bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for heterophilic adhesion of embryonic retinal cells and neuroblastoma cells to substratum-adsorbed NCAM
Authors: Murray BA , Jensen JJ
Published: May 2004

The adhesion of embryonic chicken retinal cells and mouse N2A neuroblastoma cells to purified embryonic chicken retinal NCAM adsorbed on a solid substratum was examined using a quantitative centrifugal adhesion assay. Both cell types adhered to NCAM and the adhesion was specifically inhibited by monovalent anti-NCAM antibody fragments. N2A cell adhesion depended on the amount of NCAM applied to the substratum, was cation independent, and was insensitive to treatment with the cytoskeletal perturbing drugs colchicine and cytochalasin D. These results indicated that the tubulin and actin cytoskeletons were not critically required for adhesion to NCAM and make it unlikely that the cell surface ligand for NCAM is an integrin. Adhesion was however temperature dependent, strengthening greatly after a brief incubation at 37 degrees C. CHO cells transfected with NCAM cDNAs did not adhere specifically to substratum-bound NCAM and pretreatment of N2A cells and retinal cells with anti-NCAM antibodies did not inhibit adhesion to substratum-bound NCAM. These results suggest that a heterophilic interaction between substratum-adsorbed NCAM and a non-NCAM ligand on the surface of the probe cells affects adhesion in this system and support the possibility that heterophilic adhesion may be a function of NCAM in vivo.
Steady state dynamics of intermediate filament networks.
Authors: Vikstrom K L , Lim S S , Goldman R D , Borisy G G
Published: May 2004

We have conducted experiments to examine the dynamic exchange between subunit and polymer of vimentin intermediate filaments (IF) at steady state through the use of xrhodamine-labeled vimentin in fluorescence recovery after photobleaching (FRAP) analysis. The xrhodamine-vimentin incorporated into the endogenous vimentin IF network after microinjection into fibroblasts and could be visualized with a cooled charge-coupled device (CCD) camera and digital imaging fluorescence microscopy. Bar shaped regions were bleached in the fluorescent IF network using a beam from an argon ion laser and the cells were monitored at various times after bleaching to assess recovery of fluorescence in the bleached zones. We determined that bleached vimentin fibers can recover their fluorescence over relatively short time periods. Vimentin fibers in living cells also can exhibit significant movements, but the recovery of fluorescence was not dependent upon movement of fibers. Fluorescence recovery within individual fibers did not exhibit any marked polarity and was most consistent with a steady state exchange of vimentin subunits along the lengths of IF.
Nerve growth factor transcriptional control of c-fos promoter transfected in cultured spinal sensory neurons.
Authors: Gabellini N , Minozzi M C , Leon A , Dal Toso R
Published: May 2004

High efficiency gene transfer (greater than 90%) in chicken dorsal root ganglion neurons has been obtained by DNA calcium phosphate co-precipitation, hence providing an important tool to study control of gene expression in primary neurons. Transfection with c-fos promoter sequences linked to the chloramphenicol acetyltransferase reporter gene showed that the serum responsive element functions as a strong transcriptional enhancer. Transcription from this element is developmentally regulated, and mediates the genetic response to nerve growth factor (NGF) in developing avian sensory neurons. Furthermore, NGF exerts a negative effect on transcription from the cyclic AMP responsive element, thereby supporting the involvement of tyrosine kinase activation by NGF in primary sensory neurons.
Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli.
Authors: Fujishige A , Smith K R , Silen J L , Agard D A
Published: May 2004

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.
Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex.
Authors: Lotti L V , Torrisi M R , Pascale M C , Bonatti S
Published: May 2004

We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the Golgi complex.
Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport.
Authors: Liu H , Bretscher A
Published: May 2004

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.
Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation.
Authors: Schafer D A , Mooseker M S , Cooper J A
Published: May 2004

We have localized capping protein in epithelial cells of several chicken tissues using affinity-purified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by confocal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.
Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip.
Authors: Lynch M A , Staehelin L A
Published: May 2004

Using immunocytochemical techniques and antibodies that specifically recognize xyloglucan (anti-XG), polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), and methylesterified pectins (JIM 7), we have shown that these polysaccharides are differentially synthesized and localized during cell development and differentiation in the clover root tip. In cortical cells XG epitopes are present at a threefold greater density in the newly formed cross walls than in the older longitudinal walls, and PGA/RG-I epitopes are detected solely in the expanded middle lamella of cortical cell corners, even after pretreatment of sections with pectinmethylesterase to uncover masked epitopes. These results suggest that in cortical cells XG and PGA/RG-I are differentially localized not only to particular wall domains, but also to particular cell walls. In contrast to their nonoverlapping distribution in cortical cells, XG epitopes and PGA/RG-I epitopes largely colocalize in the epidermal cell walls. The results also demonstrate that the middle lamella of the longitudinal walls shared by epidermal cells and by epidermal and cortical cells constitutes a barrier to the diffusion of cell wall and mucilage molecules. Synthesis of XG and PGA/RG-I epitope-containing polysaccharides also varies during cellular differentiation in the root cap. The differentiation of gravitropic columella cells into mucilage-secreting peripheral cells is marked by a dramatic increase in the synthesis and secretion of molecules containing XG and PGA/RG-I epitopes. In contrast, JIM 7 epitopes are present at abundant levels in columella cell walls, but are not detectable in peripheral cell walls or in secreted mucilage. There were also changes in the cisternal labeling of the Golgi stacks during cellular differentiation in the root tip. Whereas PGA/RG-I epitopes are detected primarily in cis- and medial Golgi cisternae in cortical cells (Moore, P. J., K. M. M. Swords, M. A. Lynch, and L. A. Staehelin. 1991. J. Cell Biol. 112:589-602), they are localized predominantly in the trans-Golgi cisternae and the trans-Golgi network in epidermal and peripheral root cap cells. These observations suggest that during cellular differentiation the plant Golgi apparatus can be both structurally and functionally reorganized.
Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane.
Authors: Miao G H , Hong Z , Verma D P
Published: May 2004

Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules. It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence. A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter. In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide. The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion. Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated. Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A. Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids. In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane. This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane. Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not. Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants.
Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment.
Authors: Kuliawat R , Arvan P
Published: May 2004

We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C-peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway.
Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.
Authors: Chowdhury S , Smith K W , Gustin M C
Published: May 2004

In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.
In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.
Authors: Pittenger M F , Helfman D M
Published: May 2004

Most cell types express several tropomyosin isoforms, the individual functions of which are poorly understood. In rat fibroblasts there are at least six isoforms; TM-1, TM-2, TM-3, TM-4, TM-5a, and TM-5b. TM-1 is the product of the beta gene. TM-4 is produced from the TM-4 gene, and TMs 2, 3, 5a, and 5b are the products of the alpha gene. To begin to study the localization and function of the isoforms in fibroblasts, cDNAs for TM isoforms 2, 3, 5a, and 5b were placed into bacterial expression vectors and used to produce TM isoforms. The bacterially produced TMs were determined to be full length by sequencing the amino- and carboxy termini. These TMs were found to bind to F-actin in vitro, with properties similar to that of skeletal muscle TM. In addition, competition experiments demonstrated that TM-5b was better than TM-5a in displacing other TM isoforms from F-actin in vitro. To investigate the intracellular localization of these fibroblast isoforms, each was derivatized with a fluorescent chromophore and microinjected into rat fibroblasts. TM-2, TM-3, TM-5a, and TM-5b were each found to associate along actin filaments. There was no preferred cellular location or subset of actin filaments for these isoforms. Furthermore, co-injection of two isoforms labeled with different fluorochromes showed identical staining. At the level of the light microscope, these isoforms from the alpha gene do not appear to achieve different functions by binding to particular subsets of actin filaments or locations in cells. Some alternative possibilities are discussed. The results show that bacterially produced TMs can be used to study in vitro and in vivo properties of the isoforms.
CD44H regulates tumor cell migration on hyaluronate-coated substrate.
Authors: Thomas L , Byers H R , Vink J , Stamenkovic I
Published: May 2004

CD44 is a broadly distributed cell surface glycoprotein expressed in different isoforms in various tissues and cell lines. One of two recently characterized human isoforms, CD44H, is a cell surface receptor for hyaluronate, suggesting a role in the regulation of cell-cell and cell-substrate interactions as well as of cell migration. While CD44H has been shown to mediate cell adhesion, direct demonstration that CD44H expression promotes cell motility has been lacking. In this work we show that a human melanoma cell line, stably transfected with CD44H, displays enhanced motility on hyaluronate-coated surfaces while transfectants expressing an isoform that does not bind hyaluronate, CD44E, fail to do so. Migration of CD44H-expressing transfectants is observed to be blocked by a soluble CD44-immunoglobulin fusion protein as well as by anti-CD44 antibody, and to depend on the presence of the cytoplasmic domain of CD44. However, cells expressing CD44H cytoplasmic deletion mutants retain significant binding capacity to hyaluronate-coated substrate. Taken together, our results provide direct evidence that CD44H plays a major role in regulating cell migration on hyaluronate-coated substrate.
Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod.
Authors: Hodge T P , Cross R , Kendrick-Jones J
Published: May 2004

A short nonhelical sequence at the COOH-terminus of vertebrate nonmuscle myosin has been shown to enhance myosin filament assembly. We have analyzed the role of this sequence in chicken intestinal epithelial brush border myosin, using protein engineering/site-directed mutagenesis. Clones encoding the rod region of this myosin were isolated and sequenced. They were truncated at various restriction sites and expressed in Escherichia coli, yielding a series of mutant myosin rods with or without the COOH-terminal tailpiece and with serial deletions from their NH2-termini. Deletion of the 35 residue COOH-terminal nonhelical tailpiece was sufficient to increase the critical concentration for myosin rod assembly by 50-fold (at 150 mM NaCl, pH 7.5), whereas NH2-terminal deletions had only minor effects. The only exception was the longest NH2-terminal deletion, which reduced the rod to 119 amino acids and rendered it assembly incompetent. The COOH-terminal tailpiece could be reduced by 15 amino acids and it still efficiently promoted assembly. We also found that the tailpiece promoted assembly of both filaments and segments; assemblies which have different molecular overlaps. Rod fragments carrying the COOH-terminal tailpiece did not promote the assembly of COOH-terminally deleted material when the two were mixed together. The tailpiece sequence thus has profound effects on assembly, yet it is apparently unstructured and can be bisected without affecting its function. Taken together these observations suggest that the nonhelical tailpiece may act sterically to block an otherwise dominant but unproductive molecular interaction in the self assembly process and does not, as has been previously thought, bind to a specific target site(s) on a neighboring molecule.
Association of intercellular adhesion molecule-1 (ICAM-1) with actin-containing cytoskeleton and alpha-actinin.
Authors: Carpén O , Pallai P , Staunton D E , Springer T A
Published: May 2004

We have studied the cytoskeletal association of intercellular adhesion molecule-1 (ICAM-1, CD54), an integral membrane protein that functions as a counterreceptor for leukocyte integrins (CD11/CD18). A linkage between ICAM-1 and cytoskeletal elements was suggested by studies showing a different ICAM-1 staining pattern for COS cells transfected with wild-type ICAM-1 or with an ICAM-1 construct that replaces the cytoplasmic and transmembrane domains of ICAM-1 with a glycophosphatidylinositol (GPI) anchor. Wild-type ICAM-1 appeared to localize most prominently in microvilli whereas GPI-ICAM-1 demonstrated a uniform cell surface distribution. Disruption of microfilaments with cytochalasin B (CCB) changed the localization of wild-type ICAM-1 but had no effect on GPI-ICAM-1. Some B-cell lines demonstrated a prominent accumulation of ICAM-1 into the uropod region whereas other cell surface proteins examined were not preferentially localized. CCB also induced redistribution of ICAM-1 in these cells. For characterization of cytoskeletal proteins interacting with ICAM-1, a 28-residue peptide that encompasses the entire predicted cytoplasmic domain (ICAM-1,478-505) was synthesized, coupled to Sepharose-4B, and used as an affinity matrix. One of the most predominant proteins eluted either with soluble ICAM-1,478-505-peptide or EDTA, was 100 kD, had a pI of 5.5, and in Western blots reacted with alpha-actinin antibodies. A direct association between alpha-actinin and ICAM-1 was demonstrated by binding of purified alpha-actinin to ICAM-1,478-505-peptide and to immunoaffinity purified ICAM-1 and by a strict colocalization of ICAM-1 with alpha-actinin, but not with the cytoskeletal proteins talin, tensin, and vinculin. The region of ICAM-1,478-505 interacting with alpha-actinin was mapped to the area close to the membrane spanning region. This region contains several positively charged residues and appears to mediate a charged interaction with alpha-actinin which is not highly dependent on the order of the residues.
The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation.
Authors: Wattenberg B W , Raub T J , Hiebsch R R , Weidman P J
Published: May 2004

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.
Immunocytochemical localization of alpha-protein kinase C in rat pancreatic beta-cells during glucose-induced insulin secretion.
Authors: Ganesan S , Calle R , Zawalich K , Greenawalt K , Zawalich W , Shulman G I , Rasmussen H
Published: May 2004

To investigate the role of protein kinase C (PKC) in the regulation of insulin secretion, we visualized changes in the intracellular localization of alpha-PKC in fixed beta-cells from both isolated rat pancreatic islets and the pancreas of awake unstressed rats during glucose-induced insulin secretion. Isolated, perifused rat islets were fixed in 4% paraformaldehyde, detergent permeabilized, and labeled with a mAb specific for alpha-PKC. The labeling was visualized by confocal immunofluorescent microscopy. In isolated rat pancreatic islets perifused with 2.75 mM glucose, alpha-PKC immunostaining was primarily cytoplasmic in distribution throughout the beta-cells. In islets stimulated with 20 mM glucose, there was a significant redistribution of alpha-PKC to the cell periphery. This glucose-induced redistribution was abolished when either mannoheptulose, an inhibitor of glucose metabolism, or nitrendipine, an inhibitor of calcium influx, were added to the perifusate. We also examined changes in the intracellular distribution of alpha-PKC in the beta-cells of awake, unstressed rats that were given an intravenous infusion of glucose. Immunocytochemical analysis of pancreatic sections from these rats demonstrated a glucose-induced translocation of alpha-PKC to the cell periphery of the beta-cells. These results demonstrate that the metabolism of glucose can induce the redistribution of alpha-PKC to the cell periphery of beta-cells, both in isolated islets and in the intact animal, and suggest that alpha-PKC plays a role in mediating glucose-induced insulin secretion.
The subcellular distribution of chromosome 6-encoded dystrophin-related protein in the brain.
Authors: Khurana T S , Watkins S C , Kunkel L M
Published: May 2004

Chromosome 6-encoded dystrophin-related-protein (DRP) shows significant structural similarities to dystrophin at the carboxyl terminus, though the two proteins are encoded on different chromosomes. Both DRP and dystrophin are expressed in muscle and brain and show some similarity in their subcellular localization. For example, in skeletal muscle both are expressed at neuromuscular and myotendinous junctions. However, while dystrophin is absent or severely reduced in Duchenne/Becker muscular dystrophy, DRP continues to be expressed. Within the brain, dystrophin is enriched at the postsynaptic regions of specific subsets of neurons, while the distribution of DRP is yet to be described. In this study we demonstrate a distinct though highly specific pattern of distribution of DRP in the brain. DRP is enriched in the choroid plexus, pia mater, intracerebral vasculature, and ependymal lining. Within the parenchyma proper, DRP is located at the inner plasma face of astrocytic foot processes at the abluminal aspect of the blood-brain barrier. The distribution of DRP is conserved across a large evolutionary distance, from mammals to elasmobranchs, suggesting that DRP may play a role in the maintenance of regional specializations in the brain.
Host cell factors controlling vimentin organization in the Xenopus oocyte.
Authors: Dent J A , Cary R B , Bachant J B , Domingo A , Klymkowsky M W
Published: May 2004

To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.
Calcium influx into neurons can solely account for cell contact-dependent neurite outgrowth stimulated by transfected L1.
Authors: Williams E J , Doherty P , Turner G , Reid R A , Hemperly J J , Walsh F S
Published: May 2004

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.
A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques.
Authors: Takada Y , Ylänne J , Mandelman D , Puzon W , Ginsberg M H
Published: May 2004

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation.
Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes.
Authors: Cassimeris L , Safer D , Nachmias V T , Zigmond S H
Published: May 2004

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.
Kinesin family in murine central nervous system.
Authors: Aizawa H , Sekine Y , Takemura R , Zhang Z , Nangaku M , Hirokawa N
Published: May 2004

In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family are implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members (KIF1-5), that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are murine counterparts of unc104 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central alpha-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH2-terminal globular domain, central motor domain, and COOH-terminal alpha-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIF1, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.
Identification of a transitional cell state in the developmental pathway to carrot somatic embryogenesis.
Authors: Pennell R I , Janniche L , Scofield G N , Booij H , de Vries S C , Roberts K
Published: May 2004

We have located a novel carbohydrate epitope in the cell walls of certain single cells in embryogenic, but not in non-embryogenic, suspension cultures of carrot. Expression of this epitope, recognized by the mAb JIM8, is regulated during initiation, proliferation, and prolonged growth of suspension cultures such that changes in the abundance of JIM8-reactive cells always precede equivalent changes in embryogenic potential. Therefore, a direct correlation exists between the presence of the JIM8-reactive cell wall epitope and somatic embryo formation. The JIM8-reactive cell wall epitope is expressed in the cell walls of three types of single cells and one type of cell cluster. One of the single cell types seems able to follow one of two phytohormone-controlled developmental pathways, either a cell elongation pathway that eventually leads to cell death, or a cell division pathway that gives rise to proembryogenic masses. We demonstrate that all JIM8-reactive cell types in embryogenic carrot suspension cultures are developmentally related, and that the switch by one of them to somatic embryogenesis is accompanied by the immediate dissipation of the JIM8-reactive cell wall epitope. The cell wall carbohydrate epitope recognized by JIM8 therefore represents a cell wall marker for a very early transitional cell state in the developmental pathway to carrot somatic embryogenesis.
A signal-anchor sequence selective for the mitochondrial outer membrane.
Authors: McBride H M , Millar D G , Li J M , Shore G C
Published: May 2004

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.
Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae.
Authors: Fujimoto T , Nakade S , Miyawaki A , Mikoshiba K , Ogawa K
Published: May 2004

Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.
Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores.
Authors: Sugden D , Rowe S J
Published: May 2004

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.