bluuebunny/crossref_metadata_2025 huggingface dataset:
The response to thrombin of human neutrophils: Evidence for two novel receptors
Authors: Jenkins Alison L. , Howells Gareth L. , Scott Emma , Bonniec Bernard F. Le , Curtis Michael A. , Stone Stuart R.
Published: April 2021

Human α-thrombin was a chemoattractant for human neutrophils yielding a maximal response of similar magnitude to that observed with formyl-Met-Leu-Phe. The observed chemotaxis was not due to stimulation of the proteolytically activated thrombin receptor since: (1) this receptor was not detected by flow cytometry; (2) the inactive thrombin mutant Ser195→Ala elicited a chemotactic response indistinguishable from that caused by wild-type thrombin; (3) antibodies to the cleavage site of the proteolytically activated receptor did not affect thrombin-induced chemotaxis; (4) a thrombin receptor activating peptide (TRAP) failed to stimulate chemotaxis. These data indicate the existence of a thrombin receptor for neutrophil chemotaxis which is not activated by proteolysis. In addition, although wild-type and Ser195→Ala thrombin did not cause an increase in intracellular Ca2+, a Ca2+ response to TRAP was observed with neutrophils from some donors. The TRAP-induced increase in Ca2+ was reproducible, dose dependent and specific. The use of alanine-substituted peptides demonstrated that the Ca2+ response was due to TRAP stimulation of a receptor other than the proteolytically activated thrombin receptor. Thus, it is necessary to re-evaluate the assumption made in previous studies that responses to TRAP are mediated by the proteolytically activated thrombin receptor.
Distinct heparin-binding and neurite-promoting properties of laminin isoforms isolated from chick heart
Authors: Brandenberger Ralph , Chiquet Matthias
Published: April 2021

Laminin isolated from chick heart is composed of several heterotrimeric variants of 800 and 700 kDa. Here, we used monoclonal antibodies against chick laminin to purify different laminin isoforms from this mixture. Antibody 8D3 specifically removed laminin containing α2 chain from chick heart laminin preparations, leaving behind 700 kDa variants. Using antibody C4 against the laminin β2 chain, α2 chain containing variants were further separated into α2β1γ1 and α2β2γ1 laminin, respectively. Laminins containing α2 chain and recognized by antibody 8D3 are crossshaped molecules. Their expression during embryogenesis is tightly regulated. In 5-day embryos staining with monoclonal antibody 8D3 is restricted to the dermamyotome. Older embryos (8 days) express α2 chain containing variants at myotendinous junction primordia of skeletal muscle, and only late in development these variants are generally expressed in skeletal and heart muscle basement membranes. The 700 kDa laminin variants contain β1, β2, and γ1 subunits affiliated with an immunologically distinct, shorter ax chain and appear to be T-shaped in the electron microscope. Whereas laminins with an α2 subunit bind to heparin, variants with the novel ax chain do not. Experiments using cultured sympathetic neurons showed that laminins with ax chain are less potent than α2 chain containing variants in promoting neurite outgrowth. In contrast, sympathetic neurons cannot discriminate between α2β1γ1 and α2β2γ1 laminin substrates, respectively, and show identical high rates of neurite formation.
CD44 exhibits a cell type dependent interaction with Triton X-100 insoluble, lipid rich, plasma membrane domains
Authors: Neame Stephen J. , Uff Caroline R. , Sheikh Humma , Wheatley Susan C. , Isacke Clare M.
Published: April 2021

CD44 is an abundant, widely expressed transmembrane glycoprotein which can act as a receptor for the extracellular matrix glycosaminoglycan, hyaluronan. Biochemical and morphological studies have demonstrated that in fibroblasts a significant proportion of the CD44 population is resistant to Triton X-100 extraction and that the detergent insoluble protein is co-localized with components of the cortical cytoskeleton. Surprisingly, this distribution is not abrogated upon deletion of the CD44 cytoplasmic tail indicating that mechanisms other than a direct interaction with the cytoskeleton can regulate CD44. In this manuscript, the mechanisms underlying this detergent-insoluble association are further investigated. There was no evidence that the Triton X-100 insolubility of CD44 resulted from homotypic aggregation, an association with hyaluronan or from a direct, or indirect, association with the cytoskeleton. Instead, evidence is presented that the detergent insolubility of fibroblast CD44 at 4°C results from an association of the CD44 transmembrane domain with Triton X-100 resistant, lipid rich, plasma membrane domains. The proportion of the CD44 found in these Triton X-100 insoluble structures is dependent upon cell type and cannot be altered by changing cell motility or extracellular matrix associations. These studies provide evidence for a novel mechanism regulating this adhesion protein in the plasma membrane.
Intranuclear retention of ribosomal RNAs in response to herpes simplex virus type 1 infection
Authors: Besse Sylvie , Puvion-Dutilleul Francine
Published: April 2021

The localization of ribosomal RNA (rRNA) was investigated at the ultrastructural level in herpes simplex virus type 1 infected HeLa cells using three distinct biotinylated probes which bind in sequence to three different segments of the ribosomal genes. Comparison of the above with the signal levels obtained from non-infected cells reveals information about the effects of HSV-1 infection on ribosome biogenesis. A probe specific for the 5′end portion of prerRNA labeled all nucleoli of both non-infected and infected cells in the same way, that is, it mainly labeled the dense fibrillar component and the border of the fibrillar centers but only slightly labeled the granular component. This indicates that the initial cleavage of pre-rRNA in herpes infection still occurs in the 5′ region of the 5′ external transcribed spacer. However, a probe specific for 18 S rRNA labeled the granular component of the nucleoli more intensely after infection. In addition, significant amounts of rRNA molecules were present within the intranuclear viral region, except over the enclosed viral dense bodies, and within the virus-enlarged clusters of interchromatin granules. The data indicate that the still enigmatic viral dense bodies, which are nucleolus-related structures, are excluded from the marked intranuclear retention of ribosomal RNAs and, in addition, reveal a possible role for the interchromatin granules of infected cells in the regulation of the export of the ribosomal subunits towards the cytoplasm.
Localization of Gαo to growth cones in PC12 cells: role of Gαo association with receptors and Gβγ
Authors: Nüße Oliver , Neer Eva J.
Published: April 2021

The heterotrimeric G protein Go is highly enriched in the growth cones of neuronal cells and makes up 10% of the membrane protein of growth cones from neonatal rat brain. We have used PC12 cells, a cell line that differentiates to a neuron-like phenotype, as a model with which to study the mechanism of G protein localization. First, the role of the βγ-subunit was investigated. The attachment of the βγ-subunit to the membrane depends on the isoprenylation of the γ-subunit. The drug lovastatin blocks isoprenylation by inhibiting a key enzyme in the biosynthetic pathway. After treatment of PC12 cells with 10 μM lovastatin for 48 hours 50% of the βγ-subunits were cytosolic compared with 100% membrane bound βγ in control cells, as determined by cell fractionation, gel electrophoresis and western blot. Addition of 200 μM mevalonic acid reverses this effect. However, lovastatin affects neither the membrane attachment of αo nor its localization to the growth cones as determined by immunohistochemistry. This suggests that the localization and retention of αo are independent of the membrane attachment of the full complement of βγ-subunits. Second, pertussis toxin was used to block the interaction between αo and receptors. PC12 cells were treated with 0.1 μg/ml pertussis toxin prior to and during nerve growth factor-induced differentiation. In vitro [32P]ADP-ribosylation confirmed that αo and αi were completely ADP-ribosylated by this treatment. The ADP-ribosylation by pertussis toxin did not interfere with neurite outgrowth. The localization of αo to the growth cones was indistinguishable from that in untreated cells. We conclude that G protein-receptor interaction is not necessary for the distribution of αo to growth cones.
The gelsolin-related flightless I protein is required for actin distribution during cellularisation in Drosophila
Authors: Straub Kristina L. , Stella Maria Cristina , Leptin Maria
Published: April 2021

We have analysed the developmental defects in Drosophila embryos lacking a gelsolin-related protein encoded by the gene flightless I. Such embryos have previously been reported to gastrulate abnormally. We now show that the most dramatic defects are seen earlier, in actin-dependent events during cellularisation of the syncytial blastoderm, a process with similarities to cytokinesis. The blastoderm nuclei migrate to the periphery of the egg normally but lose their precise cortical positioning during cellularisation. Cleavage membranes are initially formed, but invaginate irregularly and often fail to close at the basal end of the newly formed cells. The association of actin with the cellularisation membranes is irregular, suggesting a role for flightless I in the delivery of actin to the actin network, or in its stabilisation.
Characterization of VIP36, an animal lectin homologous to leguminous lectins
Authors: Fiedler Klaus , Simons Kai
Published: April 2021

VIP36 was isolated from MDCK cells as a component of glycolipid-enriched detergent-insoluble complexes. The protein is localized to the Golgi apparatus and the cell surface, and belongs to a new family of legume lectin homologues in the animal secretory pathway that might be involved in the trafficking of glycoproteins, glycolipids or both. Here we show that VIP36 is N-glycosylated and expressed in organs abundant in epithelial cells as well as in non-epithelial organs. Our studies demonstrate that the recombinant exoplasmic/luminal domain of VIP36 binds Ca2+ and that the protein decorates internal membrane structures of MDCK cells in vitro that are distinct from the Golgi apparatus. This binding requires Ca2+ and can be specifically inhibited by N-acetyl-D-galactosamine. The recombinant protein was used for affinity chromatography. Glycopeptides obtained from [3H]galactose-labelled cells bind to VIP36 and can be eluted with N-acetyl-D-galactosamine. Our data imply that VIP36 functions as a lectin in post-Golgi trafficking.
Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd-GFP fusion proteins
Authors: Endow Sharyn A. , Komma Donald J.
Published: April 2021

The Ncd microtubule motor protein is required for meiotic and early mitotic chromosome distribution in Drosophila. Null mutant females expressing the Ncd motor fused to the Aequorea victoria green fluorescent protein (GFP), regulated by the wild-type ncd promoter, are rescued for chromosome segregation and embryo viability. Analysis of mitosis in live embryos shows cell cycle-dependent localization of Ncd-GFP to centrosomes and spindles. The distribution of Ncd-GFP in spindles during metaphase differs strikingly from that of tubulin: the tubulin staining is excluded by the chromosomes at the metaphase plate; in contrast, Ncd-GFP forms filaments along the spindle microtubules that extend across the chromosomes. The existence of Ncd-GFP fibers that cross the metaphase plate suggests that Ncd interacts functionally with chromosomes in metaphase. Differences are no longer observed in anaphase when the chromosomes have moved off the metaphase plate. A mutant form of Ncd fused to GFP also localizes to spindles in live embryos. Mutant embryos show frequent centrosome and spindle abnormalities, including free centrosomes that dissociate from interphase nuclei, precociously split centrosomes, and spindles with microtubule spurs or bridges to nearby spindles. The precociously split and free centrosomes indicate that the Ncd motor acts in cleavage stage embryos to maintain centrosome integrity and attachment to nuclei. The frequent spindle spurs of mutant embryos are associated with mis-segregating chromosomes that partially detach from the spindle in metaphase, but can be recaptured in early anaphase. This implies that the Ncd motor functions to prevent chromosome loss by maintaining chromosome attachment to the spindle in metaphase, consistent with the Ncd-GFP fibers that cross the metaphase plate.
Depletion of lysophosphatidic acid triggers a loss of oriented detyrosinated microtubules in motile fibroblasts
Authors: Nagasaki T. , Gundersen G. G.
Published: April 2021

We reported earlier that isolated plasma membranes trigger a number of responses comprising contact inhibition of motility, including loss of oriented detyrosinated microtubules (Glu MTs) from the lamella of motile fibroblasts. In this study, we show that the membranes trigger this loss of Glu MTs, not by binding to cells, but by removing an essential component from the medium necessary to maintain oriented Glu MTs. Preincubation of membranes with medium containing serum followed by removal of the membranes by sedimentation rendered the membrane-treated medium capable of triggering the loss of oriented Glu MTs. Membrane activity was inhibited by high concentrations of serum and removal of serum from medium triggered the loss of oriented Glu MTs similar to that triggered by membranes. These results suggest that the membranes trigger the loss of Glu MTs by inactivating factors in serum that are required for the maintenance of oriented Glu MTs. By fractionating serum, we have identified lysophosphatidic acid (LPA) as the principal serum factor that is responsible for supporting oriented Glu MTs. The activity of LPA to maintain oriented Glu MTs upon serum withdrawal was half maximal at 100 nM and no activity was observed with structurally related phospholipids. Serum LPA levels were sufficient to account for the ability of serum to support oriented Glu MTs. Enzymatic degradation of serum LPA strongly reduced the ability of serum to support oriented Glu MTs. That membranes degrade LPA was shown by the ability of membranes to block LPA’s ability to maintain oriented Glu MTs, and by direct measurement of the loss of radiolabeled LPA after incubation with membranes in vitro. These results show that isolated plasma membranes trigger the loss of Glu MTs from the lamella of motile cells by degrading serum LPA. Coupled with earlier results showing that membranes trigger a number of contact inhibition responses, our data suggest a new model for contact inhibition of motility in which local degradation of LPA and/or interference with LPA-stimulated signalling pathways initiates a contact inhibition response in colliding cells.
Isolation and characterization of SYS genes from yeast, multicopy suppressors of the functional loss of the transport GTPase Ypt6p
Authors: Tsukada Miki , Gallwitz Dieter
Published: April 2021

In Saccharomyces cerevisiae, the YPT6 gene encodes the homologue of the mammalian Rab6 protein found in the Golgi apparatus. Deletion of YPT6 in yeast produces a phenotype showing temperature-sensitive growth and partial missorting of the vacuolar enzyme, carboxypeptidase Y. To identify proteins that might: (1) interact with Ypt6p; or (2) act in the same pathway, we have isolated four multicopy suppressors, named SYS1, SYS2, SYS3 and SYS5, that can complement the temperature-sensitive growth phenotype of the ypt6 null mutant. On high expression, these genes are also able to partially suppress the missorting of carboxypeptidase Y. SYS2 on a multicopy plasmid suppresses in addition the temperature-sensitive phenotype of sec7-1, a mutant defective in transport between and from the Golgi compartment. Gene disruption of SYS1 and SYS2 did not result in significant growth defects. However, deletion of SYS1 and/or SYS2 in the ypt6 null mutant enhances defects in vacuolar protein sorting and in cell growth. Whereas protein secretion was not significantly affected in these mutants, the processing of α-factor precursor by the Kex2 protease was inhibited, suggesting a function of YPT6 and its null mutant suppressors in transport between the late Golgi and a prevacuolar, endosome-like compartment.
The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development
Authors: Rivero Francisco , Köppel Bernd , Peracino Barbara , Bozzaro Salvatore , Siegert Florian , Weijer Cornelis J. , Schleicher Michael , Albrecht Richard , Noegel Angelika A.
Published: April 2021

We generated Dictyostelium double mutants lacking the two F-actin crosslinking proteins α-actinin and gelation factor by inactivating the corresponding genes via homologous recombination. Here we investigated the consequences of these deficiencies both at the single cell level and at the multicellular stage. We found that loss of both proteins severely affected growth of the mutant cells in shaking suspension, and led to a reduction of cell size from 12 μm in wild-type cells to 9 μm in mutant cells. Moreover the cells did not exhibit the typical polarized morphology of aggregating Dictyostelium cells but had a more rounded cell shape, and also exhibited an increased sensitivity towards osmotic shock and a reduced rate of phagocytosis. Development was heavily impaired and never resulted in the formation of fruiting bodies. Expression of developmentally regulated genes and the final developmental stages that were reached varied, however, with the substrata on which the cells were deposited. On phosphate buffered agar plates the cells were able to form tight aggregates and mounds and to express prespore and prestalk cell specific genes. Under these conditions the cells could perform chemotactic signalling and cell behavior was normal at the onset of multicellular development as revealed by time-lapse video microscopy. Double mutant cells were motile but speed was reduced by approximately 30% as compared to wild type. These changes were reversed by expressing the gelation factor in the mutant cells. We conclude that the actin assemblies that are formed and/or stabilized by both F-actin crosslinking proteins have a protective function during osmotic stress and are essential for proper cell shape and motility.
Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site
Authors: Hemmings L. , Rees D. J. G. , Ohanian V. , Bolton S. J. , Gilmore A. P. , Patel B. , Priddle H. , Trevithick J. E. , Hynes R. O. , Critchley D. R.
Published: April 2021

We have determined the sequence of chicken talin (2,541 amino acids, Mr 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculinbinding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.
Dynamic properties of nucleated microtubules: GTP utilisation in the sub-critical concentration regime
Authors: Symmons Martyn F. , Martin Stephen R. , Bayley Peter M.
Published: April 2021

Microtubule assembly kinetics have been studied quantitatively under solution conditions supporting microtubule dynamic instability. Purified GTP-tubulin (Tu-GTP) and covalently cross-linked short microtubule seeds (EGSseeds; Koshland et al. (1988)Nature 331, 499) were used with and without biotinylation. Under sub-critical concentration conditions ([Tu-GTP]<5.3 μM), significant microtubule growth of limited length was observed on a proportion of the EGS-seeds by immuno-electron microscopy. A sensitive fluorescence assay for microtubule GDP production was developed for parallel assessment of GTP utilisation. This revealed a correlation between the detected microtubule growth and the production of tubulin-GDP, deriving from the shortening phase of the dynamic microtubules. This correlation was confirmed by the action of nocodazole, a specific inhibitor of microtubule assembly, that was found to abolish the GDP release. The variation of the GDP release with tubulin concentration (Jh(c) plot) was determined below the critical concentration (CC). The GDP production observed was consistent with the elongation of the observed seeded microtubules with an apparent rate constant of 1.5×106M−1 second−1 above a threshold of approximately 1 μM tubulin. The form of this Jh(c) plot for elongation below CC is reproduced by the Lateral Cap model for microtubule dynamic instability adapted for seeded assembly. The behaviour of the system is contrasted with that previously studied in the absence of detectable microtubule elongation (Caplow and Shanks (1990)J. Biol. Chem. 265, 8935-8941). The approach provides a means of monitoring microtubule dynamics at concentrations inaccessible to optical microscopy, and shows that essentially the same dynamic mechanisms apply at all concentrations. Numerical simulation of the subcritical concentration regime shows dynamic growth features applicable to the initiation of microtubule growth in vivo.
Assembly of ring canals in the male germ line from structural components of the contractile ring
Authors: Hime Gary R. , Brill Julie A. , Fuller Margaret T.
Published: April 2021

Stable intercellular bridges called ring canals form following incomplete cytokinesis, and interconnect mitotically or meiotically related germ cells. We show that ring canals in Drosophila melanogaster males are surprisingly different from those previously described in females. Mature ring canal walls in males lack actin and appear to derive directly from structural proteins associated with the contractile ring. Ring canal assembly in males, as in females, initiates during cytokinesis with the appearance of a ring of phosphotyrosine epitopes at the site of the contractile ring. Following constriction, actin and myosin II disappear. However, at least four proteins present at the contractile ring remain: the three septins (Pnut, Sep1 and Sep2) and anillin. In sharp contrast, in ovarian ring canals, septins have not been detected, anillin is lost from mature ring canals and filamentous actin is a major component. In both males and females, a highly branched vesicular structure, termed the fusome, interconnects developing germ cells via the ring canals and is thought to coordinate mitotic germ cell divisions. We show that, in males, unlike females, the fusome persists and enlarges following cessation of the mitotic divisions, developing additional branches during meiosis. During differentiation, the fusome and its associated ring canals localize to the distal tip of the elongating spermatids.
The effects of cytochalasin D and phorbol myristate acetate on the apical endocytosis of ricin in polarised Caco-2 cells
Authors: Shurety Wenda , Bright Nicholas A. , Luzio J. Paul
Published: April 2021

Apical endocytosis of 125I-ricin in Caco-2 cells was inhibited >95% by hypertonic and/or acid media, consistent with the major uptake route being clathrin-mediated. The presence of apical cell surface bound ricin-gold in clathrin coated pits and vesicles was observed by electron microscopy. An electron microscopic investigation in which ricin-gold bound to the apical surface was quantitated, showed that cytochalasin D, which inhibits apical but not basolateral endocytosis, prevented movement of ricin-gold along the microvillar surface. This was consistent with an actin bound mechanochemical motor within microvilli driving the movement of membranous components towards the cell body. Cytochalasin D also caused an increase in the number of coated pits observed at the apical cell surface relative to the number observed in untreated cells. Stimulation of apical endocytosis of ricin by phorbol 12-myristate 13-acetate showed the characteristics of being mediated by protein kinase C, was not due to an effect on ricin movement along the microvillar surface, and may be explained by increases in formation and pinching off of clathrin coated pits at the apical cell surface.
GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with the TGN
Authors: Ralston Evelyn , Ploug Thorkil
Published: April 2021

There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex (α mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated in the core of the myotubes. It is primarily found around the nuclei, in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy. GLUT4 appears to be in the trans-most cisternae of the Golgi complex and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor, which forms long tubules. In untreated cells, double staining for GLUT4 and transferrin receptor by immunofluorescence shows similar but distinct patterns. Immunoelectron microscopy localizes transferrin receptor, detected by immunoperoxidase, to large vesicles, presumably endosomes, very close to the GLUT4-containing tubulovesicular elements. In brefeldin A-treated cells, a network of tubules of ∼70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing compartments. The results suggest that GLUT4 storage vesicles constitute a specialized compartment that is either a subset of the TGN, or is very closely linked to it. The link between GLUT4 vesicles and transferrin receptor containing endosomes, as revealed by brefeldin A, may be important for GLUT4 translocation in response to muscle stimulation.
Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes
Authors: Zhu Alan Jian , Watt Fiona M.
Published: April 2021

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with α-, β- and γ-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of β-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the α2β1 and α3β1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the β1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and-independent mechanisms.
The polarity of the plasma membrane protein RET-PE2 in retinal pigment epithelium is developmentally regulated
Authors: Marmorstein Alan D. , Bonilha Vera L. , Chiflet Silvia , Neill James M. , Rodriguez-Boulan Enrique
Published: April 2021

The retinal pigment epithelium (RPE) differs from other epithelia in that the apical surface is not free; instead, it interacts with both photoreceptors and a specialized extracellular material, the interphotoreceptor matrix. Biochemical characterization of the apical and basolateral surfaces of RPE in adult rat eye cups, using a novel in situ biotinylation assay, revealed very different protein compositions and identified a major surface antigen, RET-PE2, with a predominantly apical distribution (∼74%). The apical polarity of RET-PE2 was confirmed by immunofluorescence and laser scanning confocal microscopy. In striking contrast, RET-PE2 antigen was preferentially basolateral in primary cultures derived from adult rat RPE and in an immortalized RPE cell line (RPE-J). Under all conditions, RET-PE2 was highly soluble in Triton X-100 (>81% at 4°C), suggesting that its redistribution was not dependent on changes in cytoskeletal interactions. Analysis of the localization of RET-PE2 in normal rats at postnatal (PN) days 1, 7, and 14 indicated that RET-PE2 redistributes from predominantly basolateral to predominantly apical during that time. Since photoreceptors develop during the first two weeks after birth in the rat, our results suggest that the apical redistribution of RET-PE2 is dependent on the establishment of adult interactions between the RPE and the neural retina and/or the interphotoreceptor matrix, either via direct contacts or through alterations in the intracellular sorting patterns of RPE cells.
A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1
Authors: Wilson D. J. A. , Kim D.-S. , Clarke G. A. , Marshall-Clarke S. , Moss D. J.
Published: April 2021

We have previously identified a glycosylphosphatidylinosi-tol-linked glycoprotein of 55 kDa (Gp55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e. block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substratebound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a recently discovered chick protein CEPU-1. Our results suggest molecules within this family are capable of acting as cell adhesion molecules and inhibitors of neurite outgrowth.
Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor
Authors: Yao Chung-Chen , Ziober Barry L. , Sutherland Ann E. , Mendrick Donna L. , Kramer Randall H.
Published: April 2021

The α7β1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of α7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the α7 integrin. Overall expression of α7 increased as the C2C12 myoblasts differentiated; myoblasts expressed only the α7B cytoplasmic variant whereas in differentiating myotubes α7A increased markedly. Function-perturbing monoclonal antibodies generated to α7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse myoblasts on laminin 1. Other studies with MM14 myoblasts showed that α7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking antibody to α7 only partially inhibited adhesion to laminin 2/4 but almost completely blocked motility on this substrate. Finally, to assess the potential role of the α7 cytoplasmic domain, CHO cells were stably transfected to express chimeric α5 cDNA constructs containing the wildtype α5 or the α7A or α7B cytoplasmic domain; all forms of the integrin showed identical activities for adhesion, migration, proliferation, and matrix assembly on fibronectin substrates. These results established that α7β1 receptor can promote myoblast adhesion and motility on a restricted number of laminin isoforms and may be important in myogenic precursor recruitment during regeneration and differentiation.
Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase
Authors: Jackson Trevor R. , Blader Ira J. , Hammonds-Odie Latanya P. , Burga Christina R. , Cooke Frank , Hawkins Phillip T. , Wolf Andrea G. , Heldman Katherine A. , Theibert Anne B.
Published: April 2021

Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.
Fission yeast Nda1 and Nda4, MCM homologs required for DNA replication, are constitutive nuclear proteins
Authors: Okishio Nobuyuki , Adachi Yasuhisa , Yanagida Mitsuhiro
Published: April 2021

The nda1+ and nda4+ genes of the fission yeast Schizosaccharomyces pombe encode proteins similar to budding yeast MCM2 and MCM5/CDC46, respectively, which are required for the early stages of DNA replication. The budding yeast Mcm proteins display cell-cycle dependent localization. They are present in the nucleus specifically from late M phase until the beginning of S phase, so that they were suggested to be components of a replication licensing factor, a positive factor for the onset of replication, which is thought to be inactivated after use, thus restricting replication to only once in a cell cycle. In the present study, we raised antibodies against Nda1 or Nda4 and identified 115 kDa and 80 kDa proteins, respectively. Their immunolocalization was examined in wild-type cells and in various cell-cycle mutants. Both Nda1 and Nda4 proteins remained primarily in the nucleus throughout the cell cycle. In mutants arrested in Gl, S, and G2 phases, these proteins were also enriched in the nucleus. These results indicate that the dramatic change in subcellular localization as seen in budding yeast is not essential in fission yeast for the functions of Nda1 and Nda4 proteins to be executed. The histidine-tagged nda1+ gene was constructed and integrated into the chromosome to replace the wild-type nda1+ gene. The resulting His-tagged Nda1 protein was adsorbed to the Ni-affinity column, and co-eluted with the untagged Nda4 protein, suggesting that they formed a complex.
Mechanical stimulation initiates cell-to-cell calcium signaling in ovine lens epithelial cells
Authors: Churchill Grant C. , Atkinson Michael M. , Louis Charles F.
Published: April 2021

Although abnormalities in calcium regulation have been implicated in the development of most forms of cataract, the mechanisms by which Ca2+ is regulated in the cells of the ocular lens remain poorly defined. Cell-to-cell Ca2+ signaling was investigated in primary cultures of ovine epithelial cells using the Ca2+-reporter dye fura-2 and fluorescence microscopy. Mechanical stimulation of a single cell with a micropipette initiated a propagated increase in cytosolic free Ca2+ that spread from the stimulated cell through 2-8 tiers of surrounding cells. During this intercellular Ca2+ wave, cytosolic Ca2+ increased 2-to 12-fold from resting levels of ∼100 nM. Nanomolar extracellular Ca2+ did not affect the cell-to-cell propagation of the Ca2+ wave, but reduced the magnitude of the cytosolic Ca2+ increases, which was most evident in the mechanicallystimulated cell. Depletion of intracellular Ca2+ stores with thapsigargin eliminated the propagated intercellular Ca2+ wave, but did not prevent the cytosolic Ca2+ increase in the mechanically-stimulated cell, which required extracellular Ca2+ and was attenuated by the addition of the Ca2+channel blockers Ni2+, Gd3+ and La3+ to the medium. These results are most easily explained by a mechanicallyactivated channel in the plasma membrane of the stimulated cell. The propagated increase in cytosolic Ca2+ appeared to be communicated to adjacent cells by the passage of an intracellular messenger other than Ca2+ through gap junction channels. However, if the plasma membrane of the mechanically-stimulated cell was ruptured such that there was loss of cytosolic contents, the increase in cytosolic Ca2+ in the surrounding cells was elicited by both a messenger passing through gap junction channels and by a cytosolic factor(s) diffusing through the extracellular medium. These results demonstrate the existence of intercellular Ca2+ signaling in lens cells, which may play a role in regulating cytosolic Ca2+ in the intact lens.
CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium
Authors: Ryeom Sandra W. , Sparrow Janet R. , Silverstein Roy L.
Published: April 2021

Mechanisms of phagocytosis are complex and incompletely understood. The retinal pigment epithelium provides an ideal system to study the specific aspects of phagocytosis since an important function of this cell is the ingestion of packets of membranous discs that are normally discarded at the apical ends of rod and cone cells during outer segment renewal. Here we provide evidence that rod outer segment phagocytosis by retinal pigment epithelium is mediated by CD36, a transmembrane glycoprotein which has been previously characterized on hematopoietic cells as a receptor for apoptotic neutrophils and oxidized low density lipoprotein.

Immunocytochemical staining with monoclonal and polyclonal antibodies demonstrated CD36 expression by both human and rat retinal pigment epithelium in transverse cryostat sections of normal retina and in primary cultured cells. By western blot analysis of retinal pigment epithelial cell lysates, polyclonal and monoclonal antibodies to CD36 recognized an 88 kDa protein which comigrated with platelet CD36. Furthermore, the synthesis of CD36 mRNA by retinal pigment epithelium was confirmed by reverse transcriptase-PCR using specific CD36 oligonucleotides. The addition of CD36 antibodies to cultured retinal pigment epithelial cells reduced the binding and internalization of 125I-labeled rod outer segments by 60%. Immunofluorescence confocal microscopy confirmed that outer segment uptake was significantly diminished by an antibody to CD36.

Moreover, we found that transfection of a human melanoma cell line with CD36 cDNA enabled these cells to bind and internalize isolated photoreceptor outer segments as seen by double immunofluorescent staining for surface bound and total cell-associated rod outer segments, and by measurement of cell-associated 125I-labeled rod outer segments.

We conclude that the multifunctional scavenger receptor CD36 participates in the clearance of photoreceptor outer segments by retinal pigment epithelium and thus, participates in the visual process.
Nuclear architecture and ultrastructural distribution of poly(ADP-ribosyl)transferase, a multifunctional enzyme
Authors: Mosgoeller Wilhelm , Steiner Marianne , Hozák Pavel , Penner Edward , Węsierska-Gądek Józefa
Published: April 2021

A monospecific autoimmune serum for poly(ADP-ribosyl)transferase (pADPRT) was used to localise the enzyme in ultrastructural cellular compartments. We detected enzyme in mitochondria of HeLa and Sertoli cells. Within the nucleoplasm the enzyme concentration was positively correlated with the degree of chromatin condensation, with interchromatin spaces being virtually free of pADPRT. During spermatogenesis we observed a gradual increase of the chromatin associated pADPRT that parallelled chromatin condensation. The highest concentration was seen in the late stages of sperm differentiation, indicating the existence of a storage form in transcriptionally inactive nuclei.

In nucleoli pADPRT is accumulated in foci within the dense fibrillar component. Such foci are seen in close spatial relationship to sites of nucleolar transcription as revealed by high resolution immunodetection of bromouridine uptake sites. It is suggested that nucleolar pADPRT plays a role in preribosome processing via the modification of nucleolus specific proteins that bind to nascent transcripts and hence indirectly regulates polymerase I activity. The persisting binding of pADPRT to ribonucleoproteins may explain the observed disperse enzyme distribution at lower concentrations in the granular component. The fibrillar centres seem to contain no pADPRT. We conclude that known compounds of fibrillar centres like polymerase I are unlikely candidates for modification via direct covalent ADP-ribosylation.
Flexural rigidity of microtubules measured with the use of optical tweezers
Authors: Felgner Harald , Frank Rainer , Schliwa Manfred
Published: April 2021

The flexural rigidity of single microtubules is measured using optical tweezers. Two new methods are presented. In both the optical forces of the laser trap are used to directly manipulate microtubules grown off the ends of Chlamydomonas axonemes. The shapes of the microtubules are observed by video microscopy as the hydrodynamic forces of viscous flow counteract the elastic restoring forces when the microtubules are moved actively relative to the surrounding buffer medium. To determine the flexural rigidity, the bending of a microtubule is analyzed under a given velocity distribution along its length. Microtubules incubated with taxol after polymerization are measured to be more flexible than those without taxol added. On the other hand, MAPs are shown to increase microtubule stiffness.
In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae
Authors: Hauser Remy , Pypaert Marc , Häusler Thomas , Horn Elke K. , Schneider André
Published: April 2021

In eukaryotic evolution, the earliest branch of organisms to have mitochondria are the trypanosomatids. Their mitochondrial biogenesis not only includes import of most proteins, but also, unlike in other organisms, import of the whole set of tRNAs. In order to investigate these processes, we devised novel procedures for the isolation of mitochondria from two trypanosomatid species: Trypanosoma brucei and Leishmania tarentolae. Isotonic cell lysis followed by equilibrium density centrifugation in Nycodenz gradients yielded mitochondrial fractions exhibiting a membrane potential. Furthermore, we have used these fractions to reconstitute import of mitochondrial matrix proteins in vitro. Energy-dependent uptake of an artificial precursor protein, containing a trypanosomal presequence attached to mouse dihydrofolate reductase and of yeast mitochondrial alcohol dehydrogenase could be demonstrated. The presequences of both proteins were processed in T. brucei whereas only the trypanosomal one was cleaved in L. tarentolae. Trypsin pretreatment abolished the ability of the mitochondria to import proteins, indicating the involvement of proteinaceous components at the surface of mitochondria.