberrant cGMP binding activity, show no changes in MHC-PKC activities. We provide evidence that cGMP may affect MHC-PKC activities via the activation of cGMP-dependent protein kinase which, in turn, phosphorylates MHC-PKC. The results presented here indicate that cAMP-induced cGMP accumulation regulates myosin II phosphorylation and localization via the regulation of MHC-PKC.
Isolation and characterization of a Drosophila gene essential for early embryonic development and formation of cortical cleavage furrows.
Authors: Zhang C X , Lee M P , Chen A D , Brown S D , Hsieh T
Published: May 2004

We have isolated a new female sterile mutant from Drosophila melanogaster, which arrests the embryonic development during the transition from syncytial to cellular blastoderm. Cytological analysis of the mutant embryos indicates that pseudocleavage furrows in the syncytial blastoderm are abnormal but not completely disrupted. However, cleavage furrows during cellularization are totally disorganized, and no embryos can develop beyond this stage. Consistent with this observation, the expression of this gene peaks around the cellular blastoderm and not in any later developmental stages. Based on immunofluorescence experiments, the protein product of this gene is localized in both pseudocleavage furrows at the syncytial blastoderm and in the cleavage furrows during the cellularization stage. Sequence homology analysis demonstrates a modest, but statistically significant, similarity of this protein with the carboxyl-terminal domains of dystrophin and a family of proteins collectively known as apodystrophins. It is possible that this protein may play an essential role in organizing and maintaining a specialized cytoskeletal structure, a function also suggested for dystrophin and apodystrophins.
Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase.
Authors: Radhakrishna H , Klausner R D , Donaldson J G
Published: May 2004

To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.
Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase.
Authors: Saitoh S , Takahashi K , Nabeshima K , Yamashita Y , Nakaseko Y , Hirata A , Yanagida M
Published: May 2004

Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron-dense nuclear materials and contained highly compacted chromatin. The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsd1+ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsd1. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of non-chromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.
Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells.
Authors: Jin P , Gu Y , Morgan D O
Published: May 2004

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.
Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly.
Authors: Bornslaeger E A , Corcoran C M , Stappenbeck T S , Green K J
Published: May 2004

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2-terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.
Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing.
Authors: Scherthan H , Weich S , Schwegler H , Heyting C , Härle M , Cremer T
Published: May 2004

The preconditions and early steps of meiotic chromosome pairing were studied by fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes to mouse and human testis tissue sections. Premeiotic pairing of homologous chromosomes was not detected in spermatogonia of the two species. FISH with centromere- and telomere-specific DNA probes in combination with immunostaining (IS) of synaptonemal complex (SC) proteins to testis sections of prepuberal mice at days 4-12 post partum was performed to study sequentially the meiotic pairing process. Movements of centromeres and then telomeres to the nuclear envelope, and of telomeres along the nuclear envelope leading to the formation of a chromosomal bouquet were detected during mouse prophase. At the bouquet stage, pairing of a mouse chromosome-8-specific probe was observed. SC-IS and simultaneous telomere FISH revealed that axial element proteins appear as large aggregates in mouse meiocytes when telomeres are attached to the nuclear envelope. Axial element formation initiates during tight telomere clustering and transverse filament-IS indicated the initiation of synapsis during this stage. Comparison of telomere and centromere distribution patterns of mouse and human meiocytes revealed movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process. Chromosome painting in human spermatogonia revealed compacted, largely mutually exclusive chromosome territories. The territories developed into long, thin threads at the onset of meiotic prophase. Based on these results a unified model of the pairing process is proposed.
A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1.
Authors: Richards S A , Lounsbury K M , Carey K L , Macara I G
Published: May 2004

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.
Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body.
Authors: Anderson K I , Wang Y L , Small J V
Published: May 2004

We have investigated the relationship between lamellipodium protrusion and forward translocation of the cell body in the rapidly moving keratocyte. It is first shown that the trailing, ellipsoidal cell body rotates during translocation. This was indicated by the rotation of the nucleus and the movement of cytoplasmic organelles, as well as of exogenously added beads used as markers. Activated or Con A-coated fluorescent beads that were overrun by cells were commonly endocytosed and rotated with the internal organelles. Alternatively, beads applied to the rear of the cell body via a micropipette adhered to the dorsal cell surface and also moved forward, indicating that both exterior and underlying cortical elements participated in rotation. Manipulation of keratocytes with microneedles demonstrated that pushing or restraining the cell body in the direction of locomotion, and squeezing it against the substrate, which temporarily increased the intracellular pressure, did not effect the rate of lamellipodium protrusion. Rotation and translocation of the cell body continued momentarily after arrest of lamellipodium protrusion by cytochalasin B, indicating that these processes were not directly dependent on actin polymerization. The cell body was commonly flanked by phase-dense "axles," extending from the cell body into the lamellipodium. Phalloidin staining showed these to be comprised of actin bundles that splayed forward into the flanks of the lamellipodium. Disruption of the bundles on one side of the nucleus by traumatic microinjection resulted in rapid retraction of the cell body in the opposite direction, indicating that the cell body was under lateral contractile stress. Myosin II, which colocalizes with the actin bundles, presumably provides the basis of tension generation across and traction of the cell body. We propose that the basis of coupling between lamellipodium protrusion and translocation of the cell body is a flow of actin filaments from the front, where they are nucleated and engage in protrusion, to the rear, where they collaborate with myosin in contraction. Myosin-dependent force is presumably transmitted from the ends of the cell body into the flanks of the lamellipodium via the actin bundles. This force induces the spindle-shaped cell body to roll between the axles that are created continuously from filaments supplied by the advancing lamellipodium.