bluuebunny/crossref_metadata_2025 huggingface dataset:
The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle.
Authors: Jorgensen A O , Shen A C , Arnold W , McPherson P S , Campbell K P
Published: May 2004

The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T-tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma.
"Trans-differentiation" from epidermal to mesenchymal/myogenic phenotype is associated with a drastic change in cell-cell and cell-matrix adhesion molecules.
Authors: Boukamp P , Fusenig N E
Published: May 2004

Cells of the human keratinocyte line HaCaT were shifted to a mesenchymal/myogenic phenotype (DTHMZ cells) by MyoD1 transfection, 5-aza-2' deoxycytidine treatment, and selection for reduced adhesion on plastic. Since this correlated with loss of stratification (inability to form a multilayered tissue), we determined the status of cell-cell and cell-matrix adhesion molecules involved in epidermal morphogenesis. Expression of desmosomal proteins (plakoglobin, desmoglein, desmoplakin) and uvomorulin was no longer detectable at the mRNA and protein level in the DTHMZ cells while both HaCaT cells and malignant variants (transfected with c-Ha-ras oncogene) expressed uvomorulin in vitro and in transplants in vivo, the latter even in invasively growing tumor nodules. Furthermore, HaCaT cells stained positive for the integrin subunits beta 1, alpha 2, alpha 3, and alpha 5, typical for cultured keratinocytes. In contrast, the putative fibronectin receptor alpha 5 beta 1, common also in fibroblasts, was the only integrin showing strong staining in DTHMZ cells. The integrin subunits alpha v and a6, clearly expressed at the mRNA level, weakly stained HaCaT cultures and led to a dotlike fluorescence in DTHMZ cells, possibly representing focal adhesion plaques. The respective integrin status correlated well with the growth behavior on different matrices. While HaCaT cells readily attached and proliferated on collagen (type I), fibronectin-coated, and laminin-coated collagen gels, DTHMZ cells formed monolayers only on fibronectin-coated collagen. This was, however, not sufficient to allow stratification in vivo. Altogether, the status of adhesion molecules in DTHMZ cells more likely reflects that seen in mesenchymal cells as compared to the pattern of keratinocytes displayed by HaCaT cells. Moreover, since the DTHMZ cells were clearly HaCaT descendants, the results support our hypothesis of a "trans-differentiation" process from an epidermal (HaCaT) to a mesenchymal/myogenic phenotype (DTHMZ).
Role of the latent TGF-beta binding protein in the activation of latent TGF-beta by co-cultures of endothelial and smooth muscle cells.
Authors: Flaumenhaft R , Abe M , Sato Y , Miyazono K , Harpel J , Heldin C H , Rifkin D B
Published: May 2004

Transforming growth factor beta (TGF-beta) is released from cells in a latent form consisting of the mature growth factor associated with an aminoterminal propeptide and latent TGF-beta binding protein (LTBP). The endogenous activation of latent TGF-beta has been described in co-cultures of endothelial and smooth muscle cells. However, the mechanism of this activation remains unknown. Antibodies to native platelet LTBP and to a peptide fragment of LTBP inhibit in a dose-dependent manner the activation of latent TGF-beta normally observed when endothelial cells are cocultured with smooth muscle cells. Inhibition of latent TGF-beta activation was also observed when cells were co-cultured in the presence of an excess of free LTBP. These data represent the first demonstration of a function for the LTBP in the extracellular regulation of TGF-beta activity and indicate that LTBP participates in the activation of latent TGF-beta, perhaps by concentrating the latent growth factor on the cell surface where activation occurs.
Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.
Authors: Humphrey J S , Peters P J , Yuan L C , Bonifacino J S
Published: May 2004

Protein localization to the TGN was investigated by examining the subcellular distribution of chimeric proteins in which the cytoplasmic and/or transmembrane domains of the TGN protein, TGN38, were substituted for the analogous domains of the plasma membrane protein, Tac. Using immunofluorescence and immunoelectron microscopy, the COOH-terminal cytoplasmic domain of TGN38 was found to be sufficient for localization of the chimeric proteins to the TGN. Deletion analysis identified an 11-amino acid segment containing the critical sequence, YQRL, as being sufficient for TGN localization. TGN localization was abrogated by mutation of the tyrosine or leucine residues in this sequence to alanine, or of the arginine residue to aspartate. In addition to specifying TGN localization, the 11-amino acid segment was active as an internalization signal, although the property of internalization alone was insufficient to confer TGN localization. Overexpression of chimeric proteins containing TGN localization determinants resulted in their detection at the plasma membrane and in intracellular vesicles, and abolished detection of endogenous TGN38. These results suggest that discrete cytoplasmic determinants can mediate protein localization to the TGN, and reveal a novel role for tyrosine-based motifs in this process.
Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle.
Authors: North A J , Galazkiewicz B , Byers T J , Glenney J R , Small J V
Published: May 2004

The sarcolemma of the smooth muscle cell displays two alternating structural domains in the electron microscope: densely-staining plaques that correspond to the adherens junctions and intervening uncoated regions which are rich in membrane invaginations, or caveolae. The adherens junctions serve as membrane anchorage sites for the actin cytoskeleton and are typically marked by antibodies to vinculin. We show here by immunofluorescence and immunoelectron microscopy that dystrophin is specifically localized in the caveolae-rich domains of the smooth muscle sarcolemma, together with the caveolae-associated molecule caveolin. Additional labeling experiments revealed that beta 1 integrin and fibronectin are confined to the adherens junctions, as indicated by their codistribution with vinculin and tensin. Laminin, on the other hand, is distributed around the entire cell perimeter. The sarcolemma of the smooth muscle cell is thus divided into two distinct domains, featuring different and mutually exclusive components. This simple bipartite domain organization contrasts with the more complex organization of the skeletal muscle sarcolemma: smooth muscle thus offers itself as a useful system for localizing, among other components, potential interacting partners of dystrophin.
A 27,000-D core of the Dictyostelium 34,000-D protein retains Ca(2+)-regulated actin cross-linking but lacks bundling activity.
Authors: Fechheimer M , Furukawa R
Published: May 2004

Actin cross-linking proteins are important for formation of isotropic F-actin networks and anisotropic bundles of filaments in the cytoplasm of eucaryotic cells. A 34,000-D protein from the cellular slime mold Dictyostelium discoideum mediates formation of actin bundles in vitro, and is specifically incorporated into filopodia. The actin cross-linking activity of this protein is inhibited by the presence of micromolar calcium. A 27,000-D fragment obtained by digestion with alpha-chymotrypsin lacks the amino-terminal six amino acids and the carboxyl-terminal 7,000 D of the intact polypeptide. The 27,000-D fragment retains F-actin binding activity assessed by cosedimentation assays and by 125I-[F-actin] blot overlay technique, F-actin cross-linking activity as assessed by viscometry, and calcium binding activity. Ultrastructural analyses indicate that the 27,000-D fragment is deficient in the bundling activity characteristic of the intact 34,000-D protein. Actin filaments are aggregated into microdomains but not bundle in the presence of the 27,000-D fragment. A polarized light scattering assay was used to demonstrate that the 34,000-D protein increases the orientational correlation among F-actin filaments. The 27,000-D fragment does not increase the orientation of the actin filaments as assessed by this technique. A terminal segment(s) of the 34,000-D protein, lacking in the 27,000-D fragment, contributes significantly to the ability to cross-link actin filaments into bundles.
Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon.
Authors: Okabe S , Hirokawa N
Published: May 2004

We previously documented differences in the behavior of microtubules in growing axons of two types of neurons, adult mouse sensory neurons and Xenopus embryonal spinal cord neurons. Namely, the bulk of microtubules was stationary in mouse sensory neurons both by the method of photoactivation of caged-fluorescein-labeled tubulin and photobleaching of fluorescein-labeled tubulin, but the bulk of microtubules did translocate anterogradely by the method of photoactivation. Although these results indicated that the stationary nature of photobleached microtubules in mouse neurons is not an artifact derived from the high levels of energy required for the procedure, it has not yet been settled whether the photobleaching method can detect the movement of microtubules properly. Here we report photobleaching experiments on growing axons of Xenopus embryonal neurons. Anterograde movement of photobleached microtubules was observed at a frequency and translocation rate similar to the values determined by the method of photoactivation. Our results suggest that, under appropriate conditions, the photobleaching method is able to reveal the behavior of microtubules as accurately as the photoactivation method.
The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.
Authors: Kuchler K , Dohlman H G , Thorner J
Published: May 2004

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.
Mouse differentiation-specific keratins 1 and 10 require a preexisting keratin scaffold to form a filament network.
Authors: Kartasova T , Roop D R , Holbrook K A , Yuspa S H
Published: May 2004

Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.
Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA.
Authors: Perrone-Bizzozero N I , Cansino V V , Kohn D T
Published: May 2004

We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.
Structurally related class I and class II receptor protein tyrosine kinases are down-regulated by the same E3 protein coded for by human group C adenoviruses.
Authors: Kuivinen E , Hoffman B L , Hoffman P A , Carlin C R
Published: May 2004

Receptor tyrosine kinases (RTKs) are grouped into subcategories based on shared sequence and structural features. Human group C adenoviruses down-regulate EGF receptors, which are members of the class I family of RTKs, during the early stages of infection. Adenovirus appears to utilize a nonsaturable intracellular pathway since it causes EGF-R down-regulation even in cells that significantly overexpress EGF-R. Adenovirus-induced down-regulation is mediated by a small hydrophobic molecule coded for by the E3 early transcription region that has recently been localized to plasma membrane. Here we examine intracellular trafficking of other RTKs in adenovirus-infected cells, to better understand the molecular basis for the action of the E3 protein. Although p185c-neu, which is a class I RTK closely related to the EGF receptor, is down-regulated in cells expressing physiological concentrations of this molecule, it is not down-regulated in tumor cell lines that significantly overexpress p185c-neu. Cell surface receptors for insulin and IGF1, which are class II RTKs, are also reduced in cells expressing the E3 protein, although to a slightly lesser extent than the EGF receptor. Moreover, whereas EGF receptors are degraded between 3- and 9-h postinfection, insulin and IGF1 receptors are degraded between 6- and 12-h postinfection under identical conditions. In contrast to the class I and class II RTKs, there is no difference in the expression of the class III receptors for PDGF and aFGF in cells infected with a virus with an intact E3 region versus a virus mutant with an internal deletion in the relevant E3 gene. These results suggest that the E3 protein provides an internalization and degradative sorting signal for some class I and class II RTKs, although down-regulation of class II RTKs is somewhat less efficient. Molecular recognition of class I and class II RTKs during adenovirus infection may not be due strictly to amino acid structure, however, since EGF-R but not p185c-neu is down-regulated in cells where it is significantly overexpressed.
Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2.
Authors: Hennekes H , Peter M , Weber K , Nigg E A
Published: May 2004

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.
Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins.
Authors: Lew D J , Reed S I
Published: May 2004

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.
Apical secretion of apolipoproteins from enterocytes.
Authors: Danielsen E M , Hansen G H , Poulsen M D
Published: May 2004

Synthesis and secretion of apolipoproteins in pig small intestine was studied by pulse-chase labeling of jejunal segments, kept in organ culture. Apo A-1 and apo B-48 were the two major proteins released, constituting 25 and 10%, respectively, of the total amount of labeled protein in the mucosal-side medium where they appeared with a t1/2 of 50-60 min. Using tissue from fasting animals, > 85% of newly synthesized apo A-1 and about one third of apo B-48 was released to the mucosal-side medium. Newly synthesized apolipoprotein that remained associated with the intestinal segment accumulated in the soluble fraction, suggesting a basolateral secretion into the intercellular space, and both this accumulation and the release to the medium was prevented by culture at 20 degrees C. The specific radioactivity of apo A-1 and apo B-48 released to the medium was significantly higher than that of the corresponding apolipoproteins remaining associated with the intestinal tissue. Furthermore, during culture periods of up to 5 h, the enterocytes and their tight junctions largely remained intact as evidenced by the inaccessibility of the nonpermeable surface marker Ruthenium red. We therefore propose that enterocytes release most of their newly made free apo A-1 and a significant portion of apo B-48 by exocytosis via the brush border membrane into the intestinal lumen. Fat absorption reduced apolipoprotein secretion to the medium and induced the formation of chylomicrons, containing apo A-1 at their surface, as evidenced by immunogold electron microscopy. The chylomicrons were localized in the Golgi complex and near the basolateral plasma membrane, but not in the apical region of the enterocytes, indicating that only free apolipoproteins are secreted to the intestinal lumen.
Mammalian myosin I alpha, I beta, and I gamma: new widely expressed genes of the myosin I family.
Authors: Sherr E H , Joyce M P , Greene L A
Published: May 2004

A polymerase chain reaction strategy was devised to identify new members of the mammalian myosin I family of actin-based motors. Using cellular RNA from mouse granular neurons and PC12 cells, we have cloned and sequenced three 1.2-kb polymerase chain reaction products that correspond to novel mammalian myosin I genes designated MMI alpha, MMI beta, MMI gamma. The pattern of expression for each of the myosin I's is unique: messages are detected in diverse tissues including the brain, lung, kidney, liver, intestine, and adrenal gland. Overlapping clones representing full-length cDNAs for MMI alpha were obtained from mouse brain. These encode a 1,079 amino acid protein containing a myosin head, a domain with five calmodulin binding sites, and a positively charged COOH-terminal tail. In situ hybridization reveals that MMI alpha is highly expressed in virtually all neurons (but not glia) in the postnatal and adult mouse brain and in neuroblasts of the cerebellar external granular layer. Expression varies in different brain regions and undergoes developmental regulation. Myosin I's are present in diverse organisms from protozoa to vertebrates. This and the expression of three novel members of this family in brain and other mammalian tissues suggests that they may participate in critical and fundamental cellular processes.
The transport properties of axonal microtubules establish their polarity orientation.
Authors: Baas P W , Ahmad F J
Published: May 2004

It is well established that axonal microtubules (MTs) are uniformly oriented with their plus ends distal to the neuronal cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-665). However, the mechanisms by which these MTs achieve their uniform polarity orientation are unknown. Current models for axon growth differ with regard to the contributions of MT assembly and transport to the organization and elaboration of the axonal MT array. Do the transport properties or assembly properties of axonal MTs determine their polarity orientation? To distinguish between these possibilities, we wished to study the initiation and outgrowth of axons under conditions that would arrest MT assembly while maintaining substantial levels of preexisting polymer in the cell body that could still be transported into the axon. We found that we could accomplish this by culturing rat sympathetic neurons in the presence of nanomolar levels of vinblastine. In concentrations of the drug up to and including 100 nM, the neurons actively extend axons. The vinblastine-axons are shorter than control axons, but clearly contain MTs. To quantify the effects of the drug on MT mass, we compared the levels of polymer throughout the cell bodies and axons of neurons cultured overnight in the presence of 0, 16, and 50 nM vinblastine with the levels of MT polymer in freshly plated neurons before axon outgrowth. Without drug, the total levels of polymer increase by roughly twofold. At 16 nM vinblastine, the levels of polymer are roughly equal to the levels in freshly plated neurons, while at 50 nM, the levels of polymer are reduced by about half this amount. Thus, 16 nM vinblastine acts as a "kinetic stabilizer" of MTs, while 50 nM results in some net MT disassembly. At both drug concentrations, there is a progressive increase in the levels of MT polymer in the axons as they grow, and a corresponding depletion of polymer from the cell body. These results indicate that highly efficient mechanisms exist in the neuron to transport preassembled MTs from the cell body into the axon. These mechanisms are active even at the expense of the cell body, and even under conditions that promote some MT disassembly in the neuron. MT polarity analyses indicate that the MTs within the vinblastine-axons, like those in control axons, are uniformly plus-end-distal.(ABSTRACT TRUNCATED AT 400 WORDS)
Production of a unique antibody specific for membrane ruffles and its use to characterize the behavior of two distinct types of ruffles.
Authors: Hasegawa T
Published: May 2004

I have produced a new monoclonal antibody, YF-169, against membrane ruffle specific 55-kD protein. YF-169 stained membrane ruffles of chick embryo fibroblasts so definitely that it enabled clear and reliable analyses of membrane ruffles. Fibroblasts organized two distinct types of membrane ruffles. One type of the ruffles were transiently formed in serum-starved cells (Type I) when stimulated by serum or platelet-derived growth factor. After spontaneous degradation of Type I ruffles, the other type of ruffles containing many microspikes were gradually organized at leading edges (Type II). The formation of Type I ruffles was not affected by either nocodazole, a microtubule-disrupting drug, or taxol, a microtubule-stabilizing reagent. However, Type II ruffles were entirely destroyed not only by nocodazole but also by taxol, suggesting that regulated organization of microtubule network is important to maintain Type II ruffles. H8, a protein kinase inhibitor prevented the spontaneous degradation of Type I ruffles and also reduced the destructive effect of nocodazole on Type II ruffles without affecting microtubule-disrupting activity. Protein kinases may be involved in the degradation processes of both types of ruffles. W7, a calmodulin antagonist, strongly inhibited Type I ruffle formation and completely destroyed Type II ruffles. W7 was also found to induce a remarkable change of 55-kD protein localization. After degradation of Type II ruffles, most of 55-kD protein was incorporated into newly formed unusual thick fibers. These results suggest that regulated organization of microtubule network is not necessary to form Type I ruffles but is important to maintain Type II ruffles, while calmodulin function is essential for both types of membrane ruffles.
Synthesis of nerve growth factor mRNA in cultures of developing mouse whisker pad, a peripheral target tissue of sensory trigeminal neurons.
Authors: Schörnig M , Heumann R , Rohrer H
Published: May 2004

The developmental increase in the level of NGF mRNA in mouse maxillary process/whisker pad is paralleled in vivo by the biochemical and morphological differentiation of whisker pad epidermis, i.e., changes in the keratin expression pattern and the appearance of hair follicles. In cultures of maxillary processes, however, depending on the age of explanted tissue, the increase in NGF mRNA levels either precedes or follows the appearance of epithelial differentiation markers. In addition, we found that prevention of epithelial differentiation by retinoic acid did not affect the increase in NGF mRNA levels. Only in explants from E11.5 embryos was the timing of NGF mRNA production comparable to that of the in vivo situation, whereas at earlier stages (E10/10.5) NGF mRNA levels increased slowly but never reached in vivo levels, even after extended culture periods. However, the amount of NGF mRNA in E10/10.5 maxillary processes was strongly increased in the presence of medium conditioned by E11.5 explants. This effect was not mimicked by the factors IL-1 beta and TGF-beta 1 known to induce NGF mRNA in other systems. It is concluded that the developmental increase in NGF mRNA levels in developing mouse whisker pad is not linked to epidermal differentiation. Interestingly, it is strongly stimulated by a soluble factor(s) produced within the tissue.
Regulation of the differentiation of WEHI-3B D+ leukemia cells by granulocyte colony-stimulating factor receptor.
Authors: Li J , Koay D C , Xiao H , Sartorelli A C
Published: May 2004

To investigate the role of the G-CSF receptor (G-CSFR) in mediating the action of G-CSF, WEHI-3B D+ murine myelomonocytic leukemia cells were transfected with a plasmid containing the murine G-CSFR gene. Overexpression of G-CSFR in transfected clones was demonstrated by northern blotting, binding of [125I]rhG-CSF and cross-linking experiments. A high level of expression of the G-CSFR did not promote or suppress cellular proliferation or initiate differentiation; however, exposure of transfected cells to G-CSF in suspension culture caused a large percentage of the population to enter a differentiation pathway, as determined by two markers of the mature state, the ability of cells to reduce nitroblue tetrazolium (NBT) and to express the differentiation antigen Mac-1 (CD11b) on the cell surface. Thus, upon treatment with 10 ng/ml of G-CSF, 60% or more of transfected cells exhibited NBT positivity; whereas, in contrast, nontransfected cells exhibited only 6% NBT positivity in response to G-CSF. An eightfold increase in Mac-1 expression over that of the parental line was also observed in transfected cells exposed to G-CSF. The growth rate of the transfected clones was decreased by exposure to G-CSF, presumably due to terminal differentiation. The findings suggest that the predominant function of G-CSF and its receptor in WEHI-3B D+ cells is to mediate differentiation and that the level of the G-CSFR portion of the signal transduction mechanism in this malignant cell line is important for a response to the maturation inducing function of the cytokine.
Thrombin receptor peptide inhibits thrombin-induced increase in endothelial permeability by receptor desensitization.
Authors: Lum H , Andersen T T , Siflinger-Birnboim A , Tiruppathi C , Goligorsky M S , Fenton J W , Malik A B
Published: May 2004

Thrombin, a potent activator of cellular responses, proteolytically cleaves, and thereby activates its receptor. In the present study, we compared the effects of the thrombin receptor 14-amino acid peptide (TRP-14; SFLLRNPNDKYEPF), which comprises the NH2 terminus after cleavage of the thrombin receptor, and of the native alpha-thrombin on endothelial monolayer permeability. Addition of TRP-14 (1-200 microM) to bovine pulmonary artery endothelial cells increased [Ca2+]i in a dose-dependent manner. The peak increase in [Ca2+]i in response to 100 microM TRP-14 or 0.1 microM alpha-thrombin was similar (i.e., 931 +/- 74 nM and 1032 +/- 80 nM, respectively), which was followed by a slow decrease with t1/2 values of 0.73 and 0.61 min, respectively. Extracellular Ca2+ chelation with 5 mM EGTA abolished the sustained increases in [Ca2+]i induced by either TRP-14 or alpha-thrombin. alpha-thrombin (0.1 microM) increased transendothelial [125I]albumin permeability, whereas TRP-14 (1-100 microM) had no effect. Coincubation of 100 microM TRP-14 with 1 microM DIP-alpha-thrombin also did not increase permeability over control values. Stimulation of BPAEC with 0.1 microM alpha-thrombin induced translocation of protein kinase C (PKC) from the cytosol to the plasma membrane indicative of PKC activation, whereas TRP-14 had no effect at any concentration. TRP-14 at 100 microM desensitized BPAEC to thrombin-induced increases in [Ca2+]i and transendothelial permeability. The Ca2+ desensitization was reversed after approximately 60 min, and this recovery paralleled the recovery of the permeability response. These findings indicate that the TRP-14-induced Ca2+ mobilization in the absence of PKC activation is insufficient to increase endothelial permeability. In contrast, the increase in endothelial permeability after alpha-thrombin occurred in conjunction with Ca2+ mobilization as well as PKC activation. TRP-14 pretreatment prevented the alpha-thrombin-induced increase in endothelial permeability secondary to desensitization of the Ca2+ signal. The results suggest that combined cytosolic Ca2+ mobilization mediated by TRP-14 and PKC activation mediated by a TRP-14-independent pathway are dual signals responsible for the thrombin-induced increase in vascular endothelial permeability.
Interference with endogenous ras function inhibits cellular responses to wounding.
Authors: Sosnowski R G , Feldman S , Feramisco J R
Published: May 2004

Wounding of tissue induces cellular responses that ultimately result in wound repair. Studies in tissue culture model systems indicate that these responses include induction of AP-1 regulated genes, cell migration and mitogenesis which are also characteristic of cellular responses to growth factors. Investigations have identified cellular ras proteins as critical components of growth factor-stimulated signal transduction pathways, however their role in the wounding response is less clear. Investigation of the potential involvement of c-Ras in this process utilized quiescent living bovine corneal endothelium cells (BCE) which were microinjected with ras dominant interfering mutant protein (N17) and subsequently stimulated by mechanical wounding. Analysis of these cells demonstrated that microinjection of dominant-interfering ras protein, but not control proteins, inhibited the wounding response as evidenced by diminished Fos expression, lack of cell migration and a block in DNA synthesis.
Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.
Authors: Davis J Q , McLaughlin T , Bennett V
Published: May 2004

A major class of ankyrin-binding glycoproteins have been identified in adult rat brain of 186, 155, and 140 kD that are alternatively spliced products of the same pre-mRNA. Characterization of cDNAs demonstrated that ankyrin-binding glycoproteins (ABGPs) share 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides have the following features consistent with a role as ankyrin-binding proteins in vitro and in vivo: (a) ABGPs and ankyrin associate as pure proteins in a 1:1 molar stoichiometry; (b) the ankyrin-binding site is located in the COOH-terminal 21 kD of ABGP186 which contains the predicted cytoplasmic domain; (c) ABGP186 is expressed at approximately the same levels as ankyrin (15 pmoles/milligram of membrane protein); and (d) ABGP polypeptides are co-expressed with the adult form of ankyrinB late in postnatal development and are colocalized with ankyrinB by immunofluorescence. Similarity in amino acid sequence and conservation of sites of alternative splicing indicate that genes encoding ABGPs and neurofascin share a common ancestor. However, the major differences in developmental expression reported for neurofascin in embryos versus the late postnatal expression of ABGPs suggest that ABGPs and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. The predicted cytoplasmic domains of rat ABGPs and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, which includes L1, Nr-CAM, and Ng-CAM of vertebrates, and neuroglian of Drosophila. The ankyrin-binding site of rat ABGPs is localized to the C-terminal 200 residues which encompass the cytoplasmic domain, suggesting the hypothesis that ability to associate with ankyrin may be a shared feature of neurofascin and related nervous system cell adhesion molecules.
The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells.
Authors: Weidner K M , Sachs M , Birchmeier W
Published: May 2004

Depending on the target cells and culture conditions, scatter factor/hepatocyte growth factor (SF/HGF) mediates several distinct activities, i.e., cell motility, proliferation, invasiveness, tubular morphogenesis, angiogenesis, or cytotoxicity. A small isoform of SF/HGF encoded by a natural splice variant, which consists of the NH2-terminal hairpin structure and the first two kringle domains but not the protease homology region, induces cell motility but not mitogenesis. Two types of SF/HGF receptors have recently been discovered in epithelial cells, the high affinity c-Met receptor tyrosine kinase, and low affinity/high capacity binding sites, which are probably located on heparan sulfate proteoglycans. In the present study, we have addressed the question whether the various biological activities of SF/HGF are transduced into cells by a single type of receptor. We have here examined MDCK epithelial cells transfected with a hybrid cDNA encoding the ligand binding domain of the nerve growth factor (NGF) receptor and the membrane-spanning and tyrosine kinase domains of the Met receptor. We demonstrate that all biological effects of SF/HGF upon epithelial cells such as the induction of cell motility, proliferation, invasiveness, and tubular morphogenesis can now be triggered by the addition of NGF. Thus, it is likely that all known biological signals of SF/HGF are transduced through the receptor tyrosine kinase encoded by the c-Met protooncogene.
Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms.
Authors: Leavesley D I , Schwartz M A , Rosenfeld M , Cheresh D A
Published: May 2004

Human umbilical vein endothelial cell attachment, spreading and migration on collagen and vitronectin are mediated by integrins alpha 2 beta 1 and alpha v beta 3, respectively, and these events take place in the absence of cytokines, growth factors, or chemoattractants. Cell attachment and spreading on these ligands occur in the absence of extracellular calcium, as does migration on collagen. In contrast, vitronectin-mediated migration is absolutely dependent on the presence of extracellular calcium. Cell contact with immobilized vitronectin or anti-alpha v beta 3 mAbs promotes a measurable rise in [Ca2+]i which requires an extracellular calcium source, whereas collagen, or anti-alpha 2 beta 1 mAbs fail to promote this signaling event. In fact, vitronectin-mediated migration and the rise in intracellular calcium showed the same dose dependence on extracellular calcium. While vitronectin and collagen differ in their ability to induce a calcium influx both ligands or antibodies to their respective integrins promote an equivalent increase in intracellular pH consistent with activation of the Na/H antiporter an event independent of extracellular calcium. These results support two salient conclusions. Firstly, collagen and vitronectin, through their respective integrins, promote distinct intracellular signaling events. Secondly, the alpha v beta 3 specific influx of calcium is not required for cell spreading yet appears to facilitate cellular migration on vitronectin.
Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface.
Authors: Bressan G M , Daga-Gordini D , Colombatti A , Castellani I , Marigo V , Volpin D
Published: May 2004

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).
COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells.
Authors: Milgram S L , Mains R E , Eipper B A
Published: May 2004

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of bioactive peptides through a two step reaction catalyzed by separate enzymes contained within the PAM precursor. To characterize the trafficking of integral membrane PAM proteins in neuroendocrine cells, we have generated stable AtT-20 cell lines expressing full length and COOH-terminally truncated integral membrane PAM proteins. Full length integral membrane PAM was present on the cell surface in low but detectable amounts and PAM proteins which reached the cell surface were rapidly internalized but not immediately degraded in lysosomes. Internalized PAM complexed with PAM antibody was found in a subcellular compartment which overlapped with internalized transferrin and with structures binding WGA. Thus the punctate juxtanuclear staining of full length PAM represents PAM in endosomes. Endoproteolytic processing of full length PAM-1 and PAM-2 resulted in the secretion of soluble PAM proteins; the secretion of these soluble PAM proteins was stimulus dependent. Although some of the truncated PAM protein was also processed and stored in AtT-20 cells, much of the expressed protein was redistributed to the plasma membrane. Soluble proteins not observed in large amounts in cells expressing full length PAM were released from the surface of cells expressing truncated PAM and little internalization of truncated integral membrane PAM was observed. Thus, the COOH-terminal domain of PAM contains information important for its trafficking within the regulated secretory pathway as well as information necessary for its retrieval from the cell surface.