bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sperm exocytosis reconstructed in a cell-free system: Evidence for the involvement of phospholipase c and actin filaments in membrane fusion
Authors: Spungin Ben , Margalit Ilana , Breitbart Haim 
Published: April 2021

ABSTRACT
We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal membranes were labeled with chlorophyll a or DCY, respectively. The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphoproteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and Gi-like proteins, while involved in exocytosis in intact cells, are not involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin. The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholipase C at the membrane. It also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.
Expression and localization of α-adaptin isoforms
Authors: Ball Catriona L. , Hunt Stephen P. , Robinson Margaret S. 
Published: April 2021

ABSTRACT
There are two α-adaptin genes, αA and αC, which in brain encode proteins of of Mr 108×103 and 104×103, respectively. Although both mRNAs can be detected on northern blots of brain and liver, the higher molecular mass polypeptide can only be detected on western blots of brain. Here we explain these observations by showing that αA is alternatively spliced and that the protein product in most tissues is different from the one expressed in brain in that it is missing 21 amino acids within the hinge region, giving it a similar mobility to that of αC. Monospecific antibodies were raised against the various α-adaptin isoforms and used to compare their distribution in cells and tissues. Both αA and αC are co-assembled into the same coated pits, and the larger isoform of αA is co-assembled with the smaller isoforms of α-adaptin, both in cells that naturally express it and in transfected cells. Examination of brain and spinal cord sections, labelled either for the larger isoform of αA or for αC, reveals that that the two are to some extent differentially distributed, consistent with previous in situ hybridisation studies. This finding, combined with the observation that there is considerable variability in the relative expression of the two isoforms in different tissues, indicates that the two genes are switched on in response to different stimuli. Moreover, the larger isoform of αA appears to be more efficiently concentrated in the nerve terminals than αC, which is found not only at the terminals but also diffusely distributed in the cell bodies and dendrites. This suggests that αC may play more of a role in the recycling of membrane components throughout the cell.
A chromomeric model for nuclear and chromosome structure
Authors: Cook Peter R. 
Published: April 2021

ABSTRACT
The basic structural elements of chromatin and chromosomes are reviewed. Then a model involving only three architectural motifs, nucleosomes, chromatin loops and transcription factories/chromomeres, is presented. Loops are tied through transcription factors and RNA polymerases to factories during interphase and to the remnants of those factories, chromomeres, during mitosis. On entry into mitosis, increased adhesiveness between nucleosomes and between factories drives a ‘sticky-end’ aggregation to the most compact and stable structure, a cylinder of nucleosomes around an axial chromomeric core.
Arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices
Authors: Robertson E. J. , Pyke K. A. , Leech R. M. 
Published: April 2021

ABSTRACT
The arc6 (accumulation and replication of chloroplasts) mutant of Arabidopsis has only two greatly enlarged chloroplasts per mature leaf mesophyll cell compared with ninety chloroplasts per cell in the wild type. The mutation is a single nuclear gene and the plant phenotype is normal. Shoot and root apical meristems of arc6 plants have been examined to determine how early during plastid development the mutant arc6 phenotype can be recognised. In the cells of the arc6 apical meristem there are only two proplastids, which are larger than wild type with a highly variable morphology. In the cells of the leaf primordia where differentiation of proplastids to chloroplasts occurs arc6 plastids are larger and at a more advanced developmental stage than wild-type plastids. In arc6 root cells statoliths and other plastids also show grossly abnormal morphology and the statoliths are greatly increased in size. During arc6 stomatal guard cell development the perturbation in proplastid population dynamics affects plastid segregation and 30% of stomata lack plastids in one or both guard cells. Our evidence would suggest that ARC6 is expressed throughout the vegetative cells of the Arabidopsis seedling with major effects on both the proplastid phenotype and the proplastid population. ARC6 is the first gene to be identified in Arabidopsis which has a global effect on plastid development in cells arising from both the shoot and root meristems, and is of major importance in the nuclear control of plastid differentiation in higher plants.
Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster
Authors: Hao Xu Feng , Alphey Luke , Bandara Lasantha R. , Lam Eric W.-F. , Glover David , Thangue Nicholas B. La 
Published: April 2021

ABSTRACT
The cellular transcription factor DRTF1/E2F is implicated in the control of early cell cycle progression due to its interaction with important regulators of cellular proliferation, such as pocket proteins (for example, the retinoblastoma tumour suppressor gene product), cyclins and cyclin- dependent kinase subunits. In mammalian cells DRTF1/E2F is a heterodimeric DNA binding activity which arises when a DP protein interacts with an E2F protein. Here, we report an analysis of DRTF1/E2F in Drosophila cells, and show that many features of the pathway which regulate its transcriptional activity are conserved in mammalian cells, such as the interaction with pocket proteins, binding to cyclin A and cdk2, and its modulation by viral oncoproteins. We show that a Drosophila DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding component of Drosophila DRTF1/E2F. An analysis of the expression patterns of a Drosophila DP and E2F protein indicated that DmDP is developmentally regulated and in later embryonic stages preferentially expressed in proliferating cells. In contrast, the expression of DmE2F-1 in late stage embryos occurs in a restricted group of neural cells, whereas in early embryos it is widely expressed, but in a segmentally restricted fashion. Some aspects of the mechanisms which integrate early cell cycle progression with the transcription apparatus are thus conserved between Drosophila and mammalian cells. The distinct expression patterns of DmDP and DmE2F-1 suggest that the formation of DP/E2F heterodimers, and hence DRTF1/E2F, is subject to complex regulatory cues.
Ligand-stimulated β2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes
Authors: Moore Robert H. , Sadovnikoff Nicholas , Hoffenberg Simon , Liu Shaobin , Woodford Pamela , Angelides Kimon , Trial JoAnn , Carsrud N. D. Victor , Dickey Burton F. , Knoll Brian J. 
Published: April 2021

ABSTRACT
The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human β2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition &gt;50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked β2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon β2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 120 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of β2 -agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that β2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.
Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues
Authors: Frigeri Antonio , Gropper Michael A. , Umenishi Fuminori , Kawashima M. , Brown Dennis , Verkman A. S. 
Published: April 2021

ABSTRACT
It was shown recently that water channel homologs MIWC (mercurial insensitive water channel) and GLIP (glycerol intrinsic protein) colocalized in basolateral membranes of kidney collecting duct, tracheal and colonic epithelia, and in brain pia mater. We report here an extensive immunolocalization study of MIWC and GLIP in non-epithelial and glandular epithelial tissues in rat. Immunogold electron microscopy confirmed colocalization of MIWC and GLIP in basolateral membrane of principal cells in kidney collecting duct. However, in other epithelia, MIWC but not GLIP was expressed in basolateral membrane of parietal cells in stomach, and in excretory tubules of salivary and lacrimal glands; GLIP but not MIWC was expressed in transitional epithelium of urinary bladder and skin epidermis. In the central nervous system, MIWC was strongly expressed in the ependymal layer lining the aqueductal system, and in astrocytes throughout the spinal cord and in selected regions of brain. MIWC was also expressed in a plasma membrane pattern in skeletal, but not smooth or cardiac muscle. Neither protein was expressed in small intestine, testis, liver, spleen and nerve. The tissue-specific expression of MIWC suggests a role in fluid transport and/or cell volume regulation in stomach and glandular epithelia. The functional role of MIWC expression in the neuromuscular system and of GLIP expression in skin and urinary bladder is uncertain. The specific cellular sites of MIWC expression (astrocytes, trachea, sarcolemma, gastric parietal cells and kidney principal cells) correspond exactly to sites where orthogonal arrays of particles (OAPs) have been visualized by freeze-fracture electron microscopy, suggesting that MIWC may be the OAP protein.
Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus
Authors: Steensel Bas van , Brink Marijke , Meulen Krina van der , Binnendijk Erica P. van , Wansink Derick G. , Jong Luitzen de , Kloet E. Ronald de , Driel Roel van 
Published: April 2021

ABSTRACT
The cell nucleus is highly organized. Many nuclear functions are localized in discrete domains, suggesting that compartmentalization is an important aspect of the regulation and coordination of nuclear functions. We investigated the subnuclear distribution of the glucocorticoid receptor, a hormone-dependent transcription factor. By immunofluorescent labeling and confocal microscopy we found that after stimulation with the agonist dexamethasone the glucocorticoid receptor is concentrated in 1,0002,000 clusters in the nucleoplasm. This distibution was observed in several cell types and with three different antibodies against the glucocorticoid receptor. A similar subnuclear distribution of glucocorticoid receptors was found after treatment of cells with the antagonist RU486, suggesting that the association of the glucocorticoid receptor in clusters does not require transformation of the receptor to a state that is able to activate transcription. By dual labeling we found that most dexamethasone-induced receptor clusters do not colocalize with sites of pre-mRNA synthesis. We also show that RNA polymerase II is localized in a large number of clusters in the nucleus. Glucocorticoid receptor clusters did not significantly colocalize with these RNA polymerase II clusters or with domains containing the splicing factor SC-35. Taken together, these results suggest that most clustered glucocorticoid receptor molecules are not directly involved in activation of transcription.
Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: Nuclear import of nucleocapsid protein and integrase
Authors: Risco Cristina , Menéndez-Arias Luis , Copeland Terry D. , Silva Pedro Pinto da , Oroszlan Stephen 
Published: April 2021

ABSTRACT
The entry and intracellular transport of Moloney-murine leukemia virions inside mouse NIH 3T3 cells have been followed by electron microscopy techniques. Five viral proteins - matrix (MA, p15), capsid (CA, p30), nucleocapsid (NC, p10), integrase (IN), and the envelope glycoprotein (SU, gp70) - were located by immunolabeling using gold probes. After entering the cells, viral particles were frequently detected inside cytoplasmic vesicles of variable size. Their viral envelope was apparently lost during intracytoplasmic transport. When the unenveloped viral cores reached the nuclear membrane or its vicinity, they were disrupted. Two of the immunolabeled proteins, NC and IN, were detected entering the nucleus of non-dividing cells, where both were targeted to the nucleolus. However, MA and CA were found only in the cytoplasm. NC is a nucleic acid-binding protein which contains potential nuclear localization signals. We suggest that NC could enter the nucleus as part of a nucleoprotein complex, associated with IN, and possibly, also with viral DNA.
Cell adhesion in sponges: Potentiation by a cell surface 68 kDa proteoglycan-binding protein
Authors: Varner Judith A. 
Published: April 2021

ABSTRACT
Constitutive, stable intercellular adhesion is one of the distinguishing properties of metazoans, of which the sponges (Phylum Porifera) are the most primitive representatives. In sponges, intercellular adhesion is mediated by the large proteoglycan-like cell agglutinating molecule ‘aggregation factor’, which binds to cell surfaces via an oligosaccharide moiety. Previous studies indicated that this aggregation factor binds to two proteins associated with the surface of sponge cells. One of these, a 68 kDa peripheral membrane protein, was isolated by affinity chromatography on aggregation factor conjugated to Sepharose. This monomeric 68 kDa glycoprotein plays a key role in sponge cell adhesion since it potently inhibits the binding of aggregation factor to cell surfaces and completely prevents aggregation factor-mediated cell adhesion. The 68 kDa aggregation factor ligand binds with high affinity to both aggregation factor (KD = 2×109 M) and cell surfaces (KD = 6×108 M) providing evidence that it serves as an intramolecular bridge between the aggregation factor molecule and a cell surface receptor. Therefore, this early metazoan protein may represent one of the earliest extracellular matrix adhesion proteins to have arisen in the course of metazoan evolution.
Interphase phosphorylation of the Drosophila nuclear lamin: Site-mapping using a monoclonal antibody
Authors: Stuurman Nico , Maus Nancy , Fisher Paul A. 
Published: April 2021

ABSTRACT
The Drosophila nuclear lamin is highly phosphorylated during interphase. Two interphase isoforms, differing in degree of phosphorylation, can be distinguished by onedimensional SDS-polyacrylamide gel electrophoresis. One migrates with an apparent mass of 74 kDa (1amin Dm1); the other is more highly phosphorylated and migrates as a 76 kDa protein (1amin Dm2). We generated a monoclonal antibody, ADL84 which binds to lamin Dm1 but not lamin Dm2. Binding of ADL84 to lamin Dm2 was restored by phosphatase treatment of immunoblots containing lamins. Immunoprecipitation with ADL84 demonstrated that purified Drosophila nuclear lamins Dm1 and Dm2 are present as a random mixture of homo- and heterodimers. Indirect immunofluorescence experiments suggest that lamin Dm1 is present in all Drosophila cell types. The epitope for ADL84 was mapped by analyzing binding to bacterially expressed lamin deletion mutants and subsequently by screening for point mutants (randomly generated by polymerase chain reaction) which were not recognized by ADL84. The ADL84-epitope encompasses amino acids R22PPSAGP (arginine 22-proline 28). Peptide competition experiments demonstrated directly that phosphorylation of serine 25 impedes lamin binding by ADL84. This suggests that serine 25 is the lamin Dm2-specific phosphorylation site.
N-cadherin in adult rat cardiomyocytes in culture. I. Functional role of N-cadherin and impairment of cell-cell contact by a truncated N-cadherin mutant
Authors: Hertig Cecilia M. , Eppenberger-Eberhardt Monika , Koch Sabine , Eppenberger Hans M. 
Published: April 2021

ABSTRACT
N-cadherin is a transmembrane Ca2+-dependent glycoprotein that is part of adherens junctions. It functions with the cell adhesion N-terminal extracellular domain as a site of homophilic cell-cell contacts. The intracellular C-terminal domain provides via a catenin complex the interaction with the cytoskeleton.
Ectopic expression of chicken N-cadherin in adult rat cardiomyocytes (ARC) in culture was obtained after microinjection into non-dividing cardiomyocytes; it was demonstrated that the exogenous protein colocalized with the endogenous N-cadherin at the plasma membrane of the cell and formed contact sites.
A dominant negative chicken N-cadherin mutant was constructed by a large deletion of the extracellular domain. This mutant was expressed and inhibited the function of the endogenous rat N-cadherin probably by competing for the catenin complex binding domain, which is essential for the formation of a stable cell-cell contact of ARC. The injected cells lost contact with neighbouring cells and retracted; the connexons of the gap junctions were pulled out as well. This could be avoided by another N-cadherin mutation, which, in addition to the N-terminal truncation, contained a deletion of the catenin binding domain. In the case of the truncated N-cadherin at the N terminus, the sarcomeric structure of the myofibrils of ARC was also affected. Myofibrils were the most vulnerable cytoskeletal structures affected by the overexpressed dominant negative N-cadherin mutation. Similar behaviour was shown when cardiomyocytes separated following Ca2+ depletion and when new cell-cell contacts were formed after Ca2+ replenishment. N-cadherin is thought to be the essential component for establishing new cell-cell contacts which eventually led to a new formation of intercalated disc-like structures in the cardiac cell culture.
The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression
Authors: Starborg Maria , Gell Katarina , Brundell Eva , Höög Christer 
Published: April 2021

ABSTRACT
We have isolated the murine homologue of the human Ki-67 antigen. The Ki-67 antigen is used as a marker to assess the proliferative capacity of tumour cells; however, its cellular function is not known. The murine Ki-67 cDNA sequence (TSG126) was found to contain 13 tandem repeats, making up more than half of the total protein size. A comparison of this repetitive sequence block to its human counterpart, which contains 16 consecutive repeat units, revealed several conserved sequence motifs, including one motif frequently observed in proteins interacting with DNA. An antiserum developed against the product of the TSG126 cDNA clone identified a protein with an apparent molecular mass of 360 kDa, mainly expressed in proliferating cells. The TSG126 protein begins to accumulate during the late G1 stage of the cell cycle and is first seen as numerous small granules evenly distributed throughout the nucleus. During the S and the G2 phases, larger foci that overlap with the nucleoli and the heterochromatic regions are formed. At the onset of mitosis the TSG126 protein undergoes a dramatic redistribution process and becomes associated with the surface of the condensed chromosomes. The relative absence of the TSG126 protein from G1 interphase cells strongly argues against a model where the association of the TSG126 protein with mitotic chromosomes merely reflects a mechanism for the symmetrical distribution of nucleolar proteins between daughter cells. Instead, the intracellular distribution of the TSG126 protein during the cell cycle suggests that it could have a chromatin-associated function in both interphase and mitotic cells. Microinjection of anti-TSG126 antibodies into proliferating Swiss-3T3 fibroblasts was found to delay cell cycle progression, indicating that the TSG126 protein has an essential nuclear function.
Molecular characterisation of ninein, a new coiled-coil protein of the centrosome
Authors: Bouckson-Castaing Véronique , Moudjou Mohammed , Ferguson David J. P. , Mucklow Stuart , Belkaid Yasmine , Milon Geneviève , Crocker Paul R. 
Published: April 2021

ABSTRACT
We describe the cDNA cloning of ninein, a novel component of centrosomes. In the mouse, ninein is predicted to be an acidic protein (calculated pI of 4.8) with alternatively spliced forms of 245 kDa and 249 kDa that contain extensive regions of coiled-coil structure flanked by non-coiled ends. Other interesting features of this protein include an EF-hand-like domain, a potential GTP binding site and four leucine zipper domains. Specific polyclonal antisera were raised to two non-overlapping recombinant fragments of the protein and used to characterise the cellular distribution of ninein. Immunofluorescence and immunoelectron microscopy experiments with macrophage-like cells, Mm1, showed that ninein is localised specifically in the pericentriolar matrix of the centrosome. Studies with NIH3T3 fibroblasts demonstrated that ninein is associated with the centrosome throughout the cell cycle and can also be detected within nuclei at interphase. At mitosis ninein was also observed in association with the mitotic spindle. Immunocytochemical staining of mouse tissues showed that ninein was expressed in a heterogeneous fashion. Staining, if present, was always consistent with a centrosomal localisation and was never associated with nuclei. Ninein provides a new molecular tool for analysing the structure and function of the centrosome.