bluuebunny/crossref_metadata_2025_split huggingface dataset:
Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable
Authors: Franck Zsofia , Gary Ronald , Bretscher Anthony 
Published: April 2021

ABSTRACT
The band 4.1 superfamily of proteins show approx. 30% sequence identity in their amino-terminal region to the membrane binding domain of erythrocyte band 4.1. Within this superfamily are three members, ezrin, radixin and moesin, that show approx. 75% overall sequence identity. A comparison of the domain struc-ture and intracellular localization of ezrin and moesin in cultured cells is reported here.
Limited proteolytic digestion of ezrin or moesin yields a relatively stable 32 kDa domain derived from the amino-terminal region that is homologous to the pro-tease-resistant membrane binding domain of erythro-cyte band 4.1. The remaining regions of the two pro-teins give rise to very different fragments, suggesting that the secondary/tertiary structures of the two pro-teins are different in these regions.
We have generated polyclonal antibodies that dis-criminate between ezrin and moesin, and do not react with radixin. All cultured cell lines investigated contain ezrin, whereas moesin is variably expressed. Cells that contain both ezrin and moesin show a very similar pat-tern: both proteins are enriched and colocalize with actin in cell surface structures. Ezrin is also detected in the cytoplasm. In cells with few or no surface structures, both proteins show a patchy distribution in regions of the cell that contain fine networks of actin filaments. No staining of focal contacts or adherens junctions was observed. These results, together with those of others, lead to the conclusion that, of the members of this pro-tein family, only radixin is an authentic component of adherens junctions and focal contacts. Ezrin and moesin are both found in cell surface structures after treatment of human A431 cells with epidermal growth factor, and ezrin, but not moesin, becomes phosphorylated on tyro-sine. This study shows that ezrin and moesin have a sim-ilar subcellular distribution in cultured cells, yet are dis-tinguishable in their expression, structure and ability to serve as a kinase substrate.
Changing spatial patterns of DNA replication in the noise-damaged chick cochlea
Authors: Hashino Eri , Salvi Richard J. 
Published: April 2021

ABSTRACT
The purpose of the present study was to examine the spatio-temporal pattern of cell proliferation in the chick cochlea in response to the sensory hair cell loss induced by a 1.5 kHz pure tone at 120 dB SPL (1 dB=20 Pa) for 48 h. DNA replication was evaluated with the bro-modeoxyuridine (BrdU) pulse-fix technique. One group of birds was given multiple injections of BrdU (50 mg/kg) over a period of 8 h at various starting times during or after the exposure. Afterwards, their cochleas were removed and processed as whole mounts for BrdU immunohistochemistry. The cochleas of a second group of acoustically traumatized chicks were evaluated by scanning electron microscopy in order to determine the spatio-temporal pattern of hair cell loss. Hair cell loss was first observed 12 h after the start of the exposure and DNA replication started near the inferior edge of the hair cell lesion 24–32 h after the start of the expo-sure, i.e. 12–20 h after the first sign of hair cell loss. The site of hair cell loss and DNA replication shifted toward the superior edge of the basilar papilla as the exposure continued. The rate of DNA replication accelerated and reached its peak near the end of the 48 h exposure. The estimated latency of cell proliferation after hair cell loss was faster and the duration of DNA replication shorter than that observed in other sensory systems. The spatio-temporal pattern of DNA replication follows the spatio-temporal gradient of hair cell loss, suggesting that cell proliferation is triggered by hair cell loss itself rather than by intrinsic positional cues or gradients.
A mechanism for the intracellular localization of myosin II filaments in the Dictyostelium amoeba
Authors: Yumura Shigehiko , Kitanishi-Yumura Toshiko 
Published: April 2021

ABSTRACT
When ATP is added to membrane-cytoskeletons pre- pared from Dictyostelium amoebae by the method described previously (S. Yumura and T. Kitanishi- Yumura, Cell Struct. Funct. 15, 355-364, 1990), myosin II is released from the membrane-cytoskeletons after contraction. Simultaneously, the heavy chains of myosin II are phosphorylated by a putative myosin II heavy- chain kinase, at foci within the actin network, with the resultant disassembly of filaments. In this study, we examined factors that control the release of myosin II from the membrane-cytoskeletons, on the assumption that inhibition of the release of myosin II keeps the myosin II in the cortical region, and is responsible for the localization of myosin II in the cortical region. The release of myosin II is inhibited at pH values below 6.5. This effect is not due to the inhibition of heavy-chain phosphorylation but is due to the suppression of disassembly of the filaments. In the membrane-cytoskeletons of aggregating cells, the release of myosin II is inhibited by ca2+, and this effect is enhanced by pretreatment with calmodulin. In the membrane-cytoskeletons of veg- etative cells, the release of myosin II is inhibited by pre- treatment with calmodulin, and this effect is ca2+-inde- pendent. The inhibition of the release of myosin II by ca2+ and/or calmodulin is due to the inhibition of heavy- chain phosphorylation, and calmodulin is associated with the foci within the actin network. These results rep- resent a possible mechanism for the intracellular local- ization of myosin II via regulation of the release of myosin from the cortical region by changes in intracel- lular pH and/or intracellular concentrations of ca2+.
Ultrastructural distribution of DNA and RNA within the nucleolus of human Sertoli cells as seen by molecular immunocytochemistry
Authors: Thiry Marc 
Published: April 2021

ABSTRACT
The precise distribution of DNA and RNA within the human Sertoli cell nucleolus has been investigated, at the ultrastructural level, by cytochemical and molecu-lar immunocytochemical techniques. In Sertoli cells, the nucleolar components show a typical spatial distribu-tion. The fibrillar centres are not surrounded by a layer of dense fibrillar component, but come in contact only with strands of dense fibrillar component. These fibril-lar parts of strands are the extensions of granular strands connected to a large granular mass. These strands delimit numerous nucleolar interstices in which chromatin fibres are clearly obvious. Using the in situ terminal deoxynucleotidyl transferase/immunogold pro-cedure for detecting DNA, we find evident label exclusively over the chromatin fibres enclosed in the nucleo-lar interstices and over the fibrillar centres, and no sig-nificant label over the dense fibrillar component and granular component of the nucleolus. Furthermore, using the polyadenylate nucleotidyl transferase/immunogold procedure for detecting RNA, we show that label is deposited not only over the granular component and dense fibrillar component, as expected, but also quite obviously over the fibrillar centres. No label is seen over the interstices containing chromatin.
The Drosophila melanogaster dodecasatellite sequence is closely linked to the centromere and can form connections between sister chromatids during mitosis
Authors: Carmena Mar , Abad Jose P. , Villasante Alfredo , Gonzalez Cayetano 
Published: April 2021

ABSTRACT
We have used fluorescence in situ hybridisation to wild-type and rearranged mitotic chromosomes to map the Drosophila melanogaster dodecasatellite sequence. It is located at a unique site, within the pericentric hete-rochromatin of the right arm of the third chromosome, closely linked to the primary constriction. In polytene chromosomes, dodecasatellite is found as one or a few dots in the central region of the chromocentre. In untreated diploid cells, dodecasatellite sequences are found as one or two dots throughout the cell cycle. This distribution can be altered in a cell cycle-dependent manner in two ways. Firstly, in interphase cells, hypo-tonic shock promotes the decondensation of the genomic region containing this satellite, resulting in a string-like structure. Secondly, some of the precociously separated sister chromatids produced by colchicine treatment show dodecasatellite within the intervening space con-necting the main dodecasatellite signals of each chro-matid. The distribution of dodecasatellite seems to be rather constant between individuals of the same species, as indicated by the lack of any detectable variations in its pattern amongst individuals from six geographically distant strains of D. melanogaster. On the other hand, the distribution of dodecasatellite shows a remarkable degree of variation amongst closely related species of the melanogaster subgroup ranging from a non-detectable signal in Drosophila yakuba and Drosophila teissieri, to staining in the X, second and third chro-mososomes of Drosophila mauritiana.
Laminin and tenascin assembly and expression regulate HC11 mouse mammary cell differentiation
Authors: Chammas Roger , Taverna Daniela , Cella Nathalie , Santos Cecilia , Hynes Nancy E. 
Published: April 2021

ABSTRACT
HC11 is a normal mouse mammary epithelial cell line that requires certain growth factors, such as EGF or bFGF, to respond optimally to lactogenic hormones and produce the differentiation marker β-casein. Growth in insulin (Ins) or PDGF does not produce cells competent to respond to lactogenic hormones. Here we show that competency for differentiation is due at least in part to the modulation of extracellular matrix components. In particular we have studied laminin and tenascin. EGF alters endogenous laminin assembly. In addition, promotion of competency can be partially mimicked by plating HC11 cells on the E8 laminin fragment, which is able to induce lactogenic responsiveness in cells grown in the absence of EGF or bFGF. The production and assembly of tenascin is also dependent upon the growth conditions of the HC11 cells. EGF- or bFGF-grown competent cells produce tenascin but do not assemble it at the extracellular matrix as efficiently as Ins- or PDGF-grown, non-competent cells. This alteration apparently leads to a change in the cellular microenvironment that supports β-casein production. In addition, when competent cells are plated on dishes coated with tenascin, lactogenic hormone induction of β-casein is inhibited. The data suggest that tenascin assembly and β-casein production are opposing features of a coordinated differentiation program of HC11 cells.
Uric acid degrading enzymes, urate oxidase and allantoinase, are associated with different subcellular organelles in frog liver and kidney
Authors: Usuda Nobuteru , Hayashi Sueko , Fujiwara Satoko , Noguchi Tomoo , Nagata Tetsuji , Rao M. Sambasiva , Alvares Keith , Reddy Janardan K. , Yeldandi Anjana V. 
Published: April 2021

ABSTRACT
On the basis of differential and density gradient centrifugation studies, the site of the uric acid degrading enzymes, urate oxidase and allantoinase, in amphibia was previously assigned to the hepatic peroxisomes. Using specific antibodies against frog urate oxidase and allantoinase, we have undertaken an immunocytochemical study of the localization of these two proteins in frog liver and kidney, and demonstrate that whereas urate oxidase is present in peroxisomes, allantoinase is localized in mitochondria. Urate oxidase and allantoinase were detected by immunoblot analysis in both frog liver and kidney. The subcellular localization of these two enzymes was ascertained by Protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in frog liver parenchymal cells and kidney proximal tubular epithelium contained a semi-dense subcrystalloid core, which was found to be the exclusive site of urate oxidase localization. Allantoinase was detected within mitochondria, but not in peroxisomes of hepatocytes or proximal tubular epithelium. No allantoinase was detected in the mitochondria of nonhepatic parenchymal cells in liver and of the cells lining the distal convoluted tubules of the kidney. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of frog kidney contain this enzyme. Contrary to previous assumptions, these studies also clearly establish that urate oxidase and allantoinase, the first two enzymes involved in uric acid degradation, are localized in different subcellular organelles in frog liver and kidney.
Mutations in the non-helical linker segment L1-2 of keratin 5 in patients with Weber-Cockayne epidermolysis bullosa simplex
Authors: Chan Yiu-mo , Yu Qian-Chun , LeBlanc-Straceski Janine , Christiano Angela , Pulkkinen Leena , Kucherlapati Raju S. , Uitto Jouni , Fuchs Elaine 
Published: April 2021

ABSTRACT
Keratins are the major structural proteins of the epidermis. Analyzing keratin gene sequences, appreciating the switch in keratin gene expression that takes place as epidermal cells commit to terminally differentiate, and elucidating how keratins assemble into 10 nm filaments, have provided the foundation that has led to the discoveries of the genetic bases of two major classes of human skin diseases, epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EH). These diseases involve point mutations in either the basal epidermal keratin pair, K5 and K14 (EBS), or the suprabasal pair, K1 and K10 (EH). In severe cases of EBS and EH, mutations are found in the highly conserved ends of the α-helical rod domain, regions that, by random mutagenesis, had already been found to be important for 10 nm filament assembly. In order to identify regions of the keratin polypeptides that might be more subtly involved in 10 nm filament assembly and to explore the diversity in mutations within milder cases of these diseases, we have focused on Weber-Cockayne EBS, where mild blistering occurs primarily on the hands and feet in response to mechanical stress. In this report, we show that affected members of two different W-C EBS families have point mutations within 1 residue of each other in the non-helical linker segment of the K5 polypeptide. Genetic linkage analyses, the absence of this mutation in &gt;150 wild-type alleles and filament assembly studies suggest that these mutations are responsible for the W-C EBS phenotype. These findings provide the best evidence to date that the non-helical linker region in the middle of the keratin polypeptides plays a subtle but significant role in intermediate filament structure and/or intermediate filament cytoskeletal architecture.
Quantification of microtubule dynamics in living plant cells using fluorescence redistribution after photobleaching
Authors: Hush J. M. , Wadsworth P. , Callaham D. A. , Hepler P. K. 
Published: April 2021

ABSTRACT
Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is =31±6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (=67±seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all four MT arrays are more consistent with subunit exchange by the mechanism of dynamic instability than treadmilling. This is the first quantification of MT dynamics in plant cells.
An improved polarized rat hepatoma hybrid cell line: generation and comparison with its hepatoma relatives and hepatocytes in vivo
Authors: Shanks Michael R. , Cassio Doris , Lecoq Odile , Hubbard Ann L. 
Published: April 2021

ABSTRACT
Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for &gt;5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E. D., Meads, T., Shanks, M. R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761-1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not co-localize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.
The DNA binding domain of retinoic acid receptor β is required for ligand-dependent suppression of proliferation: Application of general purpose mammalian coexpression vectors
Authors: Frangioni John V. , Moghal Nadeem , Stuart-Tilley Alan , Neel Benjamin G. , Alper Seth L. 
Published: April 2021

ABSTRACT
We have developed a family of mammalian coexpression vectors that permit identification of living or fixed cells overexpressing a gene of interest by surrogate detection of a coexpressed marker protein. Using these ‘pMARK’ vectors, a fluorescence-based, single cell proliferation assay was developed and used to study the effect of retinoic acid receptor β (RAR-β) on cell cycling. We demonstrate that transient overexpression of RAR-β in the presence, but not absence, of all-trans retinoic acid results in a dramatic suppression of cell proliferation. We further show that this effect requires the DNA binding (C) domain of RAR-β. It has been previously shown that RAR-β expression is markedly altered in a variety of neoplasms and cell lines. Our data support the hypothesis that loss of RAR-β may contribute to tumor progression by removing normal restraints on proliferation. The pMARK vectors should be useful for studying other genes that putatively suppress or enhance proliferation.
Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis
Authors: Endow Sharyn A. , Chandra Rashmi , Komma Donald J. , Yamamoto Akihiko H. , Salmon Edward D. 
Published: April 2021

ABSTRACT
Nonclaret disjunctional (ncd) is a kinesin-related micro-tubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.
A chromosome breakage assay to monitor mitotic forces in budding yeast
Authors: Brock Jo-Ann K. , Bloom Kerry 
Published: April 2021

ABSTRACT
During the eukaryotic cell cycle, genetic material must be accurately duplicated and faithfully segregated to each daughter cell. Segregation of chromosomes is dependent on the centromere, a region of the chromosome which interacts with mitotic spindle microtubules during cell division. Centromere function in the budding yeast, Saccharomyces cerevisiae, can be regulated by placing an inducible promoter adjacent to centromere DNA. This conditional centromere can be integrated into chromosome III to generate a conditionally functional dicentric chromosome. Activation of the dicentric chromosome results in a transient mitotic delay followed by the generation of monocentric derivatives. The propagation of viable cells containing these monocentric derivative chromosomes is dependent upon the DNA repair gene RAD52, indicating that double-strand DNA breaks are structural intermediates in the dicentric repair pathway.
We have used these conditionally dicentric chromosomes to monitor the exertion of mitotic forces during cell division. Analysis of synchronized cells reveal that lethality in dicentric, rad52 mutant cells occurs during G2/M phase and is concomitant with the transient mitotic delay. The delay is largely dependent upon the cell cycle checkpoint gene RAD9, which is involved in monitoring DNA damage. These data demonstrate that DNA lesions resulting from dicentric activation are responsible for signalling the mitotic delay. Since the delay precedes the decline of p34cdc28 kinase activity, mitotic forces sufficient to result in dicentric chromosome breakage are generated prior to spindle elongation and anaphase onset in yeast.