bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetochore formation and behaviour following premature chromosome condensation
Authors: Rattner J. B. , Wang T. 
Published: April 2021

ABSTRACT
The potential for interphase centromeres to support kinetochore formation following premature chromosome condensation (PCC) has been investigated. We show that the centromere remains competent to initiate kinetochore formation throughout the cell cycle. PCC-kinetochores display a typical trilaminar morphology, associate with microtubules and show movement towards the centrosome. Indirect immunofluorescence studies illustrate that the centromere/kinetochore region of prematurely condensed chromosomes associates with proteins that are normally found within this region in both a cell cycle-dependent and an independent manner.
Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes
Authors: Kok Jan Willem , Hoekstra Karin , Eskelinen Sinikka , Hoekstra Dick 
Published: April 2021

ABSTRACT
Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide,6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid’s trafficking from peripheral early endosomes to centrally located late endosomes.
When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway.
Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.
Effects of the expression of mammalian annexins in yeast secretory mutants
Authors: Creutz Carl E. , Kambouris Nicholas G. , Snyder Sandra L. , Hamman Helen C. , Nelson Michael R. , Liu Wei , Rock Philip 
Published: April 2021

ABSTRACT
The hypothesis that calcium-dependent membrane-binding proteins of the annexin family can influence intracellular membrane trafficking was tested by expressing five mammalian annexins in wild-type yeast cells (Saccharomyces cerevisiae) and in 13 yeast secretory (sec) mutants. Expression of human synexin (annexin VII) inhibited the growth of sec2, sec4 and sec15 mutants at a semi-permissive temperature. These three sec mutants are defective in the final step in the secretory pathway, the process of exocytosis. The inhibition of growth correlated with reduced viability and increased accumulation of internal invertase in these mutants when expressing synexin. Bovine endonexin (annexin IV) partially suppressed the growth defect of a sec2 mutant incubated at a semi-permissive temperature. Human synexin, human lipocortin (annexin I), and murine p68 (annexin VI) reduced the lag time associated with adaptation of sec2 mutants to galactose-containing medium. These interactions suggest that the annexins may influence specific steps in membrane trafficking associated with cell growth, secretion and plasma membrane remodelling.
Contact stimulation of cell migration
Authors: Thomas Linda A. , Yamada Kenneth M. 
Published: April 2021

ABSTRACT
Mass migrations of dense cell populations occur periodically during embryonic development. It is known that extracellular matrices, through which the cells migrate, facilitate locomotion. However, this does not explain how cells, such as neural crest, can migrate as a dense cohort of cells in essentially continuous contact with one another. We report here that unique behavioral characteristics of the migrating cells may contribute to cohesive migration. We used time-lapse video microscopy to analyze the migration of quail neural crest cells and of two crest derivatives, human melanoma cells and melanocytes. These cells migrated poorly, if at all, when isolated, but could be stimulated up to 200-fold to travel following contact with migrating cells. This phenomenon, which we have termed “contact-stimulated migration,” appeared to activate and sustain migration of the mass of cells. Cells that became dissociated from the others ceased directional migration, thereby limiting aberrant cell dispersion. Fibroblasts were minimally responsive to this novel phenomenon, which may be crucial for major, mass cell migrations.
Molecular characterization of the secondary constriction region (qh) of human chromosome 9 with pericentric inversion
Authors: Luke Sunny , Verma Ram S. , Mathews Robert A. Contethomas 
Published: April 2021

ABSTRACT
Pericentric inversion of the secondary constriction region (qh) of human chromosome 9 is a frequent occurrence. This structural alteration is regarded as a normal familial variant, termed heteromorphism, and is inherited in a Mendelian fashion without any apparent phenotypic consequences. We characterized the qh region of chromosome 9 from five individuals using a series of molecular cytogenetic techniques. Four out of the five individuals have an additional area composed of alphoid DNA sequences on the inverted chromosome 9 while one case was found to have an apparently intact alphoid DNA sequence. Although the direct function(s) of alphoid DNA sequences remain unclear, the centromeric breakage involving these sequences in inverted chromsome 9 raises a series of questions pertaining to the monocentric, dicentric and pseudodicentric nature of pericentric inversions. Nevertheless, these findings have prompted us to suggest that the structural organization of alphoid DNA sequences of the centromeric region of chromosome 9 are apparently “breakage prone” and may be associated with a higher incidence of pericentric inversions. Furthermore, the hierarchical organization of various satellite DNA families (α-satellite, β-satellite and satellite III) within the primary and secondary constriction regions of chromosomes 9 are elucidated here.
Characterization of the human involucrin promoter using a transient β-galactosidase assay
Authors: Carroll Joseph M. , Taichman Lorne B. 
Published: April 2021

ABSTRACT
Involucrin, a component of the cornified cell envelope, is expressed specifically in differentiating keratinocytes of stratified squamous epithelia. To explore the regulation of involucrin expression, 3.7 kb of upstream sequences of the human involucrin gene was cloned into a plasmid containing a -galactosidase reporter gene and transfected into early passage keratinocytes and a variety of human cell types. The full-length construct gave maximal and tissue-specific expression. Deletion analysis showed that sequences between 900 and 2500 bp upstream of the transcriptional start site and the intron located between the transcriptional and translational start sites were required for maximal expression. Further analysis of the intron indicated that its effects on expression were independent of it being present in nascent RNA and suggested that sequences within the intron have regulatory activity. These results suggest that the involucrin intron operates in vivo to regulate expression in the epidermis.
Abbreviations used: ADH, alcohol dehydrogenase; β-gal, β-galactosidase; DME, Dulbecco’s modified Eagle’s medium; DDAB, dimethyldioctyldecylammonium bromide; FCS, fetal calf serum; kb, kilobase; ONPG, O-nitrophenyl β-D-galactopyranoside; PBS, phosphate buffered saline without calcium/magnesium salts; PCR, polymerase chain reaction; PtdEtn, dioleoyl-L-α-phosphatidylethanolamine; RSV, Rous sarcoma virus; SV40, simian virus 40.
Brain microtubule-associated proteins modulate microtubule dynamic instability in vitro: Real-time observations using video microscopy
Authors: Pryer Nancy K. , Walker Richard A. , Skeen Victoria Petrie , Bourns Brenda D. , Soboeiro Michael F. , Salmon Edward D. 
Published: April 2021

ABSTRACT
We used video assays to study the dynamic instability behavior of individual microtubules assembled in vitro with purified tau, purified MAP2 or a preparation of unfractionated heat-stable MAPs. Axoneme-nucleated microtubules were assembled from pure tubulin at concentrations between 4 and 9 M in the presence of MAPs, and observed by video-differential interference contrast microscopy. Microtubules co-assembled with each MAP preparation exhibited the elongation and rapid shortening phases and the abrupt transitions (catastrophe and rescue) characteristic of dynamic instability. Each MAP preparation increased the microtubule elongation rate above that for purified tubulin alone by decreasing the tubulin subunit dissociation rate during elongation. The brain MAPs used in this study reduced the rate of microtubule rapid shortening, but allowed significant loss of polymer during the shortening phase.
Purified tau and MAP2 decreased the frequency of catastrophe and increased the frequency of rescue, while the heat-stable MAPs suppressed catastrophe at all but the lowest tubulin concentrations. Thus, each of these MAPs modulates, but does not abolish, dynamic instability behavior of microtubules. We propose a model to explain how MAP2 and tau bind to the microtubule lattice at sites along protofilaments so that the MAPs promote polymerization, but do not significantly block the mechanism of rapid shortening inherent in the tubulin lattice. Rapid shortening, when it occurs, proceeds primarily by the dissociation of short fragments of protofilaments, which contain the bound MAPs.
The effect of cytochalasin D on preprophase band organization in root tip cells of Allium
Authors: Eleftheriou E. P. , Palevitz B. A. 
Published: April 2021

ABSTRACT
The relationship between microfilaments (Mfs) and microtubules (Mts) in the organization of the preprophase band (PPB) was investigated in Allium root tip cells subjected to treatment with cytochalasin D (CD). Mts and Mfs were visualized by indirect immunofluorescence and various parameters such as PPB width were analyzed quantitatively. In control samples, the PPB first appears as a wide Mt band that progressively narrows to an average width of 4 m in mid-prophase. Randomly oriented Mfs are present throughout the cytoplasm of most interphase control cells. Preprophase and prophase cells, however, contain cortical Mfs arranged parallel to the PPB. The Mfs initially occupy much of the cortex but in most cells they progressively become restricted to an area wider than the PPB. In the presence of CD, the PPB fails to narrow and remains at least two-fold wider than in control cells. PPB width expressed as a percentage of nuclear or cell length also increases compared to controls. Widening is concentration dependent, and the effect of 10 M CD is near maximal only 15 min after application of the drug. This rapid response suggests that a rebroadening of already condensed PPBs takes place. After as little as 15 min in CD, Mfs are replaced by many small specks and rods. Dual localizations of both Mts and Mfs show that prophase cells contain broad PPBs without Mfs. The rapid disorganization of Mfs, by CD, therefore coincides with the rebroadening of PPBs. CD-treated cells in metaphase, anaphase and telophase contain larger actin aggregates at the poles, as previously reported. The results indicate that Mfs play an important role in the narrowing of the PPB, which in turn is essential for determination of the exact position of the plane of division. They also indicate that movement of intact Mts is important in PPB organization.
Dynein from serotonin-activated cilia and flagella: extraction characteristics and distinct sites for cAMP-dependent protein phosphorylation
Authors: Stephens R. E. , Prior G. 
Published: April 2021

ABSTRACT
Serotonin, an activator of adenylate cyclase, stimulates motility in molluscan gill cilia and sperm flagella. To determine and compare potential targets of cAMP action, dynein was prepared from the lateral gill cilia and sperm flagella of the mussel Mytilus edulis and the clam Spisula solidissima. In the flagella of both species, high-salt extraction removes about half of the ATPase activity, half of the and heavy chains, and the outer arms. The dynein from both species sediments at 18–20 S, contains two or three intermediate chains, and three light chains. High-salt plus detergent removes most of the remaining dynein ATPase, and heavy chains, and inner arms, also yielding a stable 18–20 S particle. In gill cilia of both species, high-salt extraction removes only 12–18% of the ATPase, up to 1/3 of the heavy chains, an equivalent amount of heavy chain, and a subset of the outer arms. The dynein sediments at 18–20 S and, in Spisula, the heavy, intermediate, and light chains precisely co-sediment. High-salt plus detergent removes another 1/3 of the heavy chains, an equivalent amount of heavy chain, and the remaining outer arms. The ATPase sediments mainly as a 13–14 S form showing considerable dissociation of co-sedimenting intermediate and light chains. The inner arms and at least half of the ciliary dynein ATPase activity remain unextractable, corresponding in mass mainly to an apparent heavy chain that is vanadate-cleavable. Cyclic AMP-dependent, calcium-independent phosphorylation takes place on specific dynein light chains in cilia but on only the dynein heavy chain in flagella. Pre-activation of the flagella prevents subsequent addition of labeled phosphate. Phosphorylation has no effect on the steady-state ATPase properties. The single phosphate added to the flagellar chain is located within the LUV1 vanadate photocleavage fragment. Considering the probable locus of the light chains and the site of the heavy chain phosphorylation, both beyond the active site and toward the base of the molecule, these distinct phosphorylations may regulate dynein action by modulating arm flexibility or interaction.
Localization of the RNA polymerase I transcription factor hUBF during the cell cycle
Authors: Roussel Pascal , André Chantal , Masson Claude , Géraud Gérard , Hernandez-Verdun Danièle 
Published: April 2021

ABSTRACT
Autoantibodies directed against nucleoli that recognized a doublet of 97–94 kDa in HeLa nuclear protein extracts were identified. The two polypeptides bound equal amounts of antibody, and each was recognized by antibodies affinity purified using the other polypeptide. These antigens were localized in the secondary constriction of PtK1 cells, i.e. the nucleolar organizer regions (NORs) where ribosomal genes accumulate. They were observed in human cells in the same sites as the NOR-silver-stained proteins. The molecular mass of the antigens, their characteristics in Western blotting and their localization in nucleoli and NORs during mitosis are consistent with them being RNA polymerase I transcriptional factor, UBF. This identification was confirmed on Western blotted proteins by their identical labelling patterns, using these autoantibodies and an anti-mUBF antibody that had been previously described. We obtained definitive evidence that these autoantibodies recognize UBF by the strong positive labelling of purified hUBF (1 to 4 ng). During interphase, these autoantibodies directed against UBF labelled in a folded filament pattern as small beads that may correspond to individual transcriptional units. In electron microscopy, the antibodies were observed in the dense fibrillar component (DFC) of the nucleoli and at the periphery of the fibrillar centers (FCs). At the end of G2 phase, transcription inactivation was concomitant with the gathering of UBF at mitotic NORs. UBF was not equally distributed between NORs in human cells: some NORs scored negative (2 to 4) and the intensity of labelling of positive NORs (6 to 8) differed. In confocal microscopy, 3-dimensional analysis of mitosis indicated that UBF remained associated with NORs during all mitotic stages and that there was equal partition of UBF between the daughter cells. The relationship between proteins associated with the NORs and ribosomal gene transcription is discussed.
Composite microtubules of the axon: quantitative analysis of tyrosinated and acetylated tubulin along individual axonal microtubules
Authors: Brown Anthony , Li Yan , Slaughter Theresa , Black Mark M. 
Published: April 2021

ABSTRACT
We have shown previously, using immunoelectron microscopy, that axonal microtubules (MTs) are composite, consisting of distinct domains that differ in their content of tyrosinated α-tubulin (tyr-tubulin). Here, we extend these studies using a novel preparation that permits visualization of individual axonal MTs over distances of several tens of micrometres using conventional immunofluorescence procedures. Neurons are cultured on a substratum of poly-lysine and laminin and then extracted with a MT stabilizing solution containing Triton X-100 and NaCl. These extraction conditions cause a loosening of the axonal MT array so that individual MTs separate from each other for variable distances along their length. We call this phenomenon fraying. Within the axon shaft, individual MTs can often be traced for several tens of micrometres, but fraying is most extensive in the distal 100-200 μm of the axon, where individual MTs can frequently be traced for distances of 50 to 100 μm or more to their plus ends. In some cases MTs separate completely from the axon, permitting visualization of both of their ends. Double-staining of frayed preparations with various combinations of antibodies against tyr-tubulin, acetylated α-tubulin (Actubulin) or β-tubulin, clearly revealed the composite nature of axonal MTs. Composite MTs consisted of two distinct domains, one that was relatively rich in tyrtubulin and poor in Ac-tubulin, and the other that was relatively poor in tyr-tubulin and rich in Ac-tubulin. The transition between these domains was relatively abrupt, with the tyr-tubulin-rich domain extending from the transition to the plus-end of the MT. Quantitative analyses of fluorescence intensity along individual MTs using digital image processing revealed that the relative amount of tyr-tubulin increased by approximately 800% across the transition, whereas the relative amount of Ac-tubulin decreased by approximately 60%. Within the tyr-tubulin-rich domains, the relative amount of tyr-tubulin was generally not constant, but increased from the transition to the plus-end of the MT in a nonlinear manner. We propose that the specific pattern of variation in the extent of post-translational modification along an individual MT represents a snapshot of that polymer’s growth history.
γ-Tubulin reorganization during mouse fertilization and early development
Authors: Palacios Monica J. , Joshi Harish C. , Simerly Calvin , Schatten Gerald 
Published: April 2021

ABSTRACT
γ-Tubulin, a component of spindle pole bodies in fungal cells and pericentriolar material in vertebrate cells , is thought to play a role in the nucleation of microtubule growth and to define their polarity. In contrast to the adult somatic cells, microtubules are nucleated in the absence of centrioles in mammalian oocytes and early embryos. By studying acentriolar mouse oocytes and their early development following fertilization, we show that γ-Tubulin antibody crossreacts with a 50,000 Mr protein in unfertilized mouse oocytes and demonstrate that γ-Tubulin distribution is rearranged dramatically during fertilization. In unfertilized mouse oocytes, - tubulin is concentrated in the broad spindle poles of meiotic spindle (MII) and as the distinct foci which form the centers of the cytoplasmic microtubule asters (cytasters). The integrity of these γ-Tubulin foci and their cytoplasmic location is maintained during the drug- or cold-induced depolymerization of microtubules. γ-Tubulin is also found in the basal body of the mouse sperm. During fertilization, the γ-Tubulin is found at the cytastral centers as well as in the incorporated sperm basal body complex, and the γ-Tubulin foci coalesce at the perinuclear microtubule organizing regions of the two pronuclei at the first mitotic prophase. During mitosis, γ-Tubulin is found associated with broad bands that form the poles of the first mitotic spindle. By the late preimplantation stage, when newly generated centrioles have been reported to arise, tubulin remains localized at the centrosome of mitotic cells. The presence of γ-Tubulin in all microtubule organizing structures provides support for its proposed role as a component of microtubule organizing centers during the complex choreography of all microtubule assemblies.
A scale associated protein of Apedinella radians (Pedinellophyceae) and its possible role in the adhesion of surface components
Authors: Koutoulis Anthony , Ludwig Martha , Wetherbee Richard 
Published: April 2021

ABSTRACT
Monoclonal antibodies have been generated against cell surface components of the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). One monoclonal antibody, designated Arg 1E5/1B1, labels a scale associated protein (SAP) of 145 kDa. Immunofluorescence microscopy of whole cells as well as immunoelectron microscopy of whole cell mounts and thin sections using Arg 1E5/1B1 have shown that the SAP is located on the proximal surface of body scales and spine-scales. Its specific location suggests that the SAP may play a role in the adhesion of these surface components to the cell membrane and/or to one another. The potential of monoclonal antibody Arg 1E5/1B1 as a tool to study cell surface morphogenesis and the role of the endomembrane system in A. radians is discussed.
Three-dimensional imaging of living neurons and glia with the atomic force microscope
Authors: Parpura Vladimir , Haydon Philip G. , Henderson Eric 
Published: April 2021

ABSTRACT
The atomic force microscope (AFM) was used to directly image hippocampal neurons and glia. Using chemically fixed and living cells it was possible to reconstruct three-dimensional cell structure and detect sub-cellular fea-tures such as the nucleus, mitochondria and filaments. By repeatedly scanning a single living cell we observed the movement of filaments beneath the cell membrane. Furthermore, by controlling the force applied to the scanning tip, nanosurgery was performed to selectively remove cells from the culture substratum. Thus, the atomic force microscope offers the opportunity to gain three-dimensional information about living cells and to observe the behavior of cellular components by imaging through the intact cell membrane.