bluuebunny/crossref_metadata_2025 huggingface dataset:
Homophilic adhesion between Ig superfamily carcinoembryonic antigen molecules involves double reciprocal bonds
Authors: Zhou H , Fuks A , Alcaraz G , Bolling TJ , Stanners CP
Published: May 2004

Both carcinoembryonic antigen (CEA) and neural cell adhesion molecule (NCAM) belong to the immunoglobulin supergene family and have been demonstrated to function as homotypic Ca(++)-independent intercellular adhesion molecules. CEA and NCAM cannot associate heterotypically indicating that they have different binding specificities. To define the domains of CEA involved in homotypic interaction, hybrid cDNAs consisting of various domains from CEA and NCAM were constructed and were transfected into a CHO-derived cell line; stable transfectant clones showing cell surface expression of CEA/NCAM chimeric-proteins were assessed for their adhesive properties by homotypic and heterotypic aggregation assays. The results indicate that all five of the Ig(C)-like domains of NCAM are required for intercellular adhesion while the COOH-terminal domain containing the fibronectin-like repeats is dispensable. The results also show that adhesion mediated by CEA involves binding between the Ig(V)-like amino-terminal domain and one of the Ig(C)-like internal repeat domains: thus while transfectants expressing constructs containing either the N domain or the internal domains alone were incapable of homotypic adhesion, they formed heterotypic aggregates when mixed. Furthermore, peptides consisting of both the N domain and the third internal repeat domain of CEA blocked CEA-mediated cell aggregation, thus providing direct evidence for the involvement of the two domains in adhesion. We therefore propose a novel model for interactions between immunoglobulin supergene family members in which especially strong binding is effected by double reciprocal interactions between the V-like domains and C-like domains of antiparallel CEA molecules on apposing cell surfaces.
Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation
Authors: Brady-Kalnay SM , Flint AJ , Tonks NK
Published: May 2004

The receptor-like protein tyrosine phosphatase, PTPmu, displays structural similarity to cell-cell adhesion molecules of the immunoglobulin superfamily. We have investigated the ability of human PTPmu to function in such a capacity. Expression of PTPmu, with or without the PTPase domains, by recombinant baculovirus infection of Sf9 cells induced their aggregation. However, neither a chimeric form of PTPmu, containing the extracellular and transmembrane segments of the EGF receptor and the intracellular segment of PTPmu, nor the intracellular segment of PTPmu expressed as a soluble protein induced aggregation. PTPmu mediates aggregation via a homophilic mechanism, as judged by lack of incorporation of uninfected Sf9 cells into aggregates of PTPmu-expressing cells. Homophilic binding has been demonstrated between PTPmu-coated fluorescent beads (Covaspheres) and endogenously expressed PTPmu on MvLu cells. Additionally the PTPmu-coated beads specifically bound to a bacterially expressed glutathione-S-transferase fusion protein containing the extracellular segment of PTPmu (GST/PTPmu) adsorbed to petri dishes. Covaspheres coated with the GST/PTPmu fusion protein aggregated in vitro and also bound to PTPmu expressed endogenously on MvLu cells. These results suggest that the ligand for this transmembrane PTPase is another PTPmu molecule on an adjacent cell. Thus homophilic binding interactions may be an important component of the function of PTPmu in vivo.
Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction
Authors: Dryden KA , Wang G , Yeager M , Nibert ML , Coombs KM , Furlong DB , Fields BN , Baker TS
Published: May 2004

Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)
Molecular characterization of mammalian cylicin, a basic protein of the sperm head cytoskeleton
Authors: Hess H , Heid H , Franke WW
Published: May 2004

The cytoskeletal calyx structure surrounding part of the nucleus of the mammalian sperm head contains two major kinds of basic proteins, i.e., the approximately 60-kD calicin and a group of very basic (IEP > pH 10) polypeptides ranging in size from approximately 58 to approximately 100 kD ("multiple band proteins," MBPs). We have produced MBP-specific mAbs and have isolated a bovine and a human cDNA clone encoding one of these proteins, termed "cylicin" (from the Greek word c eta kv lambda l zeta for cup or beaker). Bovine cylicin I of a calculated molecular weight of 74,788 contains a high proportion (29%) of positively charged amino acids, resulting in an IEP of 10.55, numerous KKD tripeptides, and is characterized by an organization of the central part of the molecule in nine repeating units of maximally 41 amino acids each of which according to prediction analysis should tend to form an alpha helix. The identity of the polypeptide has been proven by direct amino acid sequencing of > 14 different fragments and by experiments using antibodies raised against a partial cDNA-derived protein segment produced in E. coli. By Northern blot analysis we have identified the 2.4-kb cylicin I mRNA only in testis. The unusual cytoskeletal protein cylicin is compared with other proteins and its possible architectural role during spermiogenesis is discussed.
Neurotrophins and their receptors in rat peripheral trigeminal system during maxillary nerve growth
Authors: Arumäe U , Pirvola U , Palgi J , Kiema TR , Palm K , Moshnyakov M , Ylikoski J , Saarma M
Published: May 2004

We examined the expression of the neurotrophins (NTFs) and their receptor mRNAs in the rat trigeminal ganglion and the first branchial arch before and at the time of maxillary nerve growth. The maxillary nerve appears first at embryonic day (E)10 and reaches the epithelium of the first branchial arch at E12, as revealed by anti-L1 immunohistochemistry. In situ hybridization demonstrates, that at E10-E11, neurotrophin-3 (NT-3) mRNA is expressed mainly in the mesenchyme, but neurotrophin-4 (NT-4) mRNA in the epithelium of the first branchial arch. NGF and brain-derived neurotrophic factor (BDNF) mRNAs start to be expressed in the distal part of the first brachial arch shortly before its innervation by the maxillary nerve. Trigeminal ganglia strongly express the mRNA of trkA at E10 and thereafter. The expression of mRNAs for low-affinity neurotrophin receptor (LANR), trkB, and trkC in trigeminal ganglia is weak at E10, but increases by E11-E12. NT-3, NT-4, and more prominently BDNF, induce neurite outgrowth from explant cultures of the E10 trigeminal ganglia but no neurites are induced by NGF, despite the expression of trkA. By E12, the neuritogenic potency of NGF also appears. The expression of NT-3 and NT-4 and their receptors in the trigeminal system prior to target field innervation suggests that these NTFs have also other functions than being the target-derived trophic factors.
Activated mouse astrocytes and T cells express similar CD44 variants. Role of CD44 in astrocyte/T cell binding
Authors: Haegel H , Tölg C , Hofmann M , Ceredig R
Published: May 2004

The CD44 adhesion molecule is expressed by astrocytes, glial-type cells which exhibit features of accessory cells for immune responses in the central nervous system. In primary cultures of mouse astrocytes, we have observed that surface expression and mRNA levels of CD44 are induced following stimulation with either PMA, or tumor necrosis factor alpha plus gamma interferon. Comparison of CD44 splice variants expressed by astrocytes and a T cell hybridoma shows that upon activation, both cell types express a similar pattern of CD44 transcripts. Thus, in both cell types, CD44 transcripts are produced which contain additional exons, including the exon v6 (known to be expressed by in vivo activated lymphocytes and by metastatic variants of tumor cells) as well as variants of larger size. In the autoimmune disease multiple sclerosis, activated T cells cross the blood-brain barrier and lead to inflammation in the central nervous system. Analysis of mice with experimental allergic encephalomyelitis, frequently used as an animal model of multiple sclerosis, shows that CD44 is induced in vivo on glial cells surrounding inflammatory lesions. Using an in vitro model for adhesion between T cells and astrocytes, we have found a correlation between the activation state of these cells and their adhesion potential. Dose-dependent inhibition of adhesion by hyaluronate and by anti-CD44 monoclonal antibody KM81 shows that CD44 is involved in the adhesive interactions between T cells and astrocytes.
Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis
Authors: French LE , Chonn A , Ducrest D , Baumann B , Belin D , Wohlwend A , Kiss JZ , Sappino AP , Tschopp J , Schifferli JA
Published: May 2004

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.
Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor
Authors: Blow JJ
Published: May 2004

Xenopus egg extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) are unable to support the initiation of DNA replication. Nuclei assembled in 6-DMAP extracts behave as though they are in G2, and will not undergo another round of DNA replication until passage through mitosis. 6-DMAP extracts are functionally devoid of a replication factor that modifies chromatin in early G1 before nuclear envelope assembly, but which is itself incapable of crossing the nuclear envelope. This chromatin modification is capable of supporting only a single round of semiconservative replication. The behavior of this replication factor is sufficient to explain why eukaryotic DNA is replicated once and only once in each cell cycle, and conforms to the previous model of a Replication Licensing Factor. Cell cycle analysis shows that this putative Licensing Factor is inactive during metaphase, but becomes rapidly activated on exit from metaphase when it can modify chromatin before nuclear envelope assembly is complete.
Beta-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro
Authors: Peter F , Plutner H , Zhu H , Kreis TE , Balch WE
Published: May 2004

Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that beta-COP, a component of non-clathrin-coated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of beta-COP specific antibodies and F(ab) fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that beta-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of beta-COP was found in a high molecular pool (> 1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rab1B could be quantitatively coprecipitated with this beta-COP containing complex and was also essential for function. We suggest that beta-COP functions in an early step during vesicle formation and that rab1B may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carriers.
Oligomerization of a membrane protein correlates with its retention in the Golgi complex
Authors: Weisz OA , Swift AM , Machamer CE
Published: May 2004

The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.
The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected
Authors: Presley JF , Mayor S , Dunn KW , Johnson LS , McGraw TE , Maxfield FR
Published: May 2004

We have characterized a new CHO cell line (12-4) derived from a parental line, TRVb-1, that expresses the human transferrin receptor. This mutant belongs to the end2 complementation group of endocytosis mutants. Like other end2 mutants, the endosomes in 12-4 cells show a partial acidification defect. These cells internalize LDL and transferrin at 70% of the rate of parental cells and externalize transferrin at 55% of the parental rate (Johnson, L. S., J. F. Presley, J. C. Park, and T. E. McGraw. J. Cell Physiol. 1993). In this report, we have used fluorescence microscopy to determine which step in receptor trafficking is affected in the mutants. Transferrin is sorted from LDL and is delivered to a peri-centriolar recycling compartment at rates similar to parental cells. However, the rate constant for exit of transferrin from the recycling compartment in mutant cells is 0.025 min-1 vs 0.062 min-1 in the parental line. We also measured the trafficking of a bulk membrane marker, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexanoyl- sphingosylphosphorylcholine (C6-NBD-SM) that labels the exofacial side of the plasma membrane. C6-NBD-SM enters the same recycling compartment as transferrin, and it exits the recycling compartment at a rate of 0.060-0.065 min-1 in both parental and 12-4 cells. We conclude that bulk membrane flow in the recycling pathway of 12-4 cells is normal, but exit of transferrin from the recycling compartment is slowed due to retention in this compartment. Thus, in the mutant cell line the recycling compartment carries out a sorting function, retaining transferrin over bulk membrane.
Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock
Authors: Lepock JR , Frey HE , Ritchie KP
Published: May 2004

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.
Synchrony of cell spreading and contraction force as phagocytes engulf large pathogens
Authors: Evans E , Leung A , Zhelev D
Published: May 2004

A simple micromechanical method has been used to directly measure the force of contraction in single mammalian phagocytes (blood granulocytes) during engulfment of large yeast pathogens. Both the time course of cell spreading over the yeast particle and increase in cell body contractile force were quantitated at three temperatures in the range of 23-35 degrees C. The surprising feature of the phagocyte response was that engulfment and cell body contraction occurred in a serial sequence: i.e., the phagocyte spread rapidly over the particle at a steady rate with no detectable cell body contraction; when spreading stopped, contraction force in the cell body then rose steadily to a plateau level that remained stationary until the next sequence of spreading and contraction. Both spreading and contraction exhibited abrupt start/stop kinetics. Also impressive, the cell contraction force stimulated by phagocytosis was quite large (approximately 10(-8) N)-two orders of magnitude larger than the force necessary to deform passive phagocytes to the same extent. If distributed uniformly over the cell cross section, the contraction force is equivalent to an average contractile stress of approximately 10(3) N/m2 (0.01 Atm). These physical measurements in situ set critical requirements for the mechanism of force generation in granulocytes, imply that a major increase in network cross-linking accompanies build-up in contractile force and that subsequent network dissolution is necessary for locomotion.
Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin
Authors: Melki R , Vainberg IE , Chow RL , Cowan NJ
Published: May 2004

The folding of actin and tubulin is mediated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin) that hydrolyzes ATP as part of the reaction whereby native proteins are ultimately released. Vertebrate actin-related protein (actin-RPV) (also termed centractin) and gamma-tubulin are two proteins that are distantly related to actin and tubulin, respectively: gamma-tubulin is exclusively located at the centrosome, while actin-RPV is conspicuously abundant at the same site. Here we show that actin-RPV and gamma-tubulin are both folded via interaction with the same chaperonin that mediates the folding of beta-actin and alpha- and beta-tubulin. In each case, the unfolded polypeptide forms a binary complex with cytoplasmic chaperonin and is released as a soluble, monomeric protein in the presence of Mg-ATP and the presence or absence of Mg-GTP. In contrast to alpha- and beta-tubulin, the folding of gamma-tubulin does not require the presence of cofactors in addition to chaperonin itself. Monomeric actin-RPV produced in in vitro folding reactions cocycles efficiently with native brain actin, while in vitro folded gamma-tubulin binds to polymerized microtubules in a manner consistent with interaction with microtubule ends. Both monomeric actin-RPV and gamma-tubulin bind to columns of immobilized nucleotide: monomeric actin-RPV has no marked preference for ATP or GTP, while gamma-tubulin shows some preference for GTP binding. We show that actin-RPV and gamma-tubulin compete with one another, and with beta-actin or alpha-tubulin, for binary complex formation with cytoplasmic chaperonin.
Differential expression of a phosphoepitope at the kinetochores of moving chromosomes
Authors: Gorbsky GJ , Ricketts WA
Published: May 2004

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.
Structure/function analysis of the integrin beta 1 subunit by epitope mapping
Authors: Shih DT , Edelman JM , Horwitz AF , Grunwald GB , Buck CA
Published: May 2004

Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti-chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.
Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.
Authors: Voos W , Gambill B D , Guiard B , Pfanner N , Craig E A
Published: May 2004

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.
Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.
Authors: Czech M P , Chawla A , Woon C W , Buxton J , Armoni M , Tang W , Joly M , Corvera S
Published: May 2004

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.
Identification of the carboxy terminus as important for the isoform-specific subcellular targeting of glucose transporter proteins.
Authors: Verhey K J , Hausdorff S F , Birnbaum M J
Published: May 2004

Differential trafficking of glucose transporters contributes significantly to the establishment of a cell's capacity for hormone-regulatable hexose uptake. In the true insulin-sensitive peripheral target tissues, muscle and adipose, the transporter isoform GLUT1 residues on the cell surface and interior of the cell whereas the highly homologous isoform GLUT4 displays virtually exclusive intracellular sequestration, allowing the latter to redistribute to the cell surface in response to hormone. These patterns are equally pronounced in cells into which the transporters have been introduced by DNA-mediated gene transfer, suggesting that signals for isoform-specific sorting are recognized in diverse cell types. To determine the primary sequences responsible for the characteristic distributions, chimeric transporters were constructed in which reciprocal domains were exchanged between GLUT1 and GLUT4. In addition, a non-disruptive, species-specific epitope "tag" was introduced into a neutral region of the transporter to allow analysis of reciprocal chimeras using a single antibody. These recombinant transporters were stably expressed in HIH 3T3 and PC12 cells by retrovirus-mediated gene transfer, and were localized by indirect immunofluorescence and laser scanning confocal microscopy, as well as by staining of plasma membrane sheets prepared from these cells. The results indicate that the carboxy-terminal 30 amino acids are primarily responsible for the differential targeting of the glucose transporter isoforms GLUT1 and GLUT4, though there is a lesser additional contribution by the amino-terminal 183 amino acids.
Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development.
Authors: Sonnenberg E , Meyer D , Weidner K M , Birchmeier C
Published: May 2004

Scatter factor/hepatocyte growth factor (SF/HGF) has potent motogenic, mitogenic, and morphogenetic activities on epithelial cells in vitro. The cell surface receptor for this factor was recently identified: it is the product of the c-met protooncogene, a receptor-type tyrosine kinase. We report here the novel and distinct expression patterns of SF/HGF and its receptor during mouse development, which was determined by a combination of in situ hybridization and RNase protection experiments. Predominantly, we detect transcripts of c-met in epithelial cells of various developing organs, whereas the ligand is expressed in distinct mesenchymal cells in close vicinity. In addition, transient SF/HGF and c-met expression is found at certain sites of muscle formation; transient expression of the c-met gene is also detected in developing motoneurons. SF/HGF and the c-met receptor might thus play multiple developmental roles, most notably, mediate a signal given by mesenchyme and received by epithelial. Mesenchymal signals are known to govern differentiation and morphogenesis of many epithelia, but the molecular nature of the signals has remained poorly understood. Therefore, the known biological activities of SF/HGF in vitro and the embryonal expression pattern reported here indicate that this mesenchymal factor can transmit morphogenetic signals in epithelial development and suggest a molecular mechanism for mesenchymal epithelial interactions.
The actin-binding protein comitin (p24) is a component of the Golgi apparatus.
Authors: Weiner O H , Murphy J , Griffiths G , Schleicher M , Noegel A A
Published: May 2004

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.
Neurons regulate Schwann cell genes by diffusible molecules.
Authors: Bolin L M , Shooter E M
Published: May 2004

Successful peripheral nerve regeneration and functional recovery require the reestablishment of the neuron-Schwann cell relationship in the regenerating rat sciatic nerve, neurons differentially regulate Schwann cell genes. The message for the low-affinity NGF receptor, p75NGFR, is induced in Schwann cells distal to the injury and is repressed as regenerating axons make contact with these cells. The inverse is true for mRNA of the myelin gene P0; expression decreases distal to injury and increases as new axons contact Schwann cells and a program of myelination is initiated. Using an in vitro co-culture paradigm in which primary neurons and adult Schwann cells are separated by a microporous membrane, we show that axon contact is not an absolute requirement for neuronal regulation of Schwann cell genes. In this system neurons but not other cell types, repress the expression of Schwann cell p75NGFR while inducing the expression of the POU domain transcription factor, suppressed cAMP inducible POU, and myelin P0. These results demonstrate that regenerating axons can direct the Schwann cell genetic program from a distance through diffusible molecules.
Genetic identification, sequence, and alternative splicing of the Caenorhabditis elegans alpha 2(IV) collagen gene.
Authors: Sibley M H , Johnson J J , Mello C C , Kramer J M
Published: May 2004

The nematode Caenorhabditis elegans has two type IV collagen genes homologous to the mammalian alpha 1(IV) and alpha 2(IV) collagen genes. We demonstrate by transgenic rescue of mutant animals that the genetic locus encoding the C. elegans alpha 2(IV) collagen gene is let-2 on the X chromosome. The most severe effect of mutations in let-2 is temperature-sensitive embryonic lethality. The embryonic lethal phenotype is similar to that seen in animals with mutations in the alpha 1(IV) collagen gene, emb-9. The sequence of the entire C. elegans alpha 2(IV) collagen gene is presented. Comparisons with mammalian type IV collagen sequences show high amino acid sequence conservation in the C-terminal NCl domain and of crosslinking residues (Cys and Lys) in the N-terminal 7S domain. RT-PCR analysis shows that transcripts of the C. elegans alpha 2(IV) collagen gene are alternatively spliced. Transcripts contain one of two mutually exclusive exons, exon 9 or 10. These exons encode very similar products, differing primarily in the sequence of a 9-10 amino acid Gly-X-Y interruption. The expression of these alternatively spliced alpha 2(IV) collagen transcripts is developmentally regulated. In embryos over 90% of the alpha 2(IV) collagen mRNA contains exon 9, while larval and adult RNAs contain 80-90% exon 10. This shift in expression of alternative alpha 2(IV) collagen transcripts suggests that C. elegans embryos may require a different form of alpha 2(IV) collagen than do larvae and adults.
Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane.
Authors: Huber L A , Pimplikar S , Parton R G , Virta H , Zerial M , Simons K
Published: May 2004

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.
Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p.
Authors: Huber L A , de Hoop M J , Dupree P , Zerial M , Simons K , Dotti C
Published: May 2004

In the companion paper (Huber, L. A., S. W. Pimplikar, R. G. Parton, H. Virta, M. Zerial, and K. Simons. J. Cell Biol. 123:35-45) we reported that the small GTPase rab8p is involved in transport from the TGN to the basolateral plasma membrane in epithelia. In the present work we investigated the localization and function of rab8p in polarized hippocampal neurons. By immunofluorescence microscopy we found that rab8p localized preferentially in the somatodendritic domain, and was excluded from the axon. Double-labeling immunofluorescence showed that some of the rab8p co-localized in the dendrites with the Semliki Forest Virus glycoprotein E2 (SFV-E2). An antisense oligonucleotide approach was used to investigate the role of rab8p in dendritic transport of newly synthesized viral glycoproteins. Antisense oligonucleotides corresponding to the initiation region of the rab8 coding sequence were added to the cultured neurons for four days. This treatment resulted in a significant decrease in cellular levels of rab8p and transport of SFV-E2 from the cell body to the dendrites was significantly reduced. However, no effect was observed on axonal transport of influenza HA. From these results we conclude that rab8p is involved in transport of proteins to the dendritic surface in neurons.
Cholesterol is required in the exit pathway of Semliki Forest virus.
Authors: Marquardt M T , Phalen T , Kielian M
Published: May 2004

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.
Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors.
Authors: Hansen S H , Sandvig K , van Deurs B
Published: May 2004

We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin-dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)-depleted cells with Con A-gold for 15 min, approximately 75% of Con A-gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)-depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.