bluuebunny/crossref_metadata_2025_split huggingface dataset:
Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor
Authors: Yao Chung-Chen , Ziober Barry L. , Sutherland Ann E. , Mendrick Donna L. , Kramer Randall H. 
Published: April 2021

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

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

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

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

ABSTRACT
Mechanisms of phagocytosis are complex and incompletely understood. The retinal pigment epithelium provides an ideal system to study the specific aspects of phagocytosis since an important function of this cell is the ingestion of packets of membranous discs that are normally discarded at the apical ends of rod and cone cells during outer segment renewal. Here we provide evidence that rod outer segment phagocytosis by retinal pigment epithelium is mediated by CD36, a transmembrane glycoprotein which has been previously characterized on hematopoietic cells as a receptor for apoptotic neutrophils and oxidized low density lipoprotein.
Immunocytochemical staining with monoclonal and polyclonal antibodies demonstrated CD36 expression by both human and rat retinal pigment epithelium in transverse cryostat sections of normal retina and in primary cultured cells. By western blot analysis of retinal pigment epithelial cell lysates, polyclonal and monoclonal antibodies to CD36 recognized an 88 kDa protein which comigrated with platelet CD36. Furthermore, the synthesis of CD36 mRNA by retinal pigment epithelium was confirmed by reverse transcriptase-PCR using specific CD36 oligonucleotides. The addition of CD36 antibodies to cultured retinal pigment epithelial cells reduced the binding and internalization of 125I-labeled rod outer segments by 60%. Immunofluorescence confocal microscopy confirmed that outer segment uptake was significantly diminished by an antibody to CD36.
Moreover, we found that transfection of a human melanoma cell line with CD36 cDNA enabled these cells to bind and internalize isolated photoreceptor outer segments as seen by double immunofluorescent staining for surface bound and total cell-associated rod outer segments, and by measurement of cell-associated 125I-labeled rod outer segments.
We conclude that the multifunctional scavenger receptor CD36 participates in the clearance of photoreceptor outer segments by retinal pigment epithelium and thus, participates in the visual process.
Nuclear architecture and ultrastructural distribution of poly(ADP-ribosyl)transferase, a multifunctional enzyme
Authors: Mosgoeller Wilhelm , Steiner Marianne , Hozák Pavel , Penner Edward , Węsierska-Gądek Józefa 
Published: April 2021

ABSTRACT
A monospecific autoimmune serum for poly(ADP-ribosyl)transferase (pADPRT) was used to localise the enzyme in ultrastructural cellular compartments. We detected enzyme in mitochondria of HeLa and Sertoli cells. Within the nucleoplasm the enzyme concentration was positively correlated with the degree of chromatin condensation, with interchromatin spaces being virtually free of pADPRT. During spermatogenesis we observed a gradual increase of the chromatin associated pADPRT that parallelled chromatin condensation. The highest concentration was seen in the late stages of sperm differentiation, indicating the existence of a storage form in transcriptionally inactive nuclei.
In nucleoli pADPRT is accumulated in foci within the dense fibrillar component. Such foci are seen in close spatial relationship to sites of nucleolar transcription as revealed by high resolution immunodetection of bromouridine uptake sites. It is suggested that nucleolar pADPRT plays a role in preribosome processing via the modification of nucleolus specific proteins that bind to nascent transcripts and hence indirectly regulates polymerase I activity. The persisting binding of pADPRT to ribonucleoproteins may explain the observed disperse enzyme distribution at lower concentrations in the granular component. The fibrillar centres seem to contain no pADPRT. We conclude that known compounds of fibrillar centres like polymerase I are unlikely candidates for modification via direct covalent ADP-ribosylation.
Flexural rigidity of microtubules measured with the use of optical tweezers
Authors: Felgner Harald , Frank Rainer , Schliwa Manfred 
Published: April 2021

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

ABSTRACT
In eukaryotic evolution, the earliest branch of organisms to have mitochondria are the trypanosomatids. Their mitochondrial biogenesis not only includes import of most proteins, but also, unlike in other organisms, import of the whole set of tRNAs. In order to investigate these processes, we devised novel procedures for the isolation of mitochondria from two trypanosomatid species: Trypanosoma brucei and Leishmania tarentolae. Isotonic cell lysis followed by equilibrium density centrifugation in Nycodenz gradients yielded mitochondrial fractions exhibiting a membrane potential. Furthermore, we have used these fractions to reconstitute import of mitochondrial matrix proteins in vitro. Energy-dependent uptake of an artificial precursor protein, containing a trypanosomal presequence attached to mouse dihydrofolate reductase and of yeast mitochondrial alcohol dehydrogenase could be demonstrated. The presequences of both proteins were processed in T. brucei whereas only the trypanosomal one was cleaved in L. tarentolae. Trypsin pretreatment abolished the ability of the mitochondria to import proteins, indicating the involvement of proteinaceous components at the surface of mitochondria.
The kinesin-homologous protein encoded by the Chlamydomonas FLA10 gene is associated with basal bodies and centrioles
Authors: Vashishtha Malini , Walther Zenta , Hall John L. 
Published: April 2021

ABSTRACT
We previously reported that the FLA10 locus on the uni linkage group of Chlamydomonas encodes a kinesin homologous protein, KHP1. The fla10 phenotype, which is a temperature-sensitive defect for flagellar assembly and maintenance, is rescued by transformation with the wild-type KHP1 gene. In the present study we identify the molecular defect associated with the fla10 mutation and examine the subcellular localization of KHP1 throughout the cell cycle. The mutation in the fla10-1 allele consists of a C to A transversion, which alters amino acid 329 in the motor domain of KHP1. This residue and the sequence of the carboxyterminal third of the motor domain in which it is located are highly conserved throughout eukaryotic evolution in a subfamily of kinesin-related proteins from mouse (KIF3), sea urchin (KRP85/95), Xenopus (XKLP3), and Drosophila (KLP68D). These data suggest a conserved function for this family of proteins. Immunofluorescence studies reveal that: (1) in interphase cells KHP1 is associated with basal bodies and with the proximal portion of the flagella; (2) in cells undergoing flagellar regeneration KHP1 occurs in punctate structures that extend to the tip of the developing axoneme; and (3) in dividing cells KHP1 remains associated with centrioles throughout mitosis and localizes to the mitotic spindle. KHP1 is the first kinesin homologous protein to be found in association with basal bodies and centrioles throughout the cell cycle. These observations provide evidence for a direct role of basal bodies in the process of flagellar development, which we propose is based on KHP1 acting as a transporter of flagellar components from the basal bodies out to the distal site of assembly. The localization of KHP1 in mitosis suggests that this protein may play an analogous role in the centriole-based assembly of the mitotic spindle.
Katanin, the microtubule-severing ATPase, is concentrated at centrosomes
Authors: McNally Francis J. , Okawa Katsuya , Iwamatsu Akihiro , Vale Ronald D. 
Published: April 2021

ABSTRACT
The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a pre-liminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin’s localization is different from that of γ-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and γ-tubulin reveals that katanin is localized in a region surrounding the γ-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.
Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium
Authors: Pullan Shirley , Wilson James , Metcalfe Anthony , Edwards Gwynneth M. , Goberdhan Nadia , Tilly Jonathan , Hickman John A. , Dive Caroline , Streuli Charles H. 
Published: April 2021

ABSTRACT
Apoptosis is an active mechanism of cell death required for normal tissue homeostasis. Cells require survival signals to avoid the engagement of apoptosis. In the mammary gland, secretory epithelial cells are removed by apoptosis during involution. This cell loss coincides with matrix metalloproteinase activation and basement membrane degradation. In this paper we describe studies that confer a new role for basement membrane in the regulation of cell phenotype. We demonstrate that first passage epithelial cells isolated from pregnant mouse mammary gland die by apoptosis in culture, but that cell death is suppressed by basement membrane. The correct type of extracellular matrix was required, since only a basement membrane, not plastic or a collagen I matrix, lowered the rate of apoptosis. Attachment to a matrix per se was not sufficient for survival, since apoptotic cells were observed when still attached to a collagen I substratum. Experiments with individually isolated cells confirmed the requirement of basement membrane for survival, and demonstrated that survival is enhanced by cell-cell contact. A function-blocking anti-β1 integrin antibody doubled the rate of apoptosis in single cells cultured with basement membrane, indicating that integrin-mediated signals contributed to survival. We examined the cell death-associated genes bcl-2 and bax in mammary epithelia, and found that although the expression of Bcl-2 did not correlate with cell survival, increased levels of Bax were associated with apoptosis. We propose that basement membrane provides a survival stimulus for epithelial cells in vivo, and that loss of interaction with this type of matrix acts as a control point for cell deletions that occur at specific times during development, such as in mammary gland involution.
The effect of wortmannin on the localisation of lysosomal type I integral membrane glycoproteins suggests a role for phosphoinositide 3-kinase activity in regulating membrane traffic late in the endocytic pathway
Authors: Reaves Barbara J. , Bright Nicholas A. , Mullock Barbara M. , Luzio J. Paul 
Published: April 2021

ABSTRACT
Addition of wortmannin to normal rat kidney cells caused a redistribution of the lysosomal type I integral membrane proteins lgp 110 and lgp120 to a swollen vacuolar compartment. This compartment did not contain the cation independent mannose 6-phosphate receptor and was depleted in acid hydrolases. It was distinct from another swollen vacuolar compartment containing the cation independent mannose 6-phosphate receptor. The swollen lgp110-positive compartment was accessible to a monoclonal antibody against lgp120 added extracellularly, showing that it had the characteristics of an endosomal compartment. Wortmannin had no gross morphological effect on the trans-Golgi network or lysosomes nor any effect on the delivery to the trans-Golgi network of endocytosed antibodies against the type I membrane protein TGN38. We propose that the observed effects of wortmannin were due to inhibition of membrane traffic between cation independent mannose 6-phosphate receptor-positive late endosomes and the trans-Golgi network and to inhibition of membrane traffic between a novel lgp120-positive, cation independent mannose 6-phosphate receptornegative late endosomal compartment and lysosomes. The effects of wortmannin suggest a function for a phosphatidylinositol 3-kinase(s) in regulating membrane traffic in the late endocytic pathway.
Different distributions of homologous chromosomes in adult human Sertoli cells and in lymphocytes signify nuclear differentiation
Authors: Chandley Ann C. , Speed R. M. , Leitch A. R. 
Published: April 2021

ABSTRACT
Using whole chromosome painting probes for human chro mosomes 3, 7, 8, 13, 17 and 21 and X and the probe pHY2.1 for the Y chromosome coupled with fluorescent in situ hybridization (FISH) analysis, the distribution of chromo somes is reported in nuclei of Sertoli cells of the adult testis and in stimulated blood lymphocytes. The distribution of chromosomes in the two cell types is significantly different. A strong tendency for each pair of homologues to pair is inferred from the observation of only a single detectable signal in the majority of Sertoli cell nuclei. The sex chromosomes, by contrast, give two clearly separated signals. Interphase nuclei in dividing blood lymphocytes, analysed as controls, also show mainly two separated signals for all non-acrocentric autosomal pairs, but acrocentric pairs no. 13 and 21 show some tendency to associate, probably reflecting satellite association.
Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons
Authors: van Steensel Bas , van Binnendijk Erica P. , Hornsby C. Diane , van der Voort Hans T. M. , Krozowski Zygmunt S. , de Kloet E. Ronald , van Driel Roel 
Published: April 2021

ABSTRACT
The glucocorticoid receptor and the mineralocorticoid receptor are hormone-dependent transcription factors. They regulate the excitability of rat hippocampus CA1 neurons in a coordinated fashion. We studied the spatial distribution of these transcription factors in nuclei of CA1 neurons by dual labeling immunocytochemistry and confocal microscopy, combined with novel image resto ration and image analysis techniques. We found that both receptors are concentrated in about one thousand clusters within the nucleus. Some clusters contain either mineralocorticoid receptors or glucocorticoid receptors, but a sig nificant number of clusters contains both receptors. These results indicate that the two receptor types are targeted to specific compartments in the nucleus. The coordinated action of the glucocorticoid and mineralocorticoid receptor on gene expression may be established in a specific set of nuclear domains that contain both receptors.