bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin.
Authors: Sánchez-Aparicio P , Dominguez-Jiménez C , Garcia-Pardo A
Published: May 2004

Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16-activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function.
Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.
Authors: Calafat J , Nijenhuis M , Janssen H , Tulp A , Dusseljee S , Wubbolts R , Neefjes J
Published: May 2004

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.
A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.
Authors: Corvera S , Chawla A , Chakrabarti R , Joly M , Buxton J , Czech M P
Published: May 2004

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.
Electric field-directed fibroblast locomotion involves cell surface molecular reorganization and is calcium independent.
Authors: Brown M J , Loew L M
Published: May 2004

Directional cellular locomotion is thought to involve localized intracellular calcium changes and the lateral transport of cell surface molecules. We have examined the roles of both calcium and cell surface glycoprotein redistribution in the directional migration of two murine fibroblastic cell lines, NIH 3T3 and SV101. These cell types exhibit persistent, cathode directed motility when exposed to direct current electric fields. Using time lapse phase contrast microscopy and image analysis, we have determined that electric field-directed locomotion in each cell type is a calcium independent process. Both exhibit cathode directed motility in the absence of extracellular calcium, and electric fields cause no detectable elevations or gradients of cytosolic free calcium. We find evidence suggesting that galvanotaxis in these cells involves the lateral redistribution of plasma membrane glycoproteins. Electric fields cause the lateral migration of plasma membrane concanavalin A receptors toward the cathode in both NIH 3T3 and SV101 fibroblasts. Exposure of directionally migrating cells to Con A inhibits the normal change of cell direction following a reversal of electric field polarity. Additionally, when cells are plated on Con A-coated substrata so that Con A receptors mediate cell-substratum adhesion, cathode-directed locomotion and a cathodal accumulation of Con A receptors are observed. Immunofluorescent labeling of the fibronectin receptor in NIH 3T3 fibroblasts suggests the recruitment of integrins from large clusters to form a more diffuse distribution toward the cathode in field-treated cells. Our results indicate that the mechanism of electric field directed locomotion in NIH 3T3 and SV101 fibroblasts involves the lateral redistribution of plasma membrane glycoproteins involved in cell-substratum adhesion.
Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization.
Authors: Ku N O , Omary M B
Published: May 2004

There is ample in vitro evidence that phosphorylation of intermediate filaments, including keratins, plays an important role in filament reorganization. In order to gain a better understanding of the function of intermediate filament phosphorylation, we sought to identify the major phosphorylation site of human keratin polypeptide 18 (K18) and study its role in filament assembly or reorganization. We generated a series of K18 ser-->ala mutations at potential phosphorylation sites, followed by expression in insect cells and comparison of the tryptic 32PO4-labeled patterns of the generated constructs. Using this approach, coupled with Edman degradation of the 32PO4-labeled tryptic peptides, and comparison with tryptic peptides analyzed after labeling normal human colonic tissues, we identified ser-52 as the major K18 physiologic phosphorylation site. Ser-52 in K18 is not glycosylated and matches consensus sequences for phosphorylation by CAM kinase, S6 kinase and protein kinase C, and all these kinases can phosphorylate K18 in vitro predominantly at that site. Expression of K18 ser-52-->ala mutant in mammalian cells showed minimal phosphorylation but no distinguishable difference in filament assembly when compared with wild-type K18. In contrast, the ser-52 mutation played a clear but nonexclusive role in filament reorganization, based on analysis of filament alterations in cells treated with okadaic acid or arrested at the G2/M stage of the cell cycle. Our results show that ser-52 is the major physiologic phosphorylation site of human K18 in interphase cells, and that its phosphorylation may play an in vivo role in filament reorganization.
Regulation of scatter factor production via a soluble inducing factor.
Authors: Rosen E M , Joseph A , Jin L , Rockwell S , Elias J A , Knesel J , Wines J , McClellan J , Kluger M J , Goldberg I D
Published: May 2004

Scatter factor (SF) (also known as hepatocyte growth factor [HGF]) is a fibroblast-derived cytokine that stimulates motility, proliferation, and morphogenesis of epithelia. SF may play major roles in development, repair, and carcinogenesis. However, the physiologic signals that regulate its production are not well delineated. We found that various human tumor cell lines that do not produce SF secrete factors that stimulate SF production by fibroblasts, suggesting a paracrine mechanism for regulation of SF production. Conditioned medium from these cell lines contained two distinct scatter factor-inducing factor SF-IF activities: a high molecular weight (> 30 kD), heat sensitive activity and a low molecular weight (< 30 kD) heat stable activity. Further studies revealed that SF-producing fibroblasts also secrete factors that stimulate their own SF production. We characterized the < 30-kD SF-IF activity from ras-3T3 (clone D4), a mouse cell line that overproduces both SF and SF-IF. The < 30-kD filtrate from ras-3T3 conditioned medium induced four- to sixfold increases in expression of SF biologic activity, immunoreactive protein, and mRNA by multiple SF-producing fibroblast lines. Ras-3T3 SF-IF activity was stable to boiling, extremes of pH, and reductive alkylation, but was destroyed by proteases. We purified ras-3T3 SF-IF about 10,000-fold from serum-free conditioned medium by a combination of ultrafiltration, cation exchange chromatography, and reverse phase chromatography. The purified protein exhibited electrophoretic mobility of about 12 kD (reduced) and 14 kD (nonreduced) by SDS-PAGE. The identity of the protein was verified by elution of biologic activity from gel slices. Purified SF-IF stimulated SF production in a physiologic concentration range (about 20-400 pM). Its properties and activities were distinct from those of IL-1 and TNF, two known inducers of SF production. We suggest that SF-IF is a physiologic regulator of SF production.
Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization.
Authors: Wickham T J , Filardo E J , Cheresh D A , Nemerow G R
Published: May 2004

Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus-mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.
LDLC encodes a brefeldin A-sensitive, peripheral Golgi protein required for normal Golgi function.
Authors: Podos S D , Reddy P , Ashkenas J , Krieger M
Published: May 2004

Two genetically distinct classes of low density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants, ldlB and ldlC, exhibit nearly identical pleiotropic defects in multiple medial and trans Golgi-associated processes (Kingsley, D., K. F. Kozarsky, M. Segal, and M. Krieger. 1986. J. Cell Biol. 102:1576-1585). In these mutants, the synthesis of virtually all N- and O-linked glycoproteins and of the major lipid-linked oligosaccharides is abnormal. The abnormal glycosylation of LDL receptors in ldlB and ldlC cells results in their dramatically reduced stability and thus very low LDL receptor activity. We have cloned and sequenced a human cDNA (LDLC) which corrects the mutant phenotypes of ldlC, but not ldlB, cells. Unlike wild-type CHO or ldlB cells, ldlC cells had virtually no detectable endogenous LDLC mRNA, indicating that LDLC is likely to be the normal human homologue of the defective gene in ldlC cells. The predicted sequence of the human LDLC protein (ldlCp, approximately 83 kD) is not similar to that of any known proteins, and contains no major common structural motifs such as transmembrane domains or an ER translocation signal sequence. We have also determined the sequence of the Caenorhabditis elegans ldlCp by cDNA cloning and sequencing. Its similarity to that of human ldlCp suggests that ldlCp mediates a well-conserved cellular function. Immunofluorescence studies with anti-ldlCp antibodies in mammalian cells established that ldlCp is a peripheral Golgi protein whose association with the Golgi is brefeldin A sensitive. In ldlB cells, ldlCp was expressed at normal levels; however, it was not associated with the Golgi. Thus, a combination of somatic cell and molecular genetics has identified a previously unrecognized protein, ldlCp, which is required for multiple Golgi functions and whose peripheral association with the Golgi is both LDLB dependent and brefeldin A sensitive.
Clathrin polymerization is not required for bulk-phase endocytosis in rat fetal fibroblasts.
Authors: Cupers P , Veithen A , Kiss A , Baudhuin P , Courtoy P J
Published: May 2004

To assess the role of clathrin in the bulk endocytic flow of rat foetal fibroblasts, the rate of internalization of fluid-phase and membrane-lipid tracers were compared, under control conditions and after inhibition of endocytic clathrin-coated pit formation. After intracellular potassium depletion or upon cell transfer into 0.35 M NaCl, the rate of internalization of receptor-bound transferrin and the residual membrane area of plasmalemmal clathrin-coated pits and vesicles were similarly decreased by approximately 90%. In contrast, the initial rate (< 5 min) of intracellular accumulation of the fluid-phase tracer HRP was not affected. Both in control and treated cells, the rate of HRP accumulation declined after approximately 5 min, and was twofold lower in treated cells, due to enhanced regurgitation. After correction for regurgitation, the endocytic rate constant was similar to measurements at shorter intervals and identical in control and treated cells. Similarly, the rate of internalization and the steady-state level of intracellular accumulation of two fluorescent lipid derivatives, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsp hingosine (C6-NBD-GlcCer) and 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), were not affected by potassium depletion, indicating that the endocytic membrane traffic was equally preserved. Finally, the size distribution of primary endocytic particles that were accessible to HRP within 15 s before glutaraldehyde fixation was also indistinguishable in control and potassium-depleted cells. The simplest explanation is that clathrin polymerization is necessary to concentrate receptor-bound ligands in primary endocytic vesicles, but superfluous to the basic endocytic machinery in rat foetal fibroblasts.
Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments.
Authors: Monteiro M J , Hicks C , Gu L , Janicki S
Published: May 2004

The mechanism by which nuclear and cytoplasmic filaments are sorted in vivo was studied by examining which lamin sequences are required to target an otherwise cytoplasmic IF protein, the small neurofilament subunit (NF-L), to the nuclear lamina. By swapping corresponding domains between NF-L and lamin A, nuclear envelope targeting of NF-L was shown to require the presence of the "head" domain, a 42-amino acid sequence unique to lamin rod domains, a nuclear localization signal and the CAAX motif. Replacement of the entire COOH-terminal tail of lamin A with that of NF-L had no discernible effect on nuclear localization of lamin A, provided the substituted NF-L tail contained a NLS and a CAAX motif. This chimeric protein exhibited characteristics more typical of lamin B than that of the parental lamin A. With regard to cytoplasmic assembly properties, substitution of the head domain of lamin A for that of NF-L did not substantially affect the ability of NF-L to coassemble with vimentin in the cytoplasm. In contrast, insertion of a 42-amino acid sequence unique to lamin rod domains into NF-L profoundly affected NF-L coassembly with vimentin indicating that the 42-amino acid insertion in lamins may be important for sorting lamins from cytoplasmic IF proteins.
The small GTP-binding protein rab6 functions in intra-Golgi transport.
Authors: Martinez O , Schmidt A , Salaméro J , Hoflack B , Roa M , Goud B
Published: May 2004

Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. Géraud, A. Santa Maria, B. Maro, V. Mayau, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type rab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Over-expression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (alpha-mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.
Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker.
Authors: de Beus E , Brockenbrough J S , Hong B , Aris J P
Published: May 2004

We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.
The role of microtubule dynamics in growth cone motility and axonal growth.
Authors: Tanaka E , Ho T , Kirschner M W
Published: May 2004

The growth cone contains dynamic and relatively stable microtubule populations, whose function in motility and axonal growth is uncharacterized. We have used vinblastine at low doses to inhibit microtubule dynamics without appreciable depolymerization to probe the role of these dynamics in growth cone behavior. At doses of vinblastine that interfere only with dynamics, the forward and persistent movement of the growth cone is inhibited and the growth cone wanders without appreciable forward translocation; it quickly resumes forward growth after the vinblastine is washed out. Direct visualization of fluorescently tagged microtubules in these neurons shows that in the absence of dynamic microtubules, the remaining mass of polymer does not invade the peripheral lamella and does not undergo the usual cycle of bundling and splaying and the growth cone stops forward movement. These experiments argue for a role for dynamic microtubules in allowing microtubule rearrangements in the growth cone. These rearrangements seem to be necessary for microtubule bundling, the subsequent coalescence of the cortex around the bundle to form new axon, and forward translocation of the growth cone.
Differential localization and functional role of calsequestrin in growing and differentiated myoblasts.
Authors: Raichman M , Panzeri M C , Clementi E , Papazafiri P , Eckley M , Clegg D O , Villa A , Meldolesi J
Published: May 2004

Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin-monensin, appear to be incompetent for Ca2+ accumulation.
CEP3 encodes a centromere protein of Saccharomyces cerevisiae.
Authors: Strunnikov A V , Kingsbury J , Koshland D
Published: May 2004

We have designed a screen to identify mutants specifically affecting kinetochore function in the yeast Saccharomyces cerevisiae. The selection procedure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative centromere protein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).
A Chinese hamster ovary cell mutant defective in the non-endocytic uptake of fluorescent analogs of phosphatidylserine: isolation using a cytosol acidification protocol.
Authors: Hanada K , Pagano R E
Published: May 2004

Transmembrane movement of phosphatidylserine (PS) and various PS analogs at the plasma membrane is thought to occur by an ATP-dependent, protein-mediated process. To isolate mutant CHO cells defective in this activity, we first obtained conditions which inhibited the endocytic, but not the non-endocytic pathway of lipid internalization since PS may enter cells by a combination of these two pathways. We found that acidic treatment of cells, which blocks clathrin-dependent endocytosis, enhanced the energy-dependent uptake of 1-palmitoyl-2-(6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl -sn- glycero-3-phosphoserine (C6-NBD-PS) in CHO cells from donor vesicles (liposomes) by about twofold. Control experiments demonstrated that the enhanced uptake of C6-NBD-PS at acidic pH was not due to: (a) an increase in the capacity of the plasma membrane to incorporate C6-NBD-PS from the donor vesicles; (b) a decrease in the rate of loss of C6-NBD-PS from the cells; or (c) fusion or engulfment of the donor vesicles. When cytosolic acidification (to pH 6.3) was imposed without acidification of the extracellular medium, C6-NBD-PS uptake by intact cells was increased by about 50% compared to control values determined in the absence of acidification. These results suggested that a protein and energy dependent system(s) for transbilayer movement of the fluorescent PS was stimulated by cytosolic acidification. A screening method for mutant cells defective in the non-endocytic uptake of fluorescent PS analogs with replica cell colonies at acidic pH was then devised. After selection of mutagenized CHO-K1 cells by in situ screening, we obtained a mutant cell line in which uptake of fluorescent PS analogs was reduced to about 25% of the wild type level at either pH 6.0 or 7.4. Control experiments demonstrated that the reduced uptake of fluorescent PS analogs in the mutant cells was unrelated to multidrug resistance, and that endocytosis of another plasma membrane lipid marker occurred normally in the mutant cells. These results suggested that a non-endocytic pathway responsible for uptake of fluorescent PS analogs was specifically affected in the mutant cells.
Protein phosphatase 1 regulates the cytoplasmic dynein-driven formation of endoplasmic reticulum networks in vitro.
Authors: Allan V
Published: May 2004

Interphase Xenopus egg extracts form extensive tubular membrane networks in vitro. These networks are identified here as endoplasmic reticulum by the presence of ER resident proteins, as shown by immunofluorescence, and by the presence of single ribosomes and polysomes, as shown by electron microscopy. The effect of phosphorylation on ER movement in interphase was tested using the phosphatase inhibitor, okadaic acid. Okadaic acid treatment resulted in an increase of up to 27-fold in the number of ER tubules moving and in the extent of ER networks formed compared to control extracts. This activation was blocked by the broad-specificity kinase inhibitor 6-dimethylaminopurine. Okadaic acid had no effect, however, on the direction of ER tubule movement, which occurred towards the minus end of microtubules, and was sensitive to low concentrations of vanadate. Inhibition of phosphatases also had no effect on the speed or duration of ER tubule extensions, and did not stimulate the activity of soluble cytoplasmic dynein. The sensitivity of ER movement to okadaic acid closely matched that of protein phosphatase 1. Although the amount of ER motility was greatly increased by inhibiting protein phosphatase 1 (PP1), the amount of cytoplasmic dynein associated with the membrane was not altered. The data support a model in which phosphorylation regulates ER movement by controlling the activity of cytoplasmic dynein bound to the ER membrane.
Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.
Authors: Ceccaldi P E , Grohovaz F , Benfenati F , Chieregatti E , Greengard P , Valtorta F
Published: May 2004

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.
Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane.
Authors: Fiedler K , Lafont F , Parton R G , Simons K
Published: May 2004

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.
High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2.
Authors: Hanneken A , Maher P A , Baird A
Published: May 2004

We recently characterized three FGF-binding proteins (FGF-BPs) which are soluble forms of the extracellular domains of the high affinity FGF receptors (Hanneken, A. M., W. Ying, N. Ling, and A. Baird. Proc. Natl. Acad. Sci. USA. 1994. 91:9170-9174). These proteins circulate in blood and have been proposed to modulate the biological activity of the FGF family of proteins. Immunohistochemical studies now demonstrate that these soluble, truncated FGF receptors are also present in the basement membranes of retinal vascular endothelial cells. These immunoreactive proteins can be detected with antibodies raised to the extracellular domain of FGFR-1 but not with antibodies raised to either the juxtamembrane domain or the cytoplasmic domain of FGFR-1. Western blotting of human retinal extracts with the antibody raised to the extracellular domain of FGFR-1 detects specific, low molecular mass proteins at 85 kD and 55 kD, corresponding in size to the FGF-BPs, which are not detected with antibodies against the cytoplasmic domain of the receptor. The interaction of this receptor with the extracellular matrix is not dependent on the presence of FGF-2. Immunoreactive receptors are still detected in vascular basement membranes after the removal of FGF-2 with heparitinase. In addition, the recombinant extracellular domain of FGFR-1 continues to bind to corneal endothelial cell matrix after endogenous FGF-2 has been removed with 2 M NaCl. Acid treatment, which has been shown to disrupt protein interactions with the extracellular matrix, leads to a significant reduction in the presence of the matrix form of the FGF receptor. This loss can be restored with exogenous incubations of the recombinant extracellular domain of FGFR-1. This report is the first demonstration that a truncated form of a high affinity growth factor receptor can be localized to the extracellular matrix. These findings add to the list of binding proteins associated with the extracellular matrix (IGFBP-5) and suggest a potentially new regulatory mechanism for controlling the biological availability of FGF, and other peptide growth factors, in the extracellular matrix.
Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues.
Authors: Taneja K L , McCurrach M , Schalling M , Housman D , Singer R H
Published: May 2004

We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochrome-conjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously hybridized to fluorochrome-conjugated probes to the 5'-end of the Mt-PK mRNA. A single oligonucleotide probe to the repeat and the sense strand each conjugated to different fluorochromes revealed the gene and the transcripts simultaneously, and indicated that these focal concentrations (up to 13 per nucleus) represented predominately posttranscriptional RNA since only a single focus contained both the DNA and the RNA. This concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.
Involvement of pRB family in TGF beta-dependent epithelial cell hypertrophy.
Authors: Franch H A , Shay J W , Alpern R J , Preisig P A
Published: May 2004

Although renal hypertrophy is often associated with the progressive loss of renal function, the mechanism of hypertrophy is poorly understood. In both primary cultures of rabbit proximal tubules and NRK-52E cells (a renal epithelial cell line), transforming growth factor beta 1 (TGF beta) converted epidermal growth factor (EGF)-induced hyperplasia into hypertrophy. TGF beta did not affect EGF-induced increases in c-fos mRNA abundance or cyclin E protein abundance, but inhibited EGF-induced entry into S, G2, and M phases. EGF alone increased the amount of hyperphosphorylated (inactive) pRB; TGF beta blocked EGF-induced pRB phosphorylation, maintaining pRB in the active form. To determine the importance of active pRB in TGF beta-induced hypertrophy, NRK-52E cells were infected with SV40 large T antigen (which inactivates pRB and related proteins and p53), HPV16 E6 (which degrades p53), HPV16 E7 (which binds and inactivates pRB and related proteins), or both HPV16 E6 and E7. In SV40 large T antigen expressing clones, the magnitude of EGF + TGF beta-induced hypertrophy was inhibited and was inversely related to the magnitude of SV40 large T antigen expression. In the HPV16-infected cells, EGF + TGF beta-induced hypertrophy was inhibited in E7- and E6E7-expressing, but not E6-expressing cells. These results suggest a requirement for active pRB in the development of EGF + TGF beta-induced renal epithelial cell hypertrophy. We suggest a model of renal cell hypertrophy mediated by EGF-induced entry into the cell cycle with TGF beta-induced blockade at G1/S, the latter due to maintained activity of pRB or a related protein.
Pmp27 promotes peroxisomal proliferation.
Authors: Marshall P A , Krimkevich Y I , Lark R H , Dyer J M , Veenhuis M , Goodman J M
Published: May 2004

Peroxisomes perform many essential functions in eukaryotic cells. The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes. This process is not understood at the molecular level. Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate. This growth substrate is metabolized by peroxisomal enzymes. We have identified a protein, Pmp27, that promotes peroxisomal proliferation. This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced. Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii. Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer. Its expression is regulated by oleate. The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid. The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type. Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation. However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes. In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes. We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission.
Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments.
Authors: Nakagawa T , Chen J , Zhang Z , Kanai Y , Hirokawa N
Published: May 2004

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross-bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl-terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.
Molecular cloning of chick beta-tectorin, an extracellular matrix molecule of the inner ear.
Authors: Killick R , Legan P K , Malenczak C , Richardson G P
Published: May 2004

The tectorial membrane is an extracellular matrix lying over the apical surface of the auditory epithelium. Immunofluorescence studies have suggested that some proteins of the avian tectorial membrane, the tectorins, may be unique to the inner ear (Killick, R., C. Malenczak, and G. P. Richardson. 1992. Hearing Res. 64:21-38). The cDNA and deduced amino acid sequences for chick beta-tectorin are presented. The cDNA encodes a protein of 36,902.6 D with a putative signal sequence, four potential N-glycosylation sites, 13 cysteines, and a hydrophobic COOH terminus. Western blots of two-dimensional gels using antibodies to a synthetic peptide confirm the identity of the cDNA. Southern and Northern analysis suggests that beta-tectorin is a single-copy gene only expressed in the inner ear. The predicted COOH terminus is similar to that of glycosylphosphatidylinositol-linked proteins, and antisera raised to this region react with in vitro translation products of the cDNA clone but not with mature beta-tectorin. These data suggest beta-tectorin is synthesized as a glycosylphosphatidyl-inositol-linked precursor, targeted to the apical surface of the sensory epithelium by the lipid moiety, and then further processed. Sequence analysis indicates the predicted protein possesses a zona pellucida domain, a sequence that is common to a limited number of other matrix-forming proteins and may be involved in the formation of filaments. In the cochlear duct, beta-tectorin is expressed in the basilar papilla, in the clear cells and the cuboidal cells, as well as in the striolar region of the lagena macula. The expression of beta-tectorin is associated with hair cells that have an apical cell surface specialization known as the 275-kD hair cell antigen restricted to the basal region of the hair bundle, suggesting that matrices containing beta-tectorin are required to drive this hair cell type.