bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin sperm axonemes
Authors: Ishijima Sumio , Kubo-Irie Miyoko , Mohri Hideo , Hamaguchi Yukihisa 
Published: April 2021

ABSTRACT
Active sliding between doublet microtubules of sea urchin sperm axonemes that were demembranated with Triton X-100 in the presence or absence of calcium was induced with ATP and elastase at various concentrations of Ca2+ to examine the effects of Ca2+ on the direction of the power stroke of the dynein arms. Dark-field light microscopy of microtubule sliding revealed that the sliding from the axonemes demembranated with Triton and millimolar calcium and disintegrated with ATP and elastase showed various patterns of sliding disintegration, including loops of doublet microtubules formed near the head or the basal body. These loops were often thicker than the remaining axonemal bundle. In contrast, only thinner loops were found from the axonemes demembranated with Triton in the absence of calcium and disintegrated with ATP and elastase at high Ca2+ concentrations. Electron microscopic examination of the direction of microtubule sliding showed that the doublet microtubules in the axonemes demem-branated in the presence of millimolar calcium moved toward the base of the axonemes by the dynein arms on the adjacent doublet microtubule as well as by their own dynein arms. Doublet microtubules in the axonemes demembranated in the absence of calcium moved toward the base of the axonemes only by their own dynein arms. Similar observations have been obtained from the axonemes from which the outer dynein arms were selectively extracted. From these observations, we can conclude that the dynein arms generate force in both directions and this feature of the dynein arms arises from at least the inner dynein arms.
An Aplysia cell adhesion molecule associated with site-directed actin filament assembly in neuronal growth cones
Authors: Thompson Corey , Lin Chi-Hung , Forscher Paul 
Published: April 2021

ABSTRACT
During neuronal growth cone-target interactions, a programmed sequence of cytoskeletal remodeling has been described, involving increased actin assembly at the target site and directed microtubule extension into it. The cell adhesion protein apCAM rapidly accumulates at such interaction sites, suggesting a possible role in regulating cytoskeletal remodeling. To test this hypothesis we crosslinked apCAM to varying degrees with antibodies. Secondary immunocomplexes exhibited a classical patching and capping response; in contrast, high density crosslinking of apCAM by antibody coated beads triggered localized actin assembly accompanied by formation of taillike actin structures referred to as inductopodia. When beads were derivatized with increasing amounts of antiapCAM they displayed three sequential dose-dependent kinetic states after binding: (1) lateral diffusion in the plane of the membrane; (2) restricted diffusion due to coupling with underlying F-actin; and (3) translocation in the plane of the membrane driven by de novo actin filament assembly local to bead binding sites, i.e. inductopodia formation. In contrast, lectin coated beads were far less efficient in triggering inductopodia formation despite demonstrated membrane protein binding. This work provides evidence that crosslinking of a diffusable membrane protein, apCAM, to threshold levels, can trigger highly localized actin filament assembly and rapid remodeling of neuronal cytoarchitecture.
Evidence for a functional link between Rab3 and the SNARE complex
Authors: Johannes Ludger , Doussau Frédéric , Clabecq Aude , Henry Jean-Pierre , Darchen François , Poulain Bernard 
Published: April 2021

ABSTRACT
Rab3 is a monomeric GTP-binding protein associated with secretory vesicles which has been implicated in the control of regulated exocytosis. We have exploited Rab3 mutant proteins to investigate the function of Rab3 in the process of neurotransmitter release from Aplysia neurons. A GTPase-deficient Rab3 mutant protein was found to inhibit acetylcholine release suggesting that GTP hydrolysis by Rab3 is rate-limiting in the exocytosis process. This effect was abolished by a mutation in the effector domain, and required the association of Rab3 with membranes. In order to determine the step at which Rab3 interferes with the secretory process, tetanus and botulinum type A neurotoxins were applied to Aplysia neurons pre-injected with the GTPase-deficient Rab3 mutant protein. These neurotoxins are Zn2+-dependent proteases that cleave VAMP/synaptobrevin and SNAP-25, two proteins which can form a ternary complex (termed the SNARE complex) with syntaxin and have been implicated in the docking of synaptic vesicles at the plasma membrane. The onset of toxin-induced inhibition of neurotransmitter release was strongly delayed in these cells, indicating that the mutant Rab3 protein led to the accumulation of a toxin-insensitive component of release. Since tetanus and botulinum type A neurotoxins cannot attack their targets, VAMP/synaptobrevin and SNAP-25, when the latter are engaged in the SNARE complex, we propose that Rab3 modulates the activity of the fusion machinery by controlling the formation or the stability of the SNARE complex.
Deficiency of protein phosphatase 2A uncouples the nuclear and centrosome cycles and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos
Authors: Snaith Hilary A. , Armstrong Christopher G. , Guo Yiquan , Kaiser Kim , Cohen Patricia T. W. 
Published: April 2021

ABSTRACT
A Drosophila strain, carrying a P[lacW] element in the promoter of the protein phosphatase 2A (PP2A) catalytic subunit gene at chromosomal location 28D, has been identified using plasmid rescue of the P element and adjoining genomic DNA in Escherichia coli. Reversion mutagenesis was employed to demonstrate that the observed phenotype of the Drosophila strain was due to a single P[lacW] element insertion at 28D and to create three deficiency strains at this locus. Drosophila heterozygous for P[lacW]28D have reduced levels of PP2A mRNA and reduced PP2A catalytic activity against four different substrates compared to wild type, while homozygotes are deduced to have approximately 20% of wild-type PP2A activity. P[lacW]28D homozygotes, termed microtubule star (mts), die in embryogenesis around the time of cellularisation, exhibiting overcondensed chromatin and a block in mitosis between prophase and the initiation of anaphase. Multiple centrosomes are visible in cellularised embryos, suggesting that PP2A may play a role in coupling the nuclear and centrosome cycles. When embryos arrest just prior to cellularisation, disorganised elongated arrays of microtubules radiate from centrosomes in all directions, but they are rarely associated with any DNA, suggesting that PP2A is required for the attachment of microtubules to chromosomal DNA at the kinetochore.
CD24 (heat stable antigen, nectadrin), a novel keratinocyte differentiation marker, is preferentially expressed in areas of the hair follicle containing the colony-forming cells
Authors: Magnaldo Thierry , Barrandon Yann 
Published: April 2021

ABSTRACT
We have isolated, by subtractive and differential hybridization from a library constructed from keratinocyte colonyforming cells (K-CFCs), a cDNA coding for the rat CD24 (nectadrin, heat stable antigen). CD24, a glycoprotein thought to be involved in cell-cell adhesion and signalling, is highly expressed in keratinocytes located in the bulge area of the rat vibrissa which contains the most K-CFCs. CD24 is also expressed in the outer epithelial sheath of human hair follicles and in glabrous epidermis. However, its expression is not restricted to K-CFCs as demonstrated by cell sorting experiments, and it is thus not a specific marker of clonogenic keratinocytes. Rather, its preferential distribution in keratinocytes located in the most innervated area of the rat vibrissal follicle, i.e. the bulge, suggests that its function could be related to the tactile role of the hair follicle.
Cadherin binding sites of plakoglobin: localization, specificity and role in targeting to adhering junctions
Authors: Troyanovsky Regina B. , Chitaev Nikolai A. , Troyanovsky Sergey M. 
Published: April 2021

ABSTRACT
Plakoglobin directly interacts with cadherins and plays an essential role in the assembly of adherens junctions and desmosomes. Recently we have reported that multiple cadherin binding sites are localized along the arm repeat region of plakoglobin. To demonstrate functionality and specificity of these sites in vivo we constructed a set of chimeric proteins containing a plakoglobin sequence fused with the transmembrane vesicular protein synaptophysin. Plakoglobin fused upstream or downstream from synaptophysin (PgSy and SyPg, chimeras, respectively) is exposed on the cytoplasmic surface of synaptic-like vesicles and is able to associate with E-cadherin, and with two desmosomal cadherins, desmoglein and desmocollin. Moreover, plakoglobin targets these vesicles to cell-cell junctions. Insertion of synaptophysin within plakoglobin (PSyG chimeras) can interfere with cadherin binding of the resulting chimeric proteins, dependent on the position of the insertion. Insertion of synaptophysin in the first three arm repeats selectively inactivates plakoglobin binding to desmoglein and desmocollin. An insertion of synaptophysin within the next two repeats inactivates E-cadherin and desmocollin binding but not desmoglein binding. This localization of the desmoglein and E-cadherin binding sites was further confirmed by replacement of plakoglobin arm repeats with the corresponding sequence derived from the plakoglobin homologue, β-catenin, and by deletion mutagenesis. Insertion of synaptophysin in most sites within arm repeats 6-13 does not change plakoglobin binding to cadherins. It does, however, strongly inhibit association of the resulting vesicles either with desmosomes and adherens junctions or with desmosomes only. Using in vitro binding assays we demonstrate that arm repeats 6-13 contain two cryptic cadherin binding sites that are masked in the intact protein. These observations suggest that the arm repeat region of plakoglobin is comprises two functionally distinct regions: the 1/5 region containing desmoglein and E-cadherin specific binding sites and the 6/13 region implicated in targeting of plakoglobin/cadherin complexes into junctional structures.
Expression and changing distribution of the human transferrin receptor in developing Drosophila oocytes and embryos
Authors: Bretscher Mark S. 
Published: April 2021

ABSTRACT
In order to understand better the membrane systems in a developing Drosophila oocyte, the human transferrin receptor has been expressed there. This was achieved using the armadillo promoter combined with K10 or oskar trailer sequences; these enable the messenger RNA to be transcribed in nurse cells and then transported to, and translated in, oocytes. This is the first exogenous protein to be expressed in oocytes. At stage 8, the transferrin receptors are mainly concentrated towards the posterior pole of the oocyte and are associated with large cytoplasmic vesicles; when combined with the shibire mutation the transferrin receptors are transferred to the oolemma, demonstrating that they participate in an endocytic cycle. At stage 10, the transferrin receptors are localised either to the anterior margin of the oocyte or to the posterior pole, depending on where the mRNA is located. In newly laid eggs, all the transferrin receptors are found in large cytoplasmic vesicles. The results reveal remarkable sorting processes which occur as oocytes mature and show that ring canals, which separate the oolemma from nurse cell plasma membranes, act as barriers to prevent components in these two compartments from intermixing.
Cleavage and inactivation of DNA-dependent protein kinase catalytic subunit during apoptosis in Xenopus egg extracts
Authors: Romancer Muriel Le , Cosulich Sabina C. , Jackson Stephen P. , Clarke Paul R. 
Published: April 2021

ABSTRACT
DNA-dependent protein kinase (DNA-PK) consists of a 460 kDa subunit that contains the catalytic domain (DNAPKcs) complexed with two polypeptides of 70 kDa and 80 kDa (Ku70 and Ku80) which comprise the Ku autoantigen. DNA-PKcs requires association with DNA via Ku for catalytic activation and is implicated in double strand break repair, V(D)J recombination and transcription. We have utilised a cell-free system of concentrated Xenopus laevis egg extracts to investigate the regulation and possible functions of DNA-PK. Recently, we have shown that this system can reproduce events of apoptosis, including activation of an apoptotic protease that cleaves poly(ADP-ribose) polymerase. Here, we report that DNA-PK is rapidly inactivated with the onset of apoptosis in this system. Loss of activity is concomitant with cleavage of the catalytic subunit, whereas the Ku subunits are stable. Cleavage and inactivation of DNA-PKcs is prevented by prior addition of the anti-apoptotic protein Bcl-2 or inhibition of an apoptotic protease that has characteristics of the CPP-32/Ced-3 family of cysteine proteases that cleave poly(ADP-ribose) polymerase. These results suggest that cleavage and inactivation of DNA-PKcs prevents this factor from functioning in DNA repair, recombination or transcriptional regulation during apoptosis.
Polar localization of a dihydropyridine receptor on living Fucus zygotes
Authors: Shaw Sidney L. , Quatrano Ralph S. 
Published: April 2021

ABSTRACT
We have used a fluorescently-labeled dihydropyridine (FL-DHP) to vitally stain living Fucus zygotes during the establishment of cell polarity. Localization of FL-DHP is primarily at the plasma membrane and FL-DHP binding is competitively blocked by an unlabeled dihydropyridine. Distribution of FL-DHP is initially symmetrical before fixation of the polar axis, but becomes asymmetrical in response to a unilateral light gradient. The distribution of FL-DHP receptors can be relocalized when the direction of the photopolarizing stimulus is changed. Treatment of cells with cytochalasin B prior to axis fixation reversibly prevents localization of FL-DHP receptors. Observation of FL-DHP labeling by time-lapse fluorescence microscopy indicates that the existing receptors are redistributed during polar axis formation. The asymmetric distribution of FL-DHP receptors coincides temporally and spatially with increased local intracellular calcium ion concentrations, as measured by calcium green dextran. Based on the site, timing, photo-reversibility, and actin dependence of the asymmetric localization of FL-DHP receptors, we conclude that FL-DHP is a vital probe for the later stage of polar axis formation in Fucus zygotes. Furthermore, we propose that FL-DHP receptors correspond to ion channels that are transported to the future site of polar growth to create the changes in local calcium concentration required for polarity establishment.
Association of thrombospondin-1 with osteogenic differentiation of retinal pericytes in vitro
Authors: Canfield Ann E. , Sutton Andrew B. , Hoyland Judith A. , Schor Ana M. 
Published: April 2021

ABSTRACT
Vascular pericytes can differentiate into osteoblast-like cells in vitro, suggesting that these cells may represent a potential source of osteoprogenitor cells in the adult. Pericyte differentiation is associated with a characteristic pattern of nodule formation and mineralisation. Nodules are formed in post-confluent cultures by the retraction of multilayered areas. Crystals of hydroxyapatite are deposited on the extracellular matrix of these nodules which then becomes mineralised. We now demonstrate that thrombospondin-1 (TSP-1) gene expression is modulated during pericyte differentiation in vitro. That is, the relative levels of TSP-1 (protein and mRNA) increased markedly during nodule formation and then decreased when mineralisation of the nodules had taken place. TSP-1 was localised throughout non-mineralised nodules but it was largely excluded from the inner mass of mineralised nodules. The production of a mineralised matrix by vascular pericytes was promoted by the presence of antibodies to TSP-1 in the culture medium and was inhibited by exogenous TSP-1. These effects did not appear to be mediated through the activation of latent TGF-β, since neither exogenous TGF-β nor neutralising antibodies to TGF-β had any effect on the rate or extent of mineralisation seen in the pericyte cultures. Taken together these results suggest that high levels of TSP-1 inhibit pericyte mineralisation, supporting the view that this protein plays a role in pericyte differentiation and bone formation.
Differential expression of epithelial sodium channel subunit mRNAs in rat skin
Authors: Roudier-Pujol C. , Rochat A. , Escoubet B. , Eugène E. , Barrandon Y. , Bonvalet J. P. , Farman N. 
Published: April 2021

ABSTRACT
Three subunits (α, β, γ) of the amiloride-sensitive epithelial sodium channel have been recently characterized. The channel subunits have significant homologies with the Caenorhabditis elegans mec-4, mec-10 and deg-1 genes, which are involved in control of cell volume and mecanotransduction. These subunits are coexpressed at equivalent levels in the renal collecting duct and the distal colon epithelium which are high resistance sodium transporting epithelia. We have investigated whether these subunits were expressed, at the mRNA level, in transporting as well as non transporting epithelial cells of rat skin. In fullthickness abdominal skin only α and γ subunit mRNAs were detected, while all three subunit mRNAs were present in sole skin, as demonstrated by RNase-protection assay. Furthermore, the level of expression of each subunit varied with the epithelial cell type as demonstrated by in situ hybridization: epidermal and follicular keratinocytes express mostly α and γ subunits (while β was low); a prevalence of β and γ was observed in sweat glands. Thus, it appeared that two out of the three subunit mRNAs predominated in each epithelial structure. In addition, mRNAs of the α, β and γ subunits of the amiloride-sensitive sodium channel were expressed at a higher level in large suprabasal epidermal keratinocytes (which undergo terminal differentiation) than in small proliferative basal keratinocytes.
Actin filaments facilitate two steps of endocytosis
Authors: Durrbach A. , Louvard D. , Coudrier E. 
Published: April 2021

ABSTRACT
Recent reports have suggested a role for microtubules in the endocytic process and pointed out the role for actin filaments for the early steps of apical endocytosis in polarized epithelial cells. However, these studies do not address the respective contribution of these two types of filaments along the endocytic pathway. In addition, several studies failed to demonstrate the role for actin filaments in clathrin dependent endocytosis in non polarized cells. In the present study we have investigated the role for both filaments in the endocytosis of two ligands internalized via clathrin coated pits (transferrin and α2-macroglobulin) in a mouse hepatoma cell line. By immunocytochemical analysis with confocal microscopy, and biochemical analysis using a temperature sensitive step at 18°C, we have shown that actin filaments are involved in two steps of the degradative pathway, and that microtubules are required at a stage in between. Actin filaments increase first, the uptake of ligands and second, their delivery to the degradative compartment, whereas microtubules are required to maintain the distribution of the late endocytic compartment in its juxtanuclear position and facilitate the delivery of the ligands to the degradative compartment upstream of the actin filament requirement. Furthermore actin filaments facilitate the recycling of transferrin from the perinuclear region to the plasma membrane. Our data indicate for the first time the sequential involvement of actin filaments and microtubules along one intracellular membrane trafficking pathway.
Glycosaminoglycans modulate cell-matrix interactions of human fibroblasts and endothelial cells in vitro
Authors: Schaefer Thomas , Roux Matthias , Stuhlsatz Helmut W. , Herken Rainer , Coulomb Bernard , Krieg Thomas , Smola Hans 
Published: April 2021

ABSTRACT
Contact of various cells with extracellular matrix molecules modulates their cellular functions and phenotype. Most investigations have employed dishes coated with purified matrix constituents or plain collagen I lattices omitting the effects of other important matrix components such as proteoglycans. In this study we analyze the effect of purified glycosaminoglycans (GAGs) on human fibroblasts and human umbilical vein endothelial cells (HUVEC) embedded within collagen I/III lattices.
HUVEC contracted collagen I/III gels far less efficiently than fibroblasts and addition of heparan sulfate and heparin almost completely inhibited contraction. In collagen gels HUVEC down-regulated collagenase mRNA while increasing collagen I, IV mRNA expression. Addition of heparin and heparan sulfate reversed the collagen IV mRNA induction whereas hyaluronic acid and chondroitin sulfate enhanced fibronectin and collagenase transcripts. Fibroblasts readily contracted collagen gels, and mRNA levels for fibronectin, collagenase and interleukin-6 were stimulated. Gel contraction was mostly unaffected by the different glycosaminoglycans. Fibroblasts responded to the addition of dermatan sulfate, heparan sulfate and heparin with a decrease in fibronectin, collagenase and interleukiN-6 mRNA. Binding studies revealed saturable binding sites on fibroblasts and HUVEC for 35S-labelled heparin, demonstrating specificity for heparin and heparan sulfate over other GAGs in competition experiments.
This study implies that glycosaminoglycans participate in cell-matrix interactions by effectively modulating the cellular phenotype via high affinity binding sites.
Antisense RNA inhibition of the putative vacuolar H+-ATPase proteolipid of Dictyostelium reduces intracellular Ca2+ transport and cell viability
Authors: Xie Yanyan , Coukell M. Barrie , Gombos Zoltan 
Published: April 2021

ABSTRACT
Transport of Ca2+ via a P-type pump into the contractile vacuole of Dictyostelium discoideum appears to be facilitated by vacuolar proton (V-H+) ATPase activity. To investigate the involvement of the V-H+-ATPase in this process using molecular techniques, we cloned a cDNA (vatP) encoding the putative proteolipid subunit of this enzyme. The deduced protein product of this cDNA is composed of 196 amino acids with a calculated Mr of 20,148 and the primary structure exhibits high amino acid sequence identity with V-H+-ATPase proteolipids from other organisms. vatP is a single-copy gene and it produces one ∼900 nt transcript at relatively constant levels during growth and development. Attempts to disrupt the endogenous gene using vatP cDNA were unsuccessful. But, expression of vatP antisense RNA reduced the levels of vatP message and V-H+-ATPase activity by 50% or more. These antisense strains grew and developed slowly, especially under acidic conditions, and the cells seemed to have difficulty forming acidic vesicles. During prolonged cultivation, all of the antisense strains either reverted to a wild-type phenotype or died. Thus in Dictyostelium, unlike yeast, the V-H+-ATPase seems to be indispensable for cell viability. When different antisense strains were analyzed for Ca2+ uptake by the contractile vacuole, they all accumulated less Ca2+ than control transformants. These results are consistent with earlier pharmacological studies which suggested that the V-H+-ATPase functions in intracellular Ca2+ transport in this organism.