bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolated plasma membranes induce the loss of oriented detyrosinated microtubules and other contact inhibition-like responses in migrating NRK cells
Authors: Nagasaki T. , Liao G. , Gundersen G. G.
Published: April 2021

We have previously shown that detyrosinated microtubules (Glu MTs), which are oriented toward the direction of locomotion in motile fibroblasts, disappear from the area adjacent to cell-cell contact soon after a cell-cell collision. To identify cell surface molecules that trigger this phenomenon, we have established a system in which this and other cellular reactions to cell-cell contact can be reproduced by the addition of isolated plasma membranes. Experimental wounds were made in confluent monolayers of NRK cells, and cells at the wound margin were allowed to develop oriented Glu MTs. Test samples were added to these cells and after a 1 hour incubation the distributions of Glu MTs, tyrosinated MTs (Tyr MTs) and microfilaments were determined by immunofluorescence. When plasma membranes isolated from NRK cells were added, oriented Glu MTs disappeared from the leading lamella of target cells and instead a small number of Glu MTs were found clustered around the nucleus. As observed for cell-cell contact, plasma membranes did not significantly affect the distribution of Tyr MTs. We also found that both cell-cell contact and membrane treatment caused the collapse of lamellipodia and loss of associated staining with antiactin antibody. Time-lapse recordings of directed locomotion of NRK cells showed that membranes suppressed the forward movement of cells. The loss of Glu MTs from the leading lamella was the most amenable response for quantification and we used it to examine the biochemical properties of the membrane activity. The ability of membranes to induce the loss of oriented Glu MTs was observed at as low as 4 μg/ml of membrane protein and was detectable 10 minutes after membrane addition. The loss of oriented Glu MTs was reversible upon removal of membranes, demonstrating that the membranes were not toxic to the cells. The oriented Glu MT reducing activity could be solubilized from the membranes by detergent, was enriched in a plasma membrane fraction, and was labile to heat and acid treatment. In summary, we have successfully reconstituted a number of responses of contact inhibition using solubilized preparations of membranes. Our preliminary results suggest that there is a specific factor in plasma membranes that is capable of triggering contact inhibition. With the assay we have developed, it should now be possible to dissect contact inhibition of motility at the molecular level.
Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited
Authors: Subtil Agathe , Hémar Agnès , Dautry-Varsat Alice
Published: April 2021

The cytokine interleukin 2 (IL2) is produced by activated helper T lymphocytes and modulates the growth and activity of cells expressing high-affinity surface IL2 receptors that transduce its signaling. After ligand binding to receptors on the plasma membrane, receptor-ligand complexes are rapidly endocytosed and IL2 is degraded in acidic compartments. The best known receptor-mediated endocytosis pathway involves clathrin-coated pits. Receptors that carry an internalization signal recognized by adaptors on the cytosolic side of the plasma membrane are clustered into the coated pits and enter cells very efficiently. Many receptors use this pathway, but other endocytic pathways have also been reported, for ricin, EGF and insulin, for instance, which seem to be less efficient than the coated one. We compared the endocytosis of IL2 and its receptors to that of transferrin, a marker of the coated pit pathway. Under normal conditions, the kinetics of entry of IL2 was two times slower than that of transferrin. When internalization via coated pits was inhibited by two different methods, potassium depletion and cytosol acidification, endocytosis of IL2 and its receptors was only partly inhibited, while transferrin entry was strongly affected. Treatment with the cationic amphiphilic drug chlorpromazine, which induces a redistribution of a clathrin-coated pit component, AP-2, to endosomes, reduced transferrin, but not IL2 internalization. Thus, unexpectedly, this cytokine and its receptors can still be rapidly endocytosed in the absence of functional clathrincoated structures. We propose a model for receptormediated endocytosis that may account for these results and published data on other receptors.
Chromatin and microtubule organization during premeiotic, meiotic and early postmeiotic stages of Drosophila melanogaster spermatogenesis*
Authors: Cenci Giovanni , Bonaccorsi Silvia , Pisano Claudio , Verni Fiammetta , Gatti Maurizio
Published: April 2021

Larval and pupal testes of Drosophila melanogaster were fixed with a methanol/acetone fixation procedure that results in good preservation of cell morphology; fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. Fixed testis preparations were treated with anti-tubulin antibodies and Hoechst 33258 to selectively stain microtubules and DNA. The combined analysis of cell morphology, chromatin and microtubule organization allowed a fine cytological dissection of gonial cell multiplication, spermatocyte development, meiosis and the early stages of spermatid differentiation. We placed special emphasis on the spermatocyte growth phase and the meiotic divisions, providing a description of these processes that is much more detailed than those previously reported. In addition, by means of bromo-deoxyuridine incorporation experiments, we were able to demonstrate that premeiotic DNA synthesis occurs very early during spermatocyte growth.
Rapid appearance of connexin 26-positive gap junctions in centrilobular hepatocytes without induction of mRNA and protein synthesis in isolated perfused liver of female rat
Authors: Kojima Takashi , Sawada Norimasa , Oyamada Masahito , Chiba Hideki , Isomura Hiroshi , Mori Michio
Published: April 2021

In the adult rat liver, the gap junction protein connexin 32 (Cx32) is evenly distributed in hepatocytes within the liver lobules, while connexin 26 (Cx26) is preferentially localized in hepatocytes in periportal zones. We report here that Cx26-positive gap junctions rapidly appear in the centrilobular hepatocytes of adult female rat livers during a 30 minute perfusion of the liver through the hepatic portal vein with a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and oxygen transport FC-43 fluid at a physiological flow rate without any changes in the distribution of Cx32. The change in the localization of Cx26 was closely related to that of E-cadherin, and there was no significant increase in the amounts of Cx26 protein and mRNA. The appearance of Cx26 in the centrilobular hepatocytes was inhibited by treatment with cytoskeleton disruptors such as colchicine and cytochalasin B, and intra-cytoplasmic transport inhibitors such as brefeldin A. The liver perfusion induced the appearance of Cx26 in the centrilobular hepatocytes only in female rats. Estrogen treatment of ovariectomized rats caused the appearance of both Cx26 and E-cadherin in centrilobular hepatocytes not only in the perfused liver but also in the non-perfused liver. Our results indicate that in the rat liver: (a) the localization of Cx26 can be modulated by a post-translational mechanism; (b) E-cadherin may play an important role in the formation of gap junctions composed of Cx26; and (c) the formation of gap junctions is regulated by female steroid hormones.
Expression of mouse LSP1/S37 isoforms S37 is expressed in embryonic mesenchymal cells
Authors: Misener V. L. , Hui C.-c. , Malapitan I. A. , Ittel M.-E. , Joyner A. L. , Jongstra J.
Published: April 2021

Mouse LSP1 is a 330 amino acid intracellular F-actin binding protein expressed in lymphocytes and macrophages but not in non-hematopoietic tissues. A 328 amino acid LSP1-related protein, designated S37, is expressed in murine bone marrow stromal cells, in fibro-blasts, and in a myocyte cell line. The two proteins differ only at their N termini, the first 23 amino acid residues of LSP1 being replaced by 21 different residues in S37. The presence of different amino termini suggests that the LSP1 and S37 proteins are encoded by transcripts arising through alternative exon splicing. Here we report the genomic organization of the Lsp1 gene and show that the distinct N termini of LSP1 and S37 are encoded by two alternatively used exons, each containing a translational start codon. We also demonstrate that alternative 3′ acceptor sites are used in the splicing of exon 5. This results in LSP1 and S37 transcripts that either do or do not contain 18 bp encoding the 6 amino acids HLIRHQ of the acidic domain. Therefore, the Lsp1 gene encodes four protein isoforms: full-length LSP1 and S37 proteins, designated LSP1-I and S37-I and the same proteins without the HLIRHQ sequence, designated LSP1-II and S37-II. By in situ hybridization analysis we show that the S37 isoforms are expressed in mesenchymal tissue, but not in adjacent epithelial tissue, of several developing organs during mouse embryogenesis. This, together with our finding that S37 is an F-actin binding protein, suggests that S37 is a cytoskeletal protein of mesenchymal cells, which may play a role in mesenchyme-induced epithelial differentiation during organogenesis.
Nuclear calmodulin/62 kDa calmodulin-binding protein complexes in interphasic and mitotic cells
Authors: Portolés Manuel , Faura Magdalena , Renau-Piqueras Jaime , José Iborra Francisco , Saez Rosana , Guerri Consuelo , Serratosa Joan , Rius Eulalia , Bachs Oriol
Published: April 2021

We report here that a 62 kDa calmodulin-binding protein (p62), recently identified in the nucleus of rat hepatocytes, neurons and glial cells, consists of four polypeptides showing pI values between 5.9 and 6.1. By using a DNA- binding overlay assay we found that the two most basic of the p62 polypeptides bind both single- and double-stranded DNA. The intranuclear distribution of calmodulin and p62 was analysed in hepatocytes and astrocyte precursor cells, and in proliferating and differentiated astrocytes in primary cultures by immunogold-labeling methods. In non-dividing cells nuclear calmodulin was mostly localized in heterochromatin although it was also present in euchro- matin and nucleoli. A similar pattern was observed for p62, with the difference that it was not located in nucleoli. p62/calmodulin complexes, mainly located over hete- rochromatin domains were also observed in interphasic cells. These complexes remained associated with the nuclear matrix after in situ sequential extraction with nucleases and high-salt containing buffers. In dividing cells, both calmodulin and p62 were found distributed over all the mitotic chromosomes but the p62/calmodulin aggregates were disrupted. These results suggest a role for calmodulin and p62 in the condensation of the chromatin.
Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells
Authors: äntti Jussi J , änen Sirkka Ker , Toikkanen Jaana , Kuismanen Esa , Ehnholm Christian , Söderlund Hans , Olkkonen Vesa M.
Published: April 2021

Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20°C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37°C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.
Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD
Authors: Vandromme Marie , Carnac Gilles , Gauthier-Rouvière Cécile , Fesquet Didier , Lamb Ned , Fernandez Anne
Published: April 2021

MyoD is a nuclear phosphoprotein that belongs to the family of myogenic regulatory factors and acts in the tran-scriptional activation of muscle-specific genes. We have investigated the role of cAMP-dependent protein kinase (A-kinase) in modulating the nuclear locale of MyoD. Purified MyoD protein microinjected into the cytoplasm of rat embryo fibroblasts is rapidly translocated into the nucleus. Inhibition of A-kinase activity through injection of the specific inhibitory peptide PKI prevents this nuclear localisation. This inhibition of nuclear location is specifi-cally reversed by injection of purified A-kinase catalytic subunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of all the putative sites for A-kinase-dependent phosphorylation on MyoD, substituting serine or threonine residues for the non-phosphorylatable amino acid alanine, had no effect on nuclear import of mutated MyoD. These data exclude the possibility that the effect of A-kinase on the nuclear translocation of MyoD is mediated by direct phosphoryla-tion of MyoD and imply that A-kinase operates through phosphorylation of components involved in the nuclear transport of MyoD.
On the molecular mechanism of the circadian clock: The 41,000 Mr clock protein of Chlorella was identified as 3-phosphoglycerate kinase
Authors: Walla Ortwin J. , Groot Egon J. de , Schweiger Manfred
Published: April 2021

A 41,000 Mr polypeptide of Chlorella exhibits a circadian rhythm in its synthesis and possesses characteristic features of a putative essential clock protein as was proposed by the coupled translation-membrane model. Purification of this polypeptide and a microsequencing analysis yielded a N-terminal sequence of 35 amino acids that showed no homology to known sequences that were thought to be involved in circadian rhythm such as the per gene of Drosophila and the frq gene of Neurospora. However, strong homology was observed to 3-phospho- glycerate kinase (PGK) of different organisms. The highest homology (83%) of this Chlorella sequence was found with the PGK of wheat chloroplast.

PGK activity and the 41,000 Mr polypeptide co-purified through differential centrifugation and gel filtration. These data, and comparison with the physical properties of other known PGK molecules, support the conclusion that the 41,000 Mr polypeptide of Chlorella, a candidate for a putative essential clock protein, is 3-phosphoglycerate kinase.
Isolation and developmental expression of the amphibian homolog of the fibroblast growth factor receptor 3
Authors: Shi De-Li , Fromentoux Valérie , Launay Catherine , Umbhauer Muriel , Boucaut Jean-Claude
Published: April 2021

Recent observations suggest that fibroblast growth factors (FGFs) and their receptors are involved in the control of embryogenesis. Several FGF receptor genes have been identified so far and their expression is differentially regulated. As part of a continuing effort to analyse the differential expression of FGF receptors and their potential role during amphibian development, we have isolated a Pleurodeles homolog of FGF receptor 3 (FGFR-3), which we designated PFR-3 because of its highest homology to human FGFR-3 (75% overall identity). PFR-3 is a maternally derived mRNA. While a low level of expression persists during the cleavage and gastrula stages, a significant increase in the mRNA was observed at the end of the gastrula stage. RNase protection analysis on dissected tissues showed that PFR-3 mRNA was mainly localized to the ectoderm at the early gastrula stage and then shifted to the embryonic neural tissues, whereas adult brain had decreased levels of PFR-3 mRNA expression. Consistent with the loss of FGF receptors during skeletal muscle terminal differentiation, PFR-3 as well as other FGF receptor mRNAs were undetectable in the adult skeletal muscle. However, highest levels of PFR-3 mRNA expression were found in the testis. In situ hybridization revealed strong expression in the germinal epithelium of the embryonic brain (especially the diencephalon and rhombencephalon) and neural tube, in the lens and the cranial ganglia. The epithelium of the developing gut, like the pharynx and esophagus, also prominently expressed PFR-3 mRNA. Other sites of expression were found in the liver and in the mesenchymal condensation sites of branchial arches. These observations further suggest that individual FGF receptor genes are differentially regulated during amphibian embryogenesis, and that PFR-3 may play a role in mesoderm induction and in other FGF-mediated developmental processes.
Expression and cellular localization of glucose transporters (GLUT1, GLUT3, GLUT4) during differentiation of myogenic cells isolated from rat fœtuses
Authors: Guillet-Deniau Isabelle , Leturque Armelle , Girard Jean
Published: April 2021

Skeletal muscle regeneration is mediated by the proliferation of myoblasts from stem cells located beneath the basal lamina of myofibres, the muscle satellite cells. They are functionally indistinguishable from embryonic myoblasts. The myogenic process includes the fusion of myoblasts into multinucleated myotubes, the biosynthesis of proteins specific for skeletal muscle and proteins that regulates glucose metabolism, the glucose transporters. We find that three isoforms of glucose transporter are expressed during fœtal myoblast differentiation: GLUT1, GLUT3 and GLUT4; their relative expression being dependent upon the stage of differentiation of the cells. GLUT1 mRNA and protein were abundant only in myoblasts from 19-day-old rat fœtuses or from adult muscles. GLUT3 mRNA and protein, detectable in both cell types, increased markedly during cell fusion, but decreased in contracting myotubes. GLUT4 mRNA and protein were not expressed in myoblasts. They appeared only in spontaneously contracting myotubes cultured on an extracellular matrix. Insulin or IGF-I had no effect on the expression of the three glucose transporter isoforms, even in the absence of glucose. The rate of glucose transport, assessed using 2-[3H]deoxyglu-cose, was 2-fold higher in myotubes than in myoblasts. Glucose deprivation increased the basal rate of glucose transport by 2-fold in myoblasts, and 4-fold in myotubes. The cellular localization of the glucose transporters was directly examined by immunofluorescence staining. GLUT1 was located on the plasma membrane of myoblasts and myotubes. GLUT3 was located intracellularly in myoblasts and appeared also on the plasma membrane in myotubes. Insulin or IGF-I were unable to target GLUT3 to the plasma membrane. GLUT4, the insulin-regulatable glucose transporter isoform, appeared only in contracting myotubes in small intracellular vesicles. It was translocated to the plasma membrane after a short exposure to insulin, as it is in skeletal muscle in vivo. These results show that there is a switch in glucose transporter isoform expression during myogenic differentiation, dependent upon the energy required by the different stages of the process. GLUT3 seemed to play a role during cell fusion, and could be a marker for the muscle’s ability to regenerate.
Cell attachment to extracellular matrix substrates is inhibited upon downregulation of expression of calreticulin, an intracellular integrin α-subunit-binding protein
Authors: Leung-Hagesteijn Chung Yee , Milankov Kosta , Michalak Marek , Wilkins John , Dedhar Shoukat
Published: April 2021

We have demonstrated recently that calreticulin, an intra-cellular calcium-binding protein, can interact with the α-subunits of integrin receptors via the highly conserved KXGFFKR amino acid sequence present in the cytoplasmic domains of all integrin α-subunits (Rojiani et al. (1991)Biochemistry 30, 9859-9866). Here we demonstrate that calreticulin can be co-localized by immunofluorescence as well as co-purified with integrins, that recombinant calreticulin can also interact with integrins, and that the interaction occurs predominantly via the N-domain of calreticulin, to a much lesser extent with the C-domain, but not at all with the proline-rich P-domain. To demonstrate a physiological role for the interaction of calreticulin with integrins, calreticulin expression was downregulated by treating cells with antisense oligonucleotides designed to inhibit the initiation of translation of calreticulin. Antisense oligonucleotides, but not sense or non-sense oligonucleotides, inhibited attachment and spreading of cells cultured in the presence of fetal bovine serum, and also of cells plated on individual extracellular matrix substrates in the absence of serum. The antisense oligonucleotide inhibited cell proliferation of anchorage-dependent cells slightly, but there was no effect on cell viability. The effect on cell attachment was similar to that achieved by treating cells with an antisense oligonucleotide designed to inhibit translation of the integrin α3 subunit, which resulted in the inhibition of cell attachment to α3β1-specific substrates. The effect of the antisense calreticulin oligonucleotide on cell attachment was demonstrated to be integrin-mediated since antisense calreticulin treatment of Jurkat cells abrogated the stimulation of collagen cell attachment achieved by attachment-stimulating signalling anti-α2 (JBS2) and anti-β1 (21C8) antibodies. The oligonucleotides did not affect the rate of cell proliferation of these cells. These results demonstrate a fundamental role of calreticulin in cell-extracellular matrix interactions.
Retinoic acid as modulator of UVB-induced melanocyte differentiation: Involvement of the melanogenic enzymes expression
Authors: Roméro Christine , Aberdam Edith , Larnier Catherine , Ortonne Jean-Paul
Published: April 2021

Retinoic acid (RA) is a hormone-like agent involved in the control of cell differentiation. The most characteristic feature of melanocyte differentiation, melanogenesis, is stimulated by UV radiations. Excessive chronic sun exposure results in irregular skin hypermelanosis that can be partially corrected by topical RA. The basic mechanisms underlying this effect of RA are unknown. To determine whether RA can directly modulate excessive melanin synthesis, we analyzed the in vitro effect of cis- and trans-RA on UVB-induced melanogenesis in S91 mouse melanoma cells and in normal human melanocytes (NHM). In both cells types, the two RA isoforms significantly decreased the UVB-stimulated melanogenesis in term of tyrosinase activity and melanin neosynthesis. To correlate changes in melanogenesis with the expression of melanogenic enzymes, we determined the neosynthesis rate of tyrosinase, tyrosinase-related protein-1 (TRP-1/gp 75) and tyrosinase-related protein-2 (TRP-2/DOPAchrome tautomerase). Here we show that UVB-induced melanogenesis in NHM is related to an increased synthesis of tyrosinase and TRP-1 and to a dramatic decrease of TRP-2 expression. RA inhibition of UVB-induced melanogenesis acts at the post-transcriptional level leading to a decreased tyrosinase and TRP-1 synthesis. We also show that in NHM, inhibition of TRP-2 following UVB-treatment is significantly reversed by RA. This demonstrates a negative correlation between melanogenesis and TRP-2 expression.
Co-localization of 1,4-dihydropyridine receptor α2/δ subunit and N-CAM during early myogenesis in vitro
Authors: Vandaele Sylvie F. , Rieger François
Published: April 2021

The surface distribution of the subunit of the 1,4-dihydropyridine receptor and its topographical relationship with the neural cell adhesion molecule (N-CAM) were investigated during early myogenesis in vitro, by double immunocytochemical labeling with the monoclonal antibody 3007 and an anti-N-CAM polyclonal antiserum. The monoclonal antibody 3007 has been previously shown to immunoprecipitate dihydropyridine receptor from skeletal muscle T-tubules. In further immunoprecipitation experiments on such preparations and muscle cell cultures, it was demonstrated here that the monoclonal antibody 3007 exclusively recognizes the α2/δ subunit of the 1,4-dihydropyridine receptor. In rabbit muscle cell cultures, the labeling for both α2/δ and N-CAM was first detected on myoblasts, in the form of spots on the membrane and per-inuclear patches. Spots of various sizes organized in aggregates were then found on the membrane of myotubes. At fusion (T0), aggregates of N-CAM spots alone were found at the junction between fusing cells. At T6and later stages, all α2/δ aggregates present on myotubes co-localized with N-CAM, while less than 3% of N-CAM aggregates did not co-localize with α2/δ. A uniform N-CAM staining also made its appearance. At T12, when myotubes showed prominent contractility, α2/δ–N-CAM aggregates diminished in size. Dispersed α2/δ spots of a small regular size spread over the whole surface of the myotubes and alignments of these spots became visible. Corresponding N-CAM spots were now occasionally seen, and uniform N-CAM staining was prominent. These results show that α2/δ and N-CAM are co-localized and that their distributions undergo concomitant changes during early myogenesis until the T-tubule network starts to be organized. This suggest that these two proteins might jointly participate in morphogenetic events preceding the formation of T-tubules.
Immuno-EM localization of the β1integrin subunit in wet-cleaved fibronectin-adherent fibroblasts
Authors: Meijne Alexandra M. L. , Casey Diane M. , Feltkamp Constance A. , Roos Ed
Published: April 2021

Using immuno-EM, we have studied the distribution of the β1integrin subunit in chicken embryo fibroblasts allowed to adhere and spread for 3 hours on a fibronectin-coated surface in serum-free medium. The cells were wet-cleaved, which removed most of the cell body, yielding ventral plasma membranes with little, and sometimes virtually no, associated cytoskeleton.

The β1integrin subunit was detected with antibodies against the cytoplasmic domain. In immune fluorescence, it colocalized with adhesion plaques, in a punctate staining pattern, and often seemed to be at the periphery of the plaque. By immuno-EM, β1was in fact found in discrete clusters, not throughout the plaque. In deep-cleaved cells from which virtually all cytoskeleton was removed, clusters could often be seen to be located on fibronectin fibrils. Furthermore, β1was present in clusters at the cell margins, and isolated or in small groups at the very edge of the cell.

When fibronectin synthesis, and consequently fibril formation, was inhibited by cycloheximide, large adhesion plaque-like structures were formed at the cell margin. This phenotype was reversed by addition of soluble fibronectin, which was incorporated into fibrils. As in normal plaques, talin and vinculin were present, the plasma membrane was very close (10-20 nm) to the substratum and the fibronectin layer underneath was removed. These plaques did contain β1integrins but they were not in clusters.

These observations indicate that the talin-vinculin network of an adhesion plaque is normally anchored to the substratum at discrete β1integrin clusters that may be located on fibronectin fibrils, and that elsewhere the plaque is not necessarily attached to the substratum by interaction of integrins with matrix proteins. In the absence of fibronectin fibrils, adhesion plaque-like structures can be formed, but these are aberrant in size, location and fine structure.
Morphogenetic response of cultured normal and transformed fibroblasts, and epitheliocytes, to a cylindrical substratum surface Possible role for the actin filament bundle pattern
Authors: Rovensky Yu. A. , Samoilovi V. I.
Published: April 2021

Morphometric characteristics such as cell area, dispersion, elongation and orientation were studied in normal and transformed fibroblasts, and in epitheliocytes cultured on flat or cylindrical substrata. Cylindrical surfaces with a high degree of curvature (12-13 or 25 μm radii) were shown to affect cell size, shape and alignment. The reaction of the cells to the curvature of cylindrical substrata was different in various cell types studied and depended on the pattern of actin microfilament bundles. The cells containing pronounced straight actin bundles (mouse embryo fibroblasts at the polarization stage of spreading, single spread cells of the ‘normal’ epithelial FBT line or the fully transformed epithelial IAR 6-1 line) were relatively resistant to bending around a cylindrical substratum, and became elongated and oriented along the cylinder. Cells with circular actin bundles as the predominant pattern (mouse embryo fibroblasts at the radial stage of spreading, single spread cells of ‘normal’ epithelial IAR 20 line) and cells with insufficient or no actin bundles (transformed fibroblastic L line) were prone to bending around a cylinder with much less pronounced elongation and orientation along its axis. The data obtained indicate that the reaction of cultured cells to the geometry of the substratum surface and, in particular, to a cylindrical surface is determined not only by the presence or absence of actin microfilament bundles but by their pattern in the cell.
A scatter factor-like factor is produced by a metastatic variant of a rat bladder carcinoma cell line
Authors: Savério Bellusci , Moens Ginette , Thiery Jean Paul , Jouanneau Jacqueline
Published: April 2021

The rat bladder carcinoma epithelial NBT-II cell line undergoes, in vitro, a morphological transition to a fibrob- last-like state in the presence of different growth factors. We have selected, in vivo, a metastatic clone, designated M- NBT-II, which has a mesenchymal phenotype and secretes into the culture medium a factor able to dissociate epithe- lial clusters of NBT-II or MDCK cells. This factor was des- ignated scatter factor-like (SFL) by analogy to the HGF/SF, which has the same dissociating effect in these two cell lines. Here, we show that SFL factor and HGF/SF are different factors: (i) no HGF/SF transcripts could be detected using either specific rat HGF/SF cDNA probes or PCR; (ii) blocking antibodies against rat HGF/SF do not inhibit the SFL activity; and (iii) crude culture medium or partially purified SFL factor-containing fractions do not induce MDCK tubulogenesis, a biological assay that is specific for HGF/SF activity in vitro. We report the partial purification of the SFL factor, based on ion exchange and reverse-phase chromatography. The results indicate that the M-NBT-II metastatic variant secretes a dissociating factor sharing some common biological properties with the HGF/SF, which suggests that the SFL factor is a member of the HGF/SF family and may be involved in tumor progression.
Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes
Authors: Véronique Dupreza , Smoljanovic Marc , Lieb Michèle , Dautry-Varsat Alice
Published: April 2021

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The α and β chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish perox- idase to probe for the whole endocytic pathway. Fraction- ation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome- containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes con- taining horseradish peroxidase.

These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.
A possible role for stable microtubules in intracellular transport from the endoplasmic reticulum to the Golgi apparatus
Authors: Mizuno Masahiro , Singer S. J.
Published: April 2021

The intracellular transport of secretory proteins involves at an early stage the formation of vesicles from transitional elements of the endoplasmic reticulum (ER) containing these proteins and the transfer of these vesicles to the cis-face of the Golgi apparatus. We propose that the latter transfer process does not occur by random diffusion, but is instead mediated by tracking along stable microtubules. To test this proposal, we have carried out double immuno-electron microscopic labeling experiments on frozen sections of HepG2 hepatoma cells secreting the protein human serum albumin (HSA). By a cycloheximide treatment protocol, the stage during which the transfer of newly synthesized HSA from the ER to the Golgi apparatus occurs in vivo was determined. Sections of the cells were then double immunolabled using primary antibodies to HSA and to glu-tubulin, the latter specifically detecting stable microtubules. We observed a significantly high frequency of HSA-containing structures between the ER and the Golgi apparatus with which stable microtubules were closely associated. These results support the proposal that stable microtubules may play a critical role in directing the transfer process from the ER to the Golgi apparatus.
Inter- and intra-site heterogeneity in the expression of fetal-like phenotypic characteristics by gingival fibroblasts: potential significance for wound healing
Authors: Irwin Christopher R. , Picardo Martino , Ellis Ian , Sloan Philip , Grey Anne Marie , McGurk Mark , Schor Seth L.
Published: April 2021

We have previously reported that fetal and adult skin fibroblasts display distinctive migratory phenotypes on 3-D collagen substrata and that these behavioural characteristics may be quantified by a function defined as the cell density migration index(CDMI). Subsequent work indicated that this difference in migratory phenotype was due to the production by fetal fibroblasts of a migration stimulating factor(MSF) that is not produced by normal adult skin fibroblasts. We now present data indicating that: (a) unselected fibroblasts obtained from 14/14 (100%) of adult gingival explants expressed fetal-like CDMI values compared to only 1/10 (10%) of similarly explanted paired skin cells; (b) 12/12 (100%) of these gingival fibroblast lines also produced detectable quantities of MSF compared to 0/9 (0%) of the tested skin cells; (c) by microdissection studies, gingival fibroblasts obtained from different anatomical microdomains consisted of behaviourally distinct subpopulations, with cells derived from the papillary tips (PAP fibroblasts) displaying fetal-like CDMI values and persistent MSF production, whilst cells obtained from the deeper reticular tissue (RET fibroblasts) were adult-like with respect to these two criteria; (d) PAP fibroblasts were also smaller and achieved higher saturation cell densities compared to paired RET cells; (e) PAP fibroblasts passaged in vitro underwent a fetal-to-adult phenotypic transition characterized by the adoption of various RET cell characteristics, including the acquisition of CDMI values falling within the adult range and cessation in MSF production; and (f) early passage PAP fibroblasts incubated in the presence of an affinity-purified anti-MSF rabbit polyclonal antibody were induced to alter their migratory phenotype and exhibited CDMI values falling within the adult range. Statistical analysis indicated a highly significant correlation between the expression of a fetal-like CDMI and production of MSF (P<0.00001, using the Fisher exact contingency test). Taken together, these observations suggest that the production of MSF by PAP fibroblasts is responsible for their characteristically fetal-like migratory behaviour. The existence of such inter- and intra-site phenotypic heterogeneity in populations of skin and gingival fibroblasts is discussed in the context of fibrob-last lineage relationships and the possible contribution of persistently fetal-like fibroblast subpopulations to connective tissue function in wound healing.
Hydra tropomyosin TROP1 is expressed in head-specific epithelial cells and is a major component of the cytoskeletal structure that anchors nematocytes
Authors: López de Haro Maria S. , Salgado Luis M. , David Charles N. , Bosch Thomas C. G.
Published: April 2021

A cDNA clone encoding a 253 amino acid tropomyosin was isolated from Hydra in a differential screen for headspecific genes. The Hydra tropomyosin gene, designated trop1, is a single copy gene, lacks introns and is strongly expressed in tentacle-specific epithelial cells. Analysis of protein synthesis in head and gastric tissue indicated a high rate of tropomyosin synthesis in head tissue. Immunolocalization of tropomyosin in tentacle tissue revealed a cushion-like tropomyosin-containing structure within battery cells at the base of nematocytes. The structure appears to form part of the cytoskeletal anchor for nematocytes. Tropomyosin cushions were also observed in epithelial cells along the body column, which contain mounted stenotele nematocytes.
RNA polymerase II transcription is concentrated outside replication domains throughout S-phase
Authors: Wansink Derick G. , Manders Erik E. M. , Kraan Ineke van der , Aten Jacob A. , Driel Roel van , Jong Luitzen de
Published: April 2021

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.
Localization of a kinesin-related protein to the central pair apparatus of the Chlamydomonas reinhardtii flagellum
Authors: Johnson Karl A. , Haas Michael A. , Rosenbaum Joel L.
Published: April 2021

Affinity-purified polyclonal antibodies raised against the conserved motor domain of Drosophila kinesin (αHD) rec- ognized a 110 kDa component of the Chlamydomonas flagellar axoneme. Whole-mount immunogold labeling of splayed axonemes showed the striking localization of this antigen along one of the two microtubules of the central pair apparatus. Interestingly, the αHD antigen was also localized along the central axis of mutant axonemes lacking the central pair microtubules. These results suggest that a 110 kDa kinesin-related protein is a structural component of the flagellar central pair apparatus and that it is correctly targeted even in the absence of the central pair microtubules.
The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells
Authors: Sarria Alfonso J. , Lieber Jonathan G. , Nordeen Steven K. , Evans Robert M.
Published: April 2021

Human SW-13 cells express the intermediate filament protein vimentin in a mosaic pattern (Hedberg, K. K. and Chen, L. B. (1986). Exp. Cell Res. 163, 509-517). We have isolated SW-13 clones that do (vim+) or do not (vim−) syn- thesize vimentin as analyzed using anti-intermediate filament immunofluorescence, electron microscopy and two-dimensional gel analysis of detergent-extracted prepa- rations. Vimentin is the only cytoplasmic intermediate filament protein present in the vim+ cells, and the vim− cells do not contain any detectable cytoplasmic intermediate filament system. The presence or absence of intermediate filaments did not observably affect the distribution of mito- chondria, endoplasmic reticulum, microtubules or actin stress fibers when these structures were visualized by fluo- rescence microscopy. However, electron microscopy and anti-lamin A/C immunofluorescence studies showed that nuclear morphology in vim− cells was frequently charac- terized by large folds or invaginations, while vim+ cells had a more regular or smooth nuclear shape. When vim− cells were transfected with a mouse vimentin expression plasmid, the synthesis of a mouse vimentin filament network restored the smooth nuclear morphology charac- teristic of vim+ cells. Conversely, when vim+ cells were transfected with a carboxy-terminally truncated mutant vimentin, expression of the mutant protein disrupted the organization of the endogenous vimentin filaments and resulted in nuclei with a prominently invaginated mor- phology. These results indicated that in SW-13 cells the vimentin filament system affects the shape of the nucleus.
Involvement of cell-cell adhesion in the expression of the cell cohesion molecule gp80 in Dictyostelium discoideum
Authors: Desbarats Laurie , Brar Simuran K. , Siu Chi-Hung
Published: April 2021

Soon after the initiation of the developmental cycle of Dictyostelium discoideum, cells acquire EDTA-sensitive cellcell binding sites mediated by the glycoprotein gp24. Cells at the aggregation stage display a second type of cell adhesion site, the EDTA-resistant cell-cell binding sites, mediated by the glycoprotein gp80. The gene encoding gp80 is first turned on to a low basal level of expression in the preaggregation stage. At the onset of the aggregation stage, cells produce pulses of low levels of cAMP, which greatly augment the expression of gp80. To investigate the role of cell-cell adhesion in the regulation of gp80 expression, cells were developed in the presence of EDTA or carnitine to block the EDTA-sensitive cell binding sites. Alternatively, cell cohesion was disrupted by shaking lowdensity cultures at high shearing forces. In all three instances, gp80 was expressed at a substantially reduced level. In addition, exogenous cAMP pulses, which normally were capable of stimulating a precocious and enhanced expression of gp80, failed to restore the high level of gp80 expression. However, if the formation of cell-cell contact was permitted, exogenous cAMP pulses were able to rescue the expression of gp80 even when the cAMP signal relay was blocked. These results indicate that previous cell-cell contact, provided by the EDTA-sensitive binding sites, is required for the activation of the cAMP-mediated signal transduction pathway producing high levels of gp80 expression.
A calcineurin-like gene ppb1
+ in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning
Authors: Yoshida Tetsuya , Toda Takashi , Yanagida Mitsuhiro
Published: April 2021

A calcineurin (type 2B)-like protein phosphatase gene designated ppb1+ was isolated from the fission yeast Schizosac-charomyces pombe. The predicted amino acid sequence was 57% identical to rat PP2Bα. ppb1 null mutant could form colonies at 33°C but the size of the colonies was small at 22°C. Cytokinesis was greatly delayed at 22°C, and a large number of multi-septate cells were produced. The cell polarity control was impaired, causing branched cells. ppb1 null was virtually sterile. These phenotypes were rescued by a plasmid carrying the ppb1+ gene. Multi-septate cells were also produced in wild type at 22°C by cyclosporin A, an inhibitor of calcineurin. This drug effect was enhanced in stst1 null mutant, which was hypersensitive to various drugs and cations. ppb1 null was not affected by cyclosporin A, consistent with the hypothesis that ppb1 is its target. Double-mutant analysis indicated that ppb1 had a function related to that of two other phosphatases, type 1-like dis2 and 2A-like ppa2. ppb1 null-sts1 null showed the severe multi-septate phenotype in the absence of cyclosporin A. ppb1+ and sts1+ gene functions are related. The double mutant ppb1-sts5 was lethal, indicating that the ppb1+ gene shared an essential function with the sts5+ gene. Overexpression of ppb1+ caused anomalies in cell and nuclear shape, microtubule arrays and spindle pole body positioning in interphase cells. Thus the ppb1+ gene appears to be involved in cytokinesis, mating, transport, nuclear and spindle pole body positioning, and cell shape.
Regulation of myosin regulatory light chain phosphorylation via cyclic GMP during chemotaxis of Dictyostelium
Authors: Liu Gang , Newell Peter C.
Published: April 2021

Previous studies on the chemotactic movement of Dictyostelium have indicated a role for cyclic GMP in regulating the association of myosin II with the cytoskeleton. In this study we have examined the part played by phosphorylation of the 18 kDa myosin regulatory light chain in this event. Using streamer F mutant NP368 (which is deficient in the structural gene for cyclic GMP-specific phosphodiesterase) we find that, for the regulatory light chain kinase, the major peak of phosphorylation is delayed compared to the parental control strain XP55, occurring at 80 seconds rather than about 30 seconds in XP55. In two independently derived mutants that are unable to increase their cellular concentration of cyclic GMP (above basal levels) in response to a chemotactic stimulus of cyclic AMP (KI-10 and SA219), no increase in the phosphorylation of the light chain occurred, or movement of myosin II to the cytoskeleton. We also find a smaller peak of light chain phosphorylation that occurs within 10 seconds of cyclic AMP stimulation of the amoebae, and which is absent in the cyclic GMP-unresponsive strains. We conclude that cyclic GMP is involved in regulating light chain phosphorylation in this system. The possible significance of these findings is discussed and a model that relates these findings to published data on cytoskeletal myosin changes during chemotaxis is presented.