bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of the role of β1 integrins in cell-cell adhesion
Authors: Weitzman Jonathan B. , Chen Annabel , Hemler Martin E.
Published: April 2021

Various β1 integrins (VLA-2, VLA-3, VLA-4) have been suggested to bind directly to themselves or to each other, thus mediating cell-cell adhesion. Here we expressed the human α2 and α3 subunits in three different cell lines (human erythroleukemia K562, human rhabdomyosar-coma RD and Chinese hamster ovary CHO cells). Although cell surface α2β1 and α3β1 in the transfectants mediated adhesion to matrix ligands (collagen or laminin 5, respec-tively), in no case did we observe enhanced cell-cell adhesion. In the presence of a range of different divalent cation concentrations, stimulatory anti-β1 antibodies or anti-α3 antibodies, VLA-2 and VLA-3 still did not appear to interact directly, through either heterophilic (i.e. VLA-3/VLA-2) or homophilic (i.e. VLA-3/VLA-3) mechanisms, to mediate cell-cell adhesion. Furthermore, in some but not all α3 transfectants we observed an unexpected decrease in cell-cell adhesion, suggesting a novel anti-adhesive function. This inhibitory effect was not observed for α2 transfection nor when the α3 cytoplasmic tail was exchanged with that of another integrin α subunit. Finally, no evidence for VLA-4/VLA-4 mediated cell-cell adhesion was observed using α4-transfected K562 and CHO cells. In conclusion, using many different combinations of cell lines, we found that cell-cell adhesion mediated by direct integrin/integrin interaction is not a widespread phenome-non, and is not observable in standard cell-cell adhesion assays. Furthermore, in some cell combinations, α3 expression may actually cause diminished cell-cell adhesion.
Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones
Authors: Blavier L. , Delaissé J. M.
Published: April 2021

A key event in bone resorption is the recruitment of osteoclasts to future resorption sites. We follow here the migration of preosteoclasts from the periosteum to the developing marrow cavity of fetal mouse metatarsals in culture, and investigate the role of proteinases and demineralization in this migration. Our approach consisted in testing inhibitors of proteinases and demineralization on the migration kinetics. Migration was monitored by histomorphometry and the (pre)osteoclasts were identified by their tartrate resistant acid phosphatase (TRAP) activity. At the time of explantation, TRAP+ cells (all mononucleated) are detected only in the periosteum, and the core of the diaphysis (future marrow cavity) consists of calcified cartilage. Upon culture, TRAP+ cells (differentiating progressively into multinucleated osteoclasts) migrate through a seam of osteoid and a very thin and discontinuous layer of mineral, invade the calcified cartilage and transform it into a ‘marrow’ cavity; despite the passage of maturing osteoclasts, the osteoid develops into a bone collar. The migration of TRAP+ cells is completely prevented by matrix metalloproteinase (MMP) inhibitors, but not by a cysteine proteinase inhibitor, an inhibitor of carbonic anhydrase, or a bisphosphonate. The latter three drugs inhibit, however, the resorptive activity of mature osteoclasts at least as efficiently as do the MMP inhibitors, as assessed in cultures of calvariae and radii. Furthermore, in situ hybridizations reveal the expression of 2 MMPs, gelatinase B (MMP-9 or 92 kDa type IV collagenase) in (pre)osteoclasts, and interstitial collagenase (MMP-13) in hypertrophic chondrocytes. It is concluded that only MMPs appear obligatory for the migration of (pre)osteoclasts, and that this role is distinct from the one MMPs may play in the subosteoclastic resorption compartment. We propose that this new role of MMPs is a major component of the mechanism that determines where and when the osteoclasts will attack the bone.
Localization of myosin II A and B isoforms in cultured neurons
Authors: Rochlin M. William , Itoh Kazuyuki , Adelstein Robert S. , Bridgman Paul C.
Published: April 2021

Tension generated by growth cones regulates both the rate and the direction of neurite growth. The most likely effectors of tension generation are actin and myosins. We are investigating the role of conventional myosin in growth cone advance. In this paper we report the localization of the two most prominent isoforms of brain myosin II in growth cones, neurites and cell bodies of rat superior cervical ganglion neurons. Affinity purified polyclonal anti-bodies were prepared against unique peptide sequences from human and rat A and B isoforms of myosin heavy chain. Although each of these antibodies brightly stained nonneuronal cells, antibodies to myosin heavy chain B stained neurons with greater intensity than antibodies to myosin heavy chain A. In growth cones, myosin heavy chain B was most concentrated in the margin bordering the thickened, organellerich central region and the thin, actinrich peripheral region. The staining colocalized with actin bundles proximal and distal to the marginal zone, though the staining was more prominent proximally. The trailing edge of growth cones and the distal portion of the neurite often had a rimmed appearance, but more proximal regions of neurites had cytoplasmic labelling. Localizing MHC-B in growth cones previously monitored during advance (using differential interference contrast microscopy) revealed a positive correlation with edges at which retraction had just occurred and a negative correlation with lamellipodia that had recently undergone protrusion. Cell bodies were brightly labelled for myosin heavy chain B. Myosin heavy chain A staining was dimmer and its colocalization with filamentous actin bundles in growth cones was less striking than that of myosin heavy chain B. Growth cones stained for both myosin heavy chain A and B revealed that the two antigens overlapped frequently, but not exclusively, and that myosin heavy chain A lacked the elevation in the marginal zone that was characteristic of myosin heavy chain B. The pattern of staining we observed is consistent with a prominent role for myosin heavy chain B in either generating tension between widely separated areas of the growth cone, or bundling of actin filaments, which would enable other motors to effect this tension. These data support the notion that conventional myosin is important in growth cone advance and turning.
The hepatocyte growth factor/scatter factor (HGF/SF) receptor, met, transduces a morphogenetic signal in renal glomerular fibromuscular mesangial cells
Authors: Kolatsi-Joannou Maria , Woolf Adrian S. , Hardman Patricia , White Stanley J. , Gordge Michael , Henderson Robert M.
Published: April 2021

Previous studies have demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) is secreted by mes-enchymal cells and that it elicits motility, morphogenesis and proliferation of epithelia expressing the met receptor. We now report that HGF/SF may act as an autocrine factor in fibromuscular renal mesangial cells. These cells mechan-ically support glomerular endothelia, control the rate of plasma ultrafiltration and are implicated in the pathogen-esis of a variety of chronic renal diseases. We detected met protein in the vascular stalk of metanephric glomeruli and in the mature mesangium. Mesangial lines from a mouse transgenic for a temperature-sensitive simian virus 40 T antigen expressed met mRNA and protein, and recombi-nant HGF/SF phosphorylated the met receptor tyrosine kinase. Cells were immortal in the permissive condition and HGF/SF enhanced proliferation in a defined medium. In the absence of the immortalising protein, division ceased and recombinant HGF/SF caused multipolar cells to become bipolar. The factor diminished stress fibres, their focal contacts and immunostaining for extracellular fibronectin, hence suggesting reduced substratum adhesion and enhanced motility. Mesangial lines also expressed HGF/SF mRNA and secreted bioactive factor; immuno-cytochemistry showed both ligand and receptor in individ-ual cells. HGF/SF blocking antibody aggregated the cells, suggesting that mesangial-derived factor affects basal cell conformation in an autocrine manner. We conclude that mesangial cells express both HGF/SF and met, and the factor induces morphogenesis of cultured mesangial cells. Therefore HGF/SF may have an autocrine role in mesangial biology but further studies are now required to investigate the potential importance of the factor in vivo.
Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology
Authors: Takegawa Kaoru , DeWald Daryll B. , Emr Scott D.
Published: April 2021

We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S. pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34Δ strain. S. pombe Vps34p-specific antiserum detects a single protein in cells of ∼90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate, but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phos-phatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.
Identification of a Ca2+-binding light chain within Chlamydomonas outer arm dynein
Authors: King Stephen M. , Patel-King Ramila S.
Published: April 2021

We describe here the molecular cloning of the Mr 18,000 dynein light chain from the outer arm of Chlamydomonas flagella. In vivo, this molecule is directly associated with the γ dynein heavy chain. Sequence analysis indicates that this light chain is a novel member of the calmodulin superfamily of Ca2+ binding regulatory proteins; this molecule is 42, 37 and 36% identical to calmodulin, centrin/caltractin and troponin C, respectively, and also shows significant similarity to myosin light chains. Although four helix-loop-helix elements are evident, only two conform precisely to the EF hand consensus and are therefore predicted to bind Ca2+ in vivo. In vitro Ca2+ binding studies indicate that this dynein light chain (expressed as a C-terminal fusion with maltose binding protein) has at least one functional Ca2+ binding site with an apparent affinity for Ca2+ of ∼3×10−5M. Within the Chlamydomonas flagellum, the transition from an assymmetric to a symmetric waveform (which implies an alteration in dynein activity) is mediated by an increase in intraflagellar Ca2+ from 10−6 to 10−4 M; this transition is altered in mutants that lack the outer arm. The data presented here suggest that a Ca2+-dependent alteration in the interaction of this dynein light chain with the motor containing heavy chain may affect outer arm function during flagellar reversal.
Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins
Authors: Obata Shuichi , Sago Haruhiko , Mori Nozomu , Rochelle Julie M. , Seldin Michael F. , Davidson Mari , John Tom St , Taketani Shigeru , Suzuki Shintaro T.
Published: April 2021

Cell adhesion and several other properties of a recently identified cadherin-related protein, protocadherin Pcdh2, were characterized. A chimeric Pcdh2 in which the original cytoplasmic domain was replaced with the cytoplasmic domain of E-cadherin was expressed in mouse L cells. The expressed protein had a molecular mass of about 150 kDa and was localized predominantly at the cell periphery, as was the wild-type Pcdh2. In a conventional cell aggregation assay, the transfectants showed cell aggregation activity comparable to that of classical cadherins. This activity was Ca2+-dependent and was inhibited by the addition of anti-Pcdh2 antibody, indicating that the chimeric Pcdh2, and probably the wild-type Pcdh2, has Ca2+-dependent cell aggregation activity. Mixed cell aggregation assay using L cells and different types of transfectants showed that the activity of Pcdh2 was homophilic and molecular type specific and that Pcdh2 transfectants did not aggregate with other types of transfectants or with L cells. In immunoprecipitation, the chimeric Pcdh2 co-precipitated with a 105 kDa and a 95 kDa protein, whereas wild-type Pcdh2 co-precipitated with no major protein. Pcdh2 was easily solubilized with non-ionic detergent, in contrast to the case of classical cadherins. On immunofluorescence microscopy, the somas of Purkinje cells were diffusely stained with anti-human Pcdh2 antibody. Mouse Pcdh1 and Pcdh2 were mapped to a small segment of chromosome 18, suggesting that various protocadherins form a gene cluster at this region. The present results suggest that Pcdh2, and possibly other protocadherins as well as proto-cadherin-related proteins such as Drosophila fat, mediate Ca2+-dependent and specific homophilic cell-cell interaction in vivo and play an important role in cell adhesion, cell recognition, and/or some other basic cell processes.
Integrin signaling: roles for the cytoplasmic tails of αIIbβ3 in the tyrosine phosphorylation of pp125FAK
Authors: Leong Lilley , Hughes Paul E. , Schwartz Martin A. , Ginsberg Mark H. , Shattil Sanford J.
Published: April 2021

pp125FAK (focal adhesion kinase) a protein tyrosine kinase that may mediate cellular responses to adhesion, is activated and tyrosine-phosphorylated when platelets adhere to fibrinogen via the integrin, αIIbβ3. To determine whether either of the cytoplasmic tails of αIIbβ3 regulates FAK phosphorylation, CHO cells were stably transfected with αIIbβ3 or various cytoplasmic tail truncation mutants. Cells expressing wild-type αIIbβ3 or αIIbβ3 that lacked the COOH-terminal 13 or 18 residues of the 20 residue αIIb tail adhered to and spread on fibrinogen or on an anti-αIIb antibody, and FAK became tyrosine-phosphorylated. FAK also became phosphorylated in adherent cells lacking the COOH-terminal 35 or 39 residues of the 47 residue β3 tail, although the extent of phosphorylation was reduced by about 50% in the latter mutant. Little or no FAK phos-phorylation was observed if 46 residues were deleted from the β3 tail. None of these β3 truncation mutants spread on the anti-αIIb antibody. When cells with wild-type αIIbβ3 or truncated β3 were detached from a surface, FAK became rapidly dephosphorylated. In contrast, FAK remained phosphorylated in the two αIIb truncation mutants for up to 90 minutes in suspension. This persistent phosphoryla-tion was not due to occupancy of αIIbβ3 by adhesive ligands because it was also observed with an αIIb tail truncation mutant that contained an additional mutation in the extra-cellular portion of the receptor that prevents ligand binding. These studies demonstrate that: (1) the β3 cyto-plasmic tail, including the membrane-proximal portion, is involved in initiation of FAK phosphorylation; (2) FAK phosphorylation can be initiated by cell adhesion in the absence of cell spreading; and (3) the membrane-distal portion of the αIIb cytoplasmic tail may normally function to dampen FAK phosphorylation in non-anchored cells.
Control of N-cadherin-mediated intercellular adhesion in migrating neural crest cells in vitro
Authors: Monier-Gavelle Frédérique , Duband Jean-Loup
Published: April 2021

Dispersion of neural crest cells and their ultimate regroup-ment into peripheral ganglia are associated with precisely coordinated regulations both in time and space of the expression and function of cell adhesion receptors. In par-ticular, the disappearance of N-cadherin from the cell surface at the onset of migration and its reexpression during cell aggregation suggest that, during migration, N-cadherin expression is repressed in neural crest cells. In the present study, we have analyzed in vitro the mechanism of control of N-cadherin expression and function in migrating neural crest cells. Although these cells moved as a dense popula-tion, each individual did not establish extensive and permanent intercellular contacts with its neighbors. However, cells synthesized and expressed mature N-cadherin molecules at levels comparable to those found in cells that exhibit stable intercellular contacts, but in contrast to them, the bulk of N-cadherin molecules was not connected with the cytoskeleton. We next determined which intracellular events are responsible for the instability of the N-cadherin junctions in neural crest cells using various chemical agents known to affect signal transduction processes. Agents that block a broad spectrum of serine-threonine kinases (6-dimethylaminopurine, H7 and stau-rosporine) or that affect selectively protein kinases C (bisin-dolylmaleimide and sphingosine), inhibitors of protein tyrosine kinases (erbstatin, herbimycin A, and tyrphostins), and inhibitors of phosphatases (vanadate) all restored tight cell-cell associations among neural crest cells, accompanied by a slight increase in the overall cellular content of N-cadherin and its accumulation to the regions of intercellu-lar contacts. The effect of the kinase and phosphatase blockers was inhibitable by agents known to affect protein synthesis (cycloheximide) and exportation (brefeldin A), indicating that the restored cell-cell contacts were mediated chiefly by an intracellular pool of N-cadherin molecules recruited to the membrane. Finally, N-cadherin molecules were constitutively phosphorylated in migrating neural crest cells, but their level and state of phosphorylation were apparently not modified in the presence of kinase and phosphatase inhibitors. These observations therefore suggest that N-cadherin-mediated cell-cell interactions are not stable in neural crest cells migrating in vitro, and that they are under the control of a complex cascade of intracellular signals involving kinases and phosphatases and probably elicited by surface receptors.
Message of nexin 1, a serine protease inhibitor, is accumulated in the follicular papilla during anagen of the hair cycle
Authors: Yu Da-Wen , Yang Tian , Sonoda Tadashige , Gaffney Kevin , Jensen Pamela J. , Dooley Tom , Ledbetter Steve , Freedberg Irwin M. , Lavker Robert , Sun Tung-Tien
Published: April 2021

A group of specialized mesenchymal cells located at the root of the mammalian hair follicle, known as the follicu-lar or dermal papillary cells, are involved in regulating the hair cycle, during which keratinocytes of the lower follicle undergo proliferation, degeneration and regrowth. Using the arbitrarily primed-PCR approach, we have identified a 1.3 kb messenger RNA that is present in large quantities in cultured rat follicular papillary cells, but not in skin fibro-blasts. This mRNA encodes nexin 1, a potent protease inhibitor that can inactivate several growth-modulating serine proteases including thrombin, urokinase and tissue plasminogen activator. In situ hybridization showed that nexin 1 message is accumulated in the follicular papilla cells of anagen follicles, but is undetectable in keratinocytes or other skin mesenchymal cells. In addition, nexin 1 message level varies widely among several immortalized rat vibrissa papillary cell lines, and these levels correlate well with the reported abilities of these cell lines to support in vivo follicular reconstitution. These results suggest a possible role of nexin 1 in regulating hair follicular growth.
Hepatocyte growth factor stimulates extensive development of branching duct-like structures by cloned mammary gland epithelial cells
Authors: Soriano J. V. , Pepper M. S. , Nakamura T. , Orci L. , Montesano R.
Published: April 2021

Although epithelial-mesenchymal (stromal) interactions are thought to play an important role in embryonic and postnatal development of the mammary gland, the underlying mechanisms are still poorly understood. To address this issue, we assessed the effect of fibroblast-derived diffusible factors on the growth and morphogenetic properties of a clonally derived subpopulation (clone TAC-2) of normal murine mammary gland (NMuMG) epithelial cells embedded in collagen gels. Under control conditions, TAC-2 mammary gland epithelial cells suspended within collagen gels formed either irregularly shaped cell aggregates or short branching cord-like structures. Addition of conditioned medium from Swiss 3T3 or MRC-5 fibroblasts dramatically stimulated cord formation by TAC-2 cells, resulting in the development of an extensive, highly arborized system of duct-like structures, which in appropriate sections were seen to contain a central lumen. The effect of fibroblast conditioned medium was completely abrogated by antibodies against hepatocyte growth factor (also known as scatter factor), a fibroblast-derived polypeptide that we have previously shown induces tubulogenesis by Madin-Darby canine kidney epithelial cells. Addition of exogenous recombinant human hepatocyte growth factor to collagen gel cultures of TAC-2 cells mimicked the tubulogenic activity of fibroblast conditioned medium by stimulating formation of branching duct-like structures in a dose-dependent manner, with a maximal 77-fold increase in cord length at 20 ng/ml. The effect of either fibroblast conditioned medium or hepatocyte growth factor was markedly potentiated by the simultaneous addition of hydrocortisone (1 μg/ml), which also enhanced lumen formation. These results demonstrate that hepatocyte growth factor promotes the formation of branching duct-like structures by mammary gland epithelial cells in vitro, and suggest that it may act as a mediator of the inducing effect of mesenchyme (or stroma) on mammary gland development.
Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs
Authors: Zernicka-Goetz Magdalena , Ciemerych Maria A. , Kubiak Jacek Z. , Tarkowski Andrzej K. , Maro Bernard
Published: April 2021

Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained arrested in M-phase, the oocytes fused with fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte activation was still present at the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involved in calcium release triggered by a sperm component at fertilization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage.
The glycophosphatidylinositol anchor affects the conformation of thy-1 protein
Authors: Barboni E. , Rivero B. Pliego , George A. J. T. , Martin S. R. , Renouf D. V. , Hounsell E. F. , Barber P. C. , Morris R. J.
Published: April 2021

Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27×10−3 s−1 to 2.39×10−3 s−1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class.
Behavior of mitochondria in synchronized cells of Chlamydomonas Reinhardtii (chlorophyta)
Authors: Ehara Tomoko , Osafune Tetsuaki , Hase Eiji
Published: April 2021

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12 hour:12 hour light:dark regimen. Behavior of mitochondria in these cells was studied by fluorescence microscopy using a mitochondrial membrane-binding fluorescent dye, dimethylaminostyrylmethylpyridiniumiodine (DASMPI), as well as by electron microscopy.

Following time courses of change in frequency of occurrence of five typical morphologies of mitochondria in synchronized cells, strikingly dynamic behavior of mitochondria was demonstrated. The five types are (A) a giant global mitochondrion with large matrix and peripherally localized cristae, a part of which is in close contact with the nucleus, (B) a mitochondrion composed of thick-corded bodies connected to each other, a part of which is in contact with the nucleus, (C) thin-corded forms with a few branches, (D) small lump forms scattered in the cytoplasm, and (E) stringy forms with intricate branchings extended throughout the cytoplasm. During the early half of the light period, changes of C→B→C→D occur, while the inversely sequential changes of D→C→B→C proceed during the later half of the light period. The appearance of the B-type mitochondrion is accompanied by a transient decrease of O2-uptake activity of cells. The early appearing B-type mitochondrion is temporarily turned into a giant A-type mitochondrion, concomitant with discharge of membranes into the cytoplasm and their retake by the A-type form in the process of reversion to B-type. In the reversion process, partitioning membranes are also formed in the large matrix of A-type mitochondrion. Toward the end of the light period, stringy E-type mitochondria are formed from C-type ones, and apportioned between two dividing protoplasts during an early phase of the dark period. The E-type forms are cut into D-type forms immediately prior to the following cell division into four daughters.

Possible significance of the formation of B- and A-type mitochondrion is discussed.
The organization of spliceosomal components in the nuclei of higher plants
Authors: Beven Alison F. , Simpson Gordon G. , Brown John W. S. , Shaw Peter J.
Published: April 2021

To analyze the organization of spliceosomal snRNPs in plant nuclei, we have used both immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B′′, and in situ hybridization with anti-sense probes to conserved regions of U1, U2 and U6 snRNAs. The organization comprises a fibrous inter-chromatin network, which may include both interchromatin fibrils and granules, and very prominent nuclear and nucleolar-associated bodies. Double labelling with an anti-p80 coilin antibody shows that these are coiled bodies. Dynamic changes in the labelling pattern were observed through the cell cycle, and in response to and on recovery from heat shock. The similarity of this organization to that observed in mammalian nuclei is strong evidence that it is fundamental to the processing of pre-mRNA in eucaryotes in general.
Expression of Interstitial Collagenase is restricted to skeletal tissue during mouse embryogenesis
Authors: Mattot Virginie , Raes Marie Berthe , Henriet Patrick , Eeckhout Yves , Stehelin Dominique , Vandenbunder Bernard , Desbiens Xavier
Published: April 2021

Collagenases are thought to be involved in physiological and pathological processes that require extracellular matrix remodeling. Using the in situ hybridization technique, we describe the expression of interstitial collagenase gene during mouse embryogenesis between E6.5 and E17. We demonstrate that interstitial collagenase expression is exclusively detected in one event, namely the onset of bone formation. Transcripts accumulate in hypertrophied chondrocytes, found in the mature cartilaginous matrix of long-bone growth plates or ribs, and in osteoblasts and/or in endothelial cells that have migrated into the shafts of developing long bones. The expression of the tissue inhibitor of metalloproteinases (TIMP-2) gene precedes the expression of interstitial collagenase in developing bones. These data suggest that interstitial collagenase plays a specific role in bone development and that the tight regulation of its activity during development is achieved not only by post-translational mechanisms with TIMPs, as previously suggested, but also at the transcriptional level.
The behavior of chick gastrula mesodermal cells under the unidirectional tractive force parallel to the substrata
Authors: Toyoizumi Ryuji , Takeuchi Shigeo
Published: April 2021

Advancement of leading lamellae of a migratory cell inevitably causes a strain inside the cell body. We investigated the effect of the tension arisen inside a mesodermal cell on its behavior by pulling the cell body unidirectionally along the substratum.

Chick gastrula mesodermal cells, known as highly migratory, were dissociated into single cells in sodium citrate buffer, conjugated with paramagnetic beads activated by tosyl-residue (4.5 μm in diameter) and seeded onto coverglasses coated with fibronectin. After the cells spread on the substratum and protruded cellular processes in all directions, they were exposed to a non-uniform magnetic field by a magnet. Thus the cells bearing the beads were pulled with a force in the order of 10−10 N. The behavior of such cells was recorded with a time-lapse video taperecorder and assessed quantitatively.

Shortly after the magnetic force was applied, the beads stuck to the cells were aligned in tandem along the line of magnetic force at the site for the magnet. Subsequently, they frequently came to extend their leading lamella precisely counter to the traction on the line of the beads. Observation with scanning electron microscope revealed that a large part of the beads attached to the cells were wrapped in the cell membrane. In this condition, the cells were stretched locally between the attachment site of the beads and adhesion plaques beneath the leading edge, which was formed in a direction away from the traction. It was proved statistically that such cells tended to locomote away from the magnet at the 0.1% significance level with Hotelling’s T2-test. In contrast, the mesodermal cells free of the artificial traction in three kinds of control experiments did not show such a preference in the direction of locomotion.

These results proved that migratory cells tended to move in the direction away from the tractive force parallel to the substratum, suggesting that advancement of a leading lamella is accelerated when it is stretched along the direction of projection by a mechanical force of sufficient strength. Implication of this finding to the mechanism of cell locomotion will be discussed.
Stimulus-dependent alterations in macrophage microtubules: increased tubulin polymerization and detyrosination
Authors: Robinson John M. , Vandré Dale D.
Published: April 2021

Murine macrophage microtubules are very dynamic. The majority of the microtubules (≈80%) exist in a rapidly depolymerizing pool ( ≈30 seconds). The remaining 20% of the microtubules are in a more slowly depolymerizing pool ( ≈7 minutes). Macrophage microtubules are responsive to cell stimulation with phorbol esters; upon cell stimulation there is a rapid increase in total microtubule polymer and number. In addition there is stimulus-induced detyrosination of α-tubulin in macrophage microtubules that is rapid and essentially complete, occurring in all microtubules rather than a subset of microtubules. Detyrosination of the macrophage microtubules in response to phorbol esters does not confer increased stability to these microtubules, since treated cells have nocodazole-induced depolymerization kinetics similar to that in non-stimulated macrophages. Regrowth of microtubules following washout of nocodazole is also rapid. Interestingly the regrown microtubules are initially in the tyrosinated form even in the presence of phorbol ester. These experiments provide in vivo support for the model that detyrosination of α-tubulin occurs in polymeric tubulin while retyrosination occurs in dimeric tubulin. Macrophage microtubules also demonstrate an unusually rapid response to extracellular stimuli and thus provide a unique model system in which to examine signal transduction events and modulation of microtubules.
Secretion from permeabilised mast cells is enhanced by addition of gelsolin: contrasting effects of endogenous gelsolin
Authors: Borovikov Yurii S. , Norman James C. , Price Leo S. , Weeds Alan , Koffer Anna
Published: April 2021

Permeabilised rat mast cells were exposed to gelsolin and its N-terminal half (S1-3), proteins that sever actin filaments in a calcium-dependent and independent manner, respectively. Gelsolin and S1-3 induced a decrease in cellular F-actin content and an increase in the extent of the secretory response. The calcium sensitivities of both these effects were consistent with the differential calcium requirements of the two proteins. Segment 1 (S1), which binds G-actin and caps filaments but does not sever them, did not show these effects. Thus, secretion of mast cells is promoted as a consequence of the severing activity of exogenous gelsolin or S1-3.

Most of the endogenous gelsolin remained within permeabilised, washed mast cells and its distribution in resting state was predominantly cortical. Addition of calcium in the absence of MgATP did not reduce the F-actin content; by contrast, calcium with MgATP induced F-actin loss that was unaffected by the presence of anti-gelsolin. Because this antibody inhibits the severing activity of gelsolin, these results indicate that in permeabilised mast cells the severing activity of the remaining endogenous gelsolin is not involved in cortical actin filaments disassembly. Upon exposure to GTP-γ-S in the absence of calcium, the content of cortical gelsolin was reduced. This parallels our previous observation of a GTP-γ-S induced reduction of cortical actin filaments followed by their relocation to the cell’s interior (Norman et al. (1994)J. Cell Biol. 126, 1005-1015) and suggests that actin redistribution may be a consequence of dissociation of gelsolin caps brought about by activation of a GTP-binding protein.
Novel centrosomal protein reveals the presence of multiple centrosomes in turkey (Meleagris Gallopavo) bnbn binucleated erythrocytes
Authors: Woods Catherine M. , Zhu Jian , Coleman Thomas , Bloom Stephen E. , Lazarides Elias
Published: April 2021

The phenotype of the bnbn hemolytic anemia mutation in the domestic turkey is manifested as binucleation specifically in the definitive erythrocyte lineage, most likely as the consequence of anomolous centrosomal activity (Bloom et al., 1970; Searle and Bloom, 1979). Here we have identified in turkey two variants of the novel, centrosomally-associated erythroid-specific protein p23. One variant is Ca2+-sensitive and is highly homologous to its chick counterpart (Zhu et al., 1995, accompanying paper). The other, p21, is a truncated form resulting from a 62 amino acid deletion from the 3′ end and a 40 amino acid insertion at the 5′ end, and appears to lack Ca2+-sensitivity. These proteins are localized at the marginal band, centrosomes and nuclear membrane of differentiated erythrocytes. Anti-p23/p21 immunofluorescence revealed the presence of multiple centrosomes in bnbn eythrocytes. We therefore undertook a detailed genetic analysis to determine whether the p21 variant represented the bn mutation. Initial tests of normal BnBn and mutant bnbn individuals suggested that the p23/p21 proteins might be encoded by the Bn/bn genes. However, further genetic tests demonstrated independent segregation for these two genetic loci. Thus, these proteins are encoded by the heretofore undescribed genes, p23/p21, mapping to an autosomal locus in the turkey genome.
Transverse pattern of microfilament bundles induced in epitheliocytes by cylindrical substrata
Authors: Svitkina Tatyana M. , Rovensky Yury A. , Bershadsky Alexander D. , Vasiliev Jury M.
Published: April 2021

Cylindrical culture substrata are known to induce longitudinal orientation of polarized fibroblasts and corresponding alignment of actin microfilament bundles in these cells. We studied microfilament bundle distribution in two cell types, fibroblasts and epitheliocytes, spread on two kinds of anisotropic substrata, quartz glass cylinders with a diameter 32 μm and narrow (25-40 μm wide) flat glass adhesive strips with non-adhesive borders. Rat embryo and human diploid fibroblasts, as expected, formed predominantly longitudinally aligned bundles on both substrata. In contrast, transverse bundles on cylinders and randomly oriented bundles on flat strips were formed in IAR-2 and MDCK epithelial cells. We interpret these data as showing that the epitheliocyte attempts to override the guiding influence of anisotropic substrata. The microfilament bundle pattern on cylinders depends on the integrity of the microtubules. Colcemid-induced microtubule depolymerization caused formation of longitudinal as well as transverse bundles both in fibroblasts and epitheliocytes, thus diminishing the differences in microfilament bundle patterns in two cell types. These results show that microtubules control the cell-type-specific distribution of microfilament bundles both in polarized fibroblasts and in discoid epitheliocytes. However, the results of this control are opposite: microtubules enhance cell polarization in fibroblasts, but prevent it in epithelial cells.
Relationship between sperm atp content and motility of carp spermatozoa
Authors: Perchec G. , Jeulin C. , Cosson J. , André F. , Billard R.
Published: April 2021

Carp spermatozoa are immotile in seminal plasma or in saline solution of high osmolality (>400 mosmol kg−1). These ‘quiescent’ spermatozoa initiate a progressive forward motility when transferred in freshwater or in saline solution with low osmolality (<160 mosmol kg−1). In this study we investigated ‘in vitro’ the relationship between sperm ATP content (measured by bioluminescence) and sperm motility (analysed by videomicroscopy). Sperm ATP content remained high in the immobilizing medium (200 mM KCl, Tris 30 mM, pH 8.0) where no flagellar movement occurs. Dilution of these spermatozoa in the activating medium (45 mM NaCl, 5 mM KCl, Tris 30 mM, pH 8.0) triggered forward motility which varied with temperature. At 20°C, sperm ATP content decreased rapidly during the progressive forward motility phase from 12 to 4 nmol/108 spermatozoa, concomitantly with decreases in velocity (130 to 10 μm s−1) and the beat frequency (50 to 7 Hz). An inhibitor of mitochondrial respiration (KCN 10 mM) produced a drop in sperm ATP content irrespective of the incubation medium (activating or immobilizing). A second phase of sperm motility in the activating medium was induced following a previous transfer of spermatozoa into a medium of high osmolality for a few minutes prior to the second phase. Within 10 minutes, spermatozoa recover 90% of the initial ATP level as well as forward motility. These results suggest that motility of carp spermatozoa depends on sperm ATP synthesized by mitochondrial respiration mainly stored before activation. In low osmolality conditions, the mitochondrial oxidative phosphorylation is unable to compensate for the ATP hydrolysis required to sustain motility. The increase in osmolality surrounding spermatozoa probably blocks the dynein ATPases and allows an ATP ‘regeneration’ as a result of mitochondrial respiration.
Atp-dependent reorganization of human sperm nuclear chromatin
Authors: Banerjee Subhasis , Smallwood Alan , Hultén Maj
Published: April 2021

Chromosomes in terminally differentiated mammalian spermatozoa are extensively condensed by protamines but a small proportion of histones remain. We examined the primary organization of somatic-type chromatin in lysolecithin-permeabilized human sperm nuclei and report that nucleosomes are closely packed with a periodicity of ∼150 bp. Incubation of nuclei in the presence of exogenous Mg2+ and ATP induced chromatin reorganization leading to an increase in spacing of the nucleosomes to ∼190 bp. This ATP-dependent chromatin rearrangement involved phosphorylation of both protamine and histone H2a. Increase in linker length between nucleosomes correlated with the phosphorylation of H2aX, the major H2a variant in human spermatozoa, predominantly at the C-terminal end. Chromatin reorganization was independent of detectable nuclear dispersion, which is an early chromosomal event in male pronuclear formation during fertilization.
Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides
Authors: Rabouille Catherine , Hui Norman , Hunte Felicia , Kieckbusch Regina , Berger Eric G. , Warren Graham , Nilsson Tommy
Published: April 2021

The distribution of β1,2 N-acetylglucosaminyltransferase I (NAGT I), α1,3-1,6 mannosidase II (Mann II), β1,4 galactosyltransferase (GalT), α2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.
Both cdc2 and cdk2 promote S phase initiation in Xenopus egg extracts
Authors: Chevalier S. , Tassan J.-P. , Cox R. , Philippe M. , Ford C.
Published: April 2021

Xenopus egg extracts induce S phase DNA replication in added sperm pronuclei in a highly regulated manner, similar to events in vivo. Removal of cyclin-dependant kinases (cdks) or cdk2 from these extracts using affinity matrices severely inhibits initiation of S phase. We have used p13suc1 beads to remove both cdk2 and cdc2 proteins from egg extracts and developed a method to replace either protein alone to assess their capacity to initiate DNA replication. Re-addition of either cdk2 or cdc2 proteins to depleted extracts, through translation of their respective mRNAs, restimulated replication, judged by both total synthesis and labelling index. An ATP-binding-site mutant cdk2 mRNA (cdk2.R33) failed to stimulate replication and inhibited S phase initiation in mock-depleted extracts. Both human and Xenopus cdc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the stimulation induced requires cdc2 catalytic activity, though not its mitotically active form. Rescue of replication by p34cdc2 is also observed in extracts depleted of cdks with a cdk2 antibody, which still retain much of their endogenous cdc2 protein. We conclude that newly synthesised p34cdc2, but not the inherited ‘old’ form, can induce S phase and in this form may overlap in function with p33cdk2.
‘Chromosomal puffing’ in diploid nuclei of Drosophila melanogaster
Authors: Gunawardena Shermali , Heddle Elizabeth , Rykowski Mary C.
Published: April 2021

In situ hybridization has become a powerful technique for dissecting nuclear structure. By localizing nucleic acids with high precision, it is possible to infer the native structure of chromosomes, replication factories and transcript processing complexes. To increase the value of this technique, we have established the limits of resolution of two-color in situ hybridization to chromosomal DNA in diploid chromosomes of Drosophila embryonic nuclei. Using high-resolution 3-dimensional optical microscopy and computational image analysis, we establish that we can distinguish the location of chromosomal sequences that lie 27-29 kb apart within a 40 kb transcription unit with an accuracy of about 100 nm. Contrary to observations made in mammalian tissue culture cells, we find that when the gene is expressed, it assumes an open configuration, and that the extent of decondensation is variable from chromosome to chromosome. Further experiments suggest that variation in gene structure results from asynchrony in transcriptional elongation. We suggest that the phenomenon we observe is the diploid analog to chromosomal puffing that occurs in the transcriptionally active regions of Drosophila polytene chromosomes.