bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substratum-dependent stimulation of fibroblast migration by the gelatin-binding domain of fibronectin
Authors: Schor S. L. , Ellis I. , Dolman C. , Banyard J. , Humphries M. J. , Mosher D. F. , Grey A. M. , Mould A. P. , Sottile J. , Schor A. M. 
Published: April 2021

ABSTRACT
Nanomolar concentrations of native fibronectin and its RGDS-containing cell-binding domain have previously been reported to stimulate fibroblast migration in the transmembrane (or ‘Boyden chamber’) assay; in contrast, the gelatin-binding domain (GBD) of fibronectin has consistently been reported to be devoid of migration-stimulating activity in this assay. We have examined the effects of fibronectin and several of its purified functional domains on the migration of human skin fibroblasts in what is presumably a more physiologically relevant assay involving the movement of cells into a 3-D matrix of native type I collagen fibrils. We report that: (a) femtomolar concentrations of GBD stimulate fibroblast migration into such collagen matrices; and (b) fibronectin, as well as peptides containing all other of its functional domains, do not exhibit migration-stimulating activity when tested in the femtomolar to nanomolar concentration range (i.e. 0.1 pg/ml to 1 µg/ml). The correct assignment of migrationstimulating activity to GBD, rather than to a contaminant, was confirmed by: (a) the use of several fibronectin and GBD purification protocols; (b) the neutralization of GBD migration-stimulating activity by monoclonal antibodies directed against epitopes present in this domain; (c) the time-dependent generation of migration-stimulating activity by the proteolytic degradation of native fibronectin; and (d) obtaining an identical dose-response curve with a genetically engineered GBD peptide. The cryptic migration-stimulating activity of GBD was not affected by the presence of serum or native fibronectin, but was inhibited by TGF-β1. Parallel experiments using the transmembrane assay confirmed that GBD was devoid of migration-stimulating activity in this assay when membranes coated with gelatin were used, but revealed that significant stimulation of migration was achieved with membranes coated with native type I collagen. Cells preincubated with GBD for 24 hours whilst growing on plastic tissue culture dishes and then plated onto native collagen matrices in the absence of further GBD also displayed an elevated migration compared to controls. Taken together, these observations suggest that: (a) the interaction of GBD with a putative cell surface receptor (and not the collagen substratum) initiates a persistent alteration in cell phenotype which is manifest by an increase in migratory activity when these cells are cultured on a native collagen substratum; and (b) GBD may play a hitherto unrecognised role in the control of cell migration in response to the local release of proteases during pathological processes, such as tumour invasion and wound repair.
A maternal requirement for glutamine synthetase I for the mitotic cycles of syncytial Drosophila embryos
Authors: Frenz Lisa M. , Glover David M. 
Published: April 2021

ABSTRACT
We describe the maternal effect phenotype of a hypomorphic mutation in the Drosophila gene for glutamine synthetase I (GSI). The extent of development of embryos derived from homozygous mutant females is variable, although most mutant embryos fail to survive past germband elongation and none develop into larvae. These embryos are characterised by an increase in the number of yolk-like nuclei following nuclear migration to the cortex. These nuclei appear to fall into the interior of the embryo from the cortex at blastoderm. As they do so, the majority continue to show association with PCNA in synchrony with nuclei at the cortex, suggesting some continuity of the synchrony of DNA replication. However, the occurrence of nuclei that have lost cell cycle synchrony with their neighbours is not uncommon. Immunostaining of mutant embryos revealed a range of mitotic defects, ultimately resulting in nuclear fusion events, division failure or other mitotic abnormalities. A high proportion of these mitotic figures show chromatin bridging at anaphase and telophase consistent with progression through mitosis in the presence of incompletely replicated DNA. GSI is responsible for the ATP-dependent amination of glutamate to produce glutamine, which is required in the formation of amino acids, purines and pyrimidines. We discuss how the loss of glutamine could depress both protein and DNA synthesis and lead to a variety of mitotic defects in this embryonic system that lacks certain checkpoint controls.
Intracellular free calcium responses during chemotaxis of Dictyostelium cells
Authors: Yumura Shigehiko , Furuya Kishio , Takeuchi Ikuo 
Published: April 2021

ABSTRACT
A calcium ion indicator, fura-2 bovine serum albumin, was introduced into Dictyostelium discoideum cells by electroporation. The concentration of intracellular calcium ions ([Ca2+]i) increased transiently in vegetative cells upon stimulation with submicromolar concentrations of folic acid, a chemoattractant for this organism at the vegetative stage. Similar [Ca2+]i responses were also observed in aggregation-competent cells upon stimulation with subnanomolar concentrations of cAMP, a chemoattractant at the aggregation stage. The [Ca2+]i response caused by cAMP was 2.1 times higher than that caused by folic acid. The magnitude of these responses depended on the concentration of Ca2+ in the external buffer. The presence of magnesium ions inhibited the [Ca2+]i responses in a dose-dependent manner. [Ca2+]i was higher in the rear region than in the anterior region of cells freely migrating on the surface, although such a gradient was not always maintained. When aggregation competent cells were locally stimulated by the application of a microcapillary containing cAMP, the cells extended pseudopods toward the microcapillary. In these cases, an increase in [Ca2+]i was transiently observed in the region opposite to the tip of the capillary. At the slug stage, [Ca2+]i was higher in prestalk cells than in prespore cells of slugs. The possibility that the [Ca2+]i is spatially regulated within a cell was discussed.
Dual functions of transglutaminase in novel cell adhesion
Authors: Ueki Shoko , Takagi Junichi , Saito Yuji 
Published: April 2021

ABSTRACT
Transglutaminases (TGases) are enzymes which catalyze cross-link formation between glutamine residues and lysine residues in substrate proteins. In the present study, we report for the first time that a representative enzyme, blood coagulation factor XIIIa (FXIIIa), is capable of mediating adhesion of various cells. When coated on plastic surfaces FXIIIa promoted adhesion and spreading of various cells of both normal and tumor origin, in a concentration-dependent manner. The adhesion was not inhibited by antibodies against possible contaminants in the enzyme preparation such as fibronectin and vitronectin, but was completely inhibited by a polyclonal antibody against the enzyme. Therefore, if there were any contaminating cell adhesive substrates in the enzyme preparation, they cannot account for the observed cell adhesion to the enzyme; FXIIIa itself mediates the cell adhesion. Furthermore, phosphorylation of tyrosine residues in 120 kDa and 70 kDa proteins was clearly shown in human fibroblasts adhering to the enzyme. Formation of actin stress fibers was also unambiguously observed in the adhering cells. These biochemical reactions, which are also observed when cells adhere to a typical cell adhesion protein, fibronectin, are believed to be of importance in the process of cell adhesion. This adhesion activity of FXIIIa was dependent on its TGase activity, because both a modification of the active center cysteine with iodoacetamide and the addition of ammonium ion abolished the cell adhesion activity along with the enzyme activity. The cell adhesion to fibronectin, however, was not affected by these treatments. The effects of various anti-integrin antibodies suggested that both αvβ3 and β1 family integrins participated in the cell adhesion to FXIIIa. Taken together, these data demon-strate for the first time that there is a unique TGase activity-mediated cell adhesion. This novel function of the enzyme may be of physiological importance.
Immortalised mouse submandibular epithelial cell lines retain polarised structural and functional properties
Authors: Laoide Bríd M. , Courty Yves , Gastinne Isabelle , Thibaut Cécile , Kellermann Odile , Rougeon François 
Published: April 2021

ABSTRACT
The mouse submandibular gland (SMG) is an excellent model for the study of many important biological phenomena such as hormonal regulation of differentiation, neurotransmitter control of secretion, epithelial transport, exocytosis and endocytosis as well as the regulation of mouse SMG specific gene expression, in particular, NGF, EGF and renin. The postnatal development and sexual dimorphism of the mouse gland permits the isolation of male SMGs of different ages, corresponding to different stages of differentiation, particularily with respect to the cytodifferentiation of ductal cell types. We have immortalized SMG epithelial cell lines using mice transgenic for the large T antigen of SV40 or polyoma viruses. Epithelial clusters from the dissected glands were placed in culture and cell lines were established from the immortalized population. Two cell lines, SIMS and SIMP, which retain structural and functional characteristics, are described here. The cell lines are immortalised but not transformed, as judged by the absence of anchorage independent growth potential and the lack of tumour formation in athymic nude mice. Confocal and electron microscopy examination demonstrate that SIMP and SIMS cells express E-cadherin and ZO-1 and have features of polarised epithelial cells. In addition, they form spherical cysts with a wide lumen when grown in type I collagen gels. When grown on a filter support SIMS cells form a tight monolayer, exhibit vectorial transport function and show exclusive Na+,K+-ATPase localisation to the basolateral domain. We determined the cell type restricted expression of cytokeratin markers in the mouse SMG in vivo and we demonstrate that SIMS and SIMP cell lines express duct-specific cytokeratins. Finally, the expression of a set of differentiation markers, including EGF, NGF and renin, was detected by RT-PCR and by indirect immunofluorescence staining in these lines. Thus, these polarised ductal cell lines, as well as having important intrinsic properties, represent well characterised mouse epithelial models which, until now, have not been readily available for cellular studies.
Integrin α6Bβ1 is involved in kidney tubulogenesis in vitro
Authors: Falk Mats , Salmivirta Katriina , Durbeej Madeleine , Larsson Erik , Ekblom Marja , Vestweber Dietmar , Ekblom Peter 
Published: April 2021

ABSTRACT
Laminin-1 has previously been shown to be of major importance for the development of kidney tubules. Antibodies against fragments E8 and E3 of laminin-1 perturb kidney development in vitro. We here studied expression of integrins α6β1 and α6β4, two known laminin receptors, during kidney development. Integrin β1 subunit could be detected by immunofluorescence on all cell types of embryonic mouse kidney, but we could not detect integrin β4 subunit in embryonic kidney by immunofluorescence or by in situ hybridization. The presence of integrin α6 subunit in all epithelia of embryonic kidney was demonstrated by immunofluorescence and by in situ hybridization. RT-PCR showed that α6B is the major splice variant in embryonic kidney. During in vitro conversion of nephrogenic mesenchyme to epithelial tubules, a strong increase in the expression of the 6 kb mRNA for α6 integrin subunit was seen by northern blotting at the onset of epithelial morphogenesis, on day two of culture. Immunoprecipitation of extracts from embryonic kidney with antibodies against α6 subunit yielded bands corresponding to the expected size of β1 integrin subunit but not of β4 subunit. Monoclonal antibodies against either α6 or β1 subunit but not against E-cadherin blocked kidney tubulogenesis in vitro. This suggests that integrin α6Bβ1 is involved in kidney tubulogenesis in vitro. Another possibility is that the antibodies against integrin α6 and β1 subunit cause abnormal signalling by the integrin.
p230 is associated with vesicles budding from the trans-Golgi network
Authors: Gleeson Paul A. , Anderson Timothy J. , Stow Jennifer L. , Griffiths Gareth , Toh Ban Hock , Matheson Fiona 
Published: April 2021

ABSTRACT
Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using α2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTPγS or AlF4− resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4−, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (∼10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.
Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix
Authors: Sasaki Takako , Wiedemann Hanna , Matzner Monika , Chu Mon-Li , Timpl Rupert 
Published: April 2021

ABSTRACT
The extracellular matrix protein fibulin-2 was shown to be a typical product of cultured human and mouse fibroblasts by several immunological assays. It is secreted and deposited in cells and tissues as a disulfide-bonded oligomer identical in size to the previously described recombinant fibulin-2. Most of the fibroblast fibulin-2 is deposited into a dense fibrillar meshwork which requires treatment with EDTA and/or 6 M urea for solubilization. Fibulin-2 and fibronectin are synthesized at equivalent levels and both colocalize in the fibrils as shown by immunofluorescence. Metabolic labelling and pulse-chase studies demonstrated fibulin-2 oligomers in detergent extracts of cells and their rapid translocation to extracellular EDTA-sensitive assembly forms. Unlike for fibronectin and fibulin-1 only a little fibulin-2 was found in the cell culture medium. Immunogold staining of confluent human fibroblasts showed localization of fibulin-2 to a fine meshwork or bundles of amorphous microfibrils in the matrix. This also demonstrated a distinct colocalization of fibulin-2 and fibronectin at the electron microscope level, indicating that the interaction between these two protein shown in in vitro assays may also exist in situ. No distinct colocalization of both proteins could, however, be observed with crossstriated fibrils of collagen I and collagen VI microfibrils.
Subcellular localization of Trypanosoma cruzi glycoprotein Gp72
Authors: Haynes Paul A. , Russell David G. , Cross George A. M. 
Published: April 2021

ABSTRACT
We have investigated the subcellular location of the Trypanosoma cruzi surface glycoprotein, Gp72, by introducing epitope-tagged copies of gp72 into gp72 null-mutant cells. A tagged Gp72, containing three tandemly repeated copies of a human influenza hemagglutinin nonapeptide (HA) adjacent to the mature Gp72 amino terminus, was able to complement the null mutant phenotype, as well as being recognized in western blots by both anti-HA antibody and the carbohydrate-specific monoclonal antibody WIC29.26. Integration of this epitope-tagged gp72 into the chromosomal gp72 locus produced a clonal cell line, 72HAN3.1G7, which was used for studies of the subcellular location of the epitope-tagged Gp72. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 epimastigotes showed that Gp72 was evenly distributed over the cell body and somewhat concentrated in the proximal region of the flagellum. No fluorescence could be detected in the distal tip of the flagellum. Immunoelectron microscopy of fixed 72HAN3.1G7 epimastigotes revealed that Gp72 was predominantly membrane-associated and located on the cell surface. Indirect immunofluorescence microscopy of live 72HAN3.1G7 epimastigote cells showed a similar pattern of fluorescence on the flagellum, but no fluorescence was detected on the cell body, which was attributed to masking by other cell-surface components. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 amastigotes revealed that Gp72, which has long been considered to be expressed only in epimastigotes and metacyclic trypomastigotes, can be expressed in amastigotes, but it no longer contains the WIC29.26 carbohydrate epitope.
A membrane-associated protein complex with selective binding to the clathrin coat adaptor AP1
Authors: Mallet William G. , Brodsky Frances M. 
Published: April 2021

ABSTRACT
Adaptors are the membrane-binding components of clathrin-coated vesicles. The interaction of the trans-Golgi coat adaptor AP1 with membrane-associated proteins was analyzed by affinity chromatography. Proteins of 83 and 52 kDa bound specifically to the core domain of AP1 and showed no interaction with AP2 or other clathrin-coated vesicle proteins. The AP1-binding proteins were tightly membrane-associated, though behaved as peripheral membrane proteins. They were detected in membranes depleted of clathrin-coated vesicles and not in coated vesicles, suggesting that the interaction of these proteins with AP1 may precede coated vesicle budding. Co-fractionation of the AP1-binding proteins with trans-Golgi network membrane was also observed. Upon gel filtration, both AP1-binding proteins eluted in a high molecular mass complex which was labile at high concentrations of Tris. The 83 kDa protein bound to AP1 affinity resin in the absence of the 52 kDa protein. In contrast, the separated 52 kDa protein did not bind AP1, suggesting that the 83 kDa protein is the AP1-binding component of the complex. Characterization of this protein complex defines a novel membrane-associated component that specifically interacts with AP1 and may contribute to its function in forming clathrin-coated vesicles.
Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles
Authors: Paoletti Anne , Moudjou Mohammed , Paintrand Michel , Salisbury Jeffrey L. , Bornens Michel 
Published: April 2021

ABSTRACT
Centrin is a member of the calcium-binding EF-hand protein superfamily present in centrosomes of widely divergent species. Investigating the cellular distribution of human centrin by both immunofluorescence and cell fractionation, we report that centrin is biochemically complex in human cells, displaying as much as ten isoforms in 2-D electrophoresis. This suggests that centrin may be subject to multiple regulations. Strikingly, more than 90% of centrin is not associated with the centrosome fraction. The centrosome-associated centrin, however, displays a specific pattern in 2-D electrophoresis and is concentrated within the distal lumen of the centrioles, where a complex structure has been previously described. This precise localization allows the resolution of centrioles at the optical level throughout the cell cycle and provides a valuable tool for monitoring centriole duplication. To get insights on centrin function, we performed injection experiments of recombinant heterologous centrin in two-cell stage frog embryos in an attempt to produce dominant negative effects. We report that green algae and human centrin delay cleavage and promote the formation of abnormal blastomeres in which the distribution of microtubule asters and of nuclei is dramatically impaired. This suggests that centrin could be involved in the centrosome reproduction cycle, in the coordination of cytoplasmic and nuclear division or in cytokinesis.
Bone-resorbing osteoclasts reveal a dynamic division of basal plasma membrane into two different domains
Authors: Salo Jari , Metsikkö Kalervo , Palokangas Harri , Lehenkari Petri , Väänänen H. Kalervo 
Published: April 2021

ABSTRACT
Bone-resorbing multinucleate osteoclasts exhibit a ruffled border membrane apposing the bone and a basal membrane contacting the circulation. A junctional complex called the sealing zone separates these two membrane domains, but the defined nature of these membrane domains has remained obscure. We now show, using enveloped viral glycoproteins and lectins as tools, that osteoclasts exhibit a novel membrane domain in the basal surface when they are polarized for resorption. Influenza haemagglutinin, which is apically targeted in epithelial cells, is targeted to a restricted area at the top of the basal surface, while vesicular stomatitis virus G-protein which is basolaterally targeted in epithelia, occupies the rest of the basal surface. Neither of these viral glycoproteins is gathered to the ruffled border nor sealing zone area, but they share in a specific way the basal surface. To show that the division of basal membrane into two different domains also occurs in non-infected cells, we have analyzed the distribution of receptors for these viruses and binding sites of some lectins. Both of these methods show that also some endogenous proteins are located in different domains in the basal surface in active osteoclasts. We also show that these two different membrane domains can be distinquished in scanning electron microscopy level due to the villus appearance of the central basal domain.