bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis
Authors: Samejima Itaru , Matsumoto Tomohiro , Nakaseko Yukinobu , Beach David , Yanagida Mitsuhiro
Published: April 2021

Fission yeast cut mutants cause cytokinesis in the absence of normal nuclear division. These mutants show abnormal uncoupled mitosis and are known to be the result of mutations in the genes encoding DNA topoiso-merase II, proteins related to spindle pole duplication, and a kinesin-related mitotic motor. We have screened 717 temperature-sensitive (ts) mutants by individually observing their cytological phenotypes at the restrictive temperature, and have newly isolated 25 cut mutants. Genetic analyses indicate that 14 of them fall into five previously identified loci, namely, top2, cut1, cut5, cut7 and cut9, whereas nine have been mapped onto seven new loci, designated cut13 to cut19. The cytological phe-notypes of the newly identified cut mutants can be clas-sified into three groups. One group consists of mutants in which a portion of the nuclear chromatin is stretched by the elongated spindle but the entire nucleus is not separated, reminiscent of, but not identical to, the phe-notypes of top2 and cut1; mutants cut14-208, cut15-85, cut16-267 and cut17-275 display such a phenotype. Another group exhibits non-disjunctioned and con-densed chromosomes in the presence of the spindle; cut13-131 belongs to this group. The cut19-708 mutant has also been found to have condensed chromosomes. The remaining group has a mixed phenotype of the above two groups; namely, stretched chromatin and condensed chromosomes; cut18-447 exhibits such a phenotype. The isolation and characterization of the mutated genes will be the subjects of future investiga-tions.
Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI, and β1 integrin receptors
Authors: Lee Tien-Ling , Lin Ying-Chun , Mochitate Katsumi , Grinnell Frederick
Published: April 2021

To learn about the effects of tension on fibroblast func-tion, we have been studying initial cellular responses to stress-relaxation. Human foreskin fibroblasts were cul-tured in anchored collagen matrices for 2 days, during which time mechanical stress developed. Subsequently, the matrices were dislodged; thereby allowing stress to dissipate. Within 5 min after initiating stress-relaxation, fibroblasts retracted their pseudopodia. At this time, we observed the disappearance of cellular stress fibers and the formation of actin clusters along the cell margins. The actin was found to be located inside 200 nm diam-eter vesicles that were budding from the cell surface. Vesicles isolated from the matrix after stress-relaxation contained prominent 24 kDa, 36 kDa (doublet), 45 kDa, and 135 kDa polypeptides. The 45 kDa polypeptide was the major component in the Triton-insoluble vesicle fraction and appeared to be actin. The 36 kDa (doublet) polypeptide, which was found predominantly in the Triton-soluble vesicle fraction, was identified as annexin II. Vesicles also contained annexin VI and 11 integrin receptors but not tubulin, vimentin, vinculin or annexin I. The results suggest that stress-relaxation of fibrob-lasts induces a novel ectocytotic process involving tran-sient budding of intact, plasma membrane vesicles from the cell cortex. On the basis of their morphological and biochemical features, these vesicles may be analogous to the ‘matrix vesicles’ released by chondrocytes and could play a role in extracellular matrix remodeling after wound contraction.
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

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

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

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

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

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

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

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

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 >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

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

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 >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

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

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

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.
Cellular localization of RNA14p and RNA15p, two yeast proteins involved in mRNA stability
Authors: Bonneaud Nathalie , Minvielle-Sebastia Lionel , Cullin Christophe , Lacroute François
Published: April 2021

RNA14 and RNA15 were originally identified by temperature-sensitive mutations that cause a rapid decrease in poly(A)-tail length and overall mRNA levels at the restrictive temperature. We have raised antibodies to the RNA14 and RNA15 proteins, and used subcellular fractionation and immunofluorescence to localize these proteins within the yeast cell. RNA14p is a 73 kDa protein found in both the nucleus and the cytoplasm, whilst RNA15p is a 42 kDa protein detected only in the nucleus. The observation that both proteins are found in the nucleus is in agreement with previous genetic data which suggest an interaction between RNA14p and RNA15p. Also the joint nuclear localization is consistent with the biochemical data suggesting a role in polyadenylation. The detection of significant amounts of RNA14p in the cytoplasm opens the possibility of a second function for this protein, either in cytoplasmic regulation of mRNA deadenylation or, more interestingly, in mRNA stability.
Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells
Authors: Wagner Martina , Rajasekaran Ayyappan K. , Hanzel David K. , Mayor Satyajit , Rodriguez-Boulan Enrique
Published: April 2021

The trans-Golgi network (TGN) of MDCK cells is exquisitely sensitive to the fungal metabolite brefeldin A (BFA), in contrast to the refractory Golgi stack of these cells. At a concentration of 1 μg/ml, BFA promoted extensive tubulation of the TGN while the medial Golgi marker α-mannosidase II was not affected. Tubules emerging minutes after addition of the drug contained both the apical marker influenza hemagglutinin (HA), previously accumulated at 20°C, and the fusion protein interleukin receptor/TGN38 (TGG), a TGN marker that recycles basolaterally, indicating that, in contrast to TGN vesicles, TGN-derived tubules cannot sort apical and basolateral proteins. After 60 minutes treatment with BFA, HA and TGG tubules formed extensive networks widely spread throughout the cell, different from the focused centrosomal localization previously described in non-polarized cells. The TGG network partially codistributed with an early endosomal tubular network loaded with transferrin, suggesting that the TGG and endosomal networks had fused or that TGG had entered the endosomal network via surface recycling and endocytosis. The extensive structural alterations of the TGN were accompanied by functional disruptions, such as the extensive mis-sorting of influenza HA, and by the release of the TGN marker γ-adaptin. Our results suggest the involvement of BFA-sensitive adaptor proteins in TGNrsurface transport.
Conformation dependence of integrin-type II collagen binding: Inability of collagen peptides to support α2β1 binding, and mediation of adhesion to denatured collagen by a novel α5β1-fibronectin bridge
Authors: Tuckwell Danny S. , Ayad Shirley , Grant Michael E. , Takigawa Masaharu , Humphries Martin J.
Published: April 2021

The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin α2β1 while binding to denatured collagen was mediated by a novel α5β1-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.
Intracellular free calcium oscillations in normal and cleavage-blocked embryos and artificially activated eggs of Xenopus laevis
Authors: Keating Thomas J. , Cork R. John , Robinson Kenneth R.
Published: April 2021

We have measured levels of intracellular free calcium ([Ca2+]i) in albino Xenopus laevis embryos using recombinant aequorin and a photon-counting system. We observed sinusoidal oscillations in [Ca2+]i that had the same frequency as cleavage, with cleavage occurring when [Ca2+]i was lowest. An increase in calcium was seen to precede first cleavage. The cyclic changes in calcium were superimposed on a secondary pattern that increased, peaked between third and fifth cleavages and then slowly declined to a level similar to that measured before first cleavage. The amplitude of the oscillations was small during the first few cleavages but became larger with each cycle, with the largest oscillations occurring when the secondary pattern peaked (between third and fifth cleavage). As the secondary pattern declined, the amplitude of the oscillations also became smaller. The oscillations are due to release of calcium from intracellular stores, since the signal was the same in calcium-free solution as in normal medium.

When cleavage was blocked with the microtubule-disrupting drugs colchicine or nocodazole, the [Ca2+]i oscillations persisted. Calcium oscillations of a similar magnitude and frequency were also present in artificially activated eggs. The secondary pattern was different in cleavage-blocked embryos and artificially activated eggs, the baseline increasing until about the third cycle and then remaining elevated for the rest of the recording (>8 hours).

By fixing embryos at various points in the calcium cycle, we determined that mitosis began shortly after calcium levels reached their peak and was complete before the calcium level dropped to its lowest point. The fact that the calcium oscillations persist when nuclear division is not occurring suggests that either they operate independently of any downstream events that they might control or they are related to other cyclic activities in the eggs and embryos such as cycling of pHi, MPF, or surface contraction waves.

When aequorin was injected into individual blastomeres of 64-cell embryos, the shape of the signal was substantially different from that seen in recordings from whole embryos. Compared to the whole-embryo recordings, the signal from a subregion of the embryo rose more quickly and had a slower, biphasic decline. These differences indicate that [Ca2+]i increases are not occurring uniformly across the embryo but are spatially localized, perhaps progressing as waves.
Band 6 protein, a major constituent of desmosomes from stratified epithelia, is a novel member of the armadillo multigene family
Authors: Hatzfeld Mechthild , Kristjansson Gunnar Ingi , Plessmann Uwe , Weber Klaus
Published: April 2021

Desmosomes are intercellular adhering junctions characteristic of epithelial cells. Several constitutive proteins-desmoplakin, plakoglobin and the transmembrane glycoproteins desmoglein and desmocollin-have been identified as fundamental constituents of desmosomes in all tissues. A number of additional and cell type-specific constituents also contribute to desmosomal plaque formation. Among these proteins is the band 6 polypeptide (B6P). This positively charged, non-glycosylated protein is a major constituent of the plaque in stratified and complex glandular epithelia. Using an overlay assay we show that purified keratins bind in vitro to B6P. Thus B6P may play a role in ordering intermediate filament networks of adjacent epithelial cells. To characterize the structure of B6P in the desmosome we have isolated cDNA clones representing the entire coding sequence. The predicted amino acid sequence of human B6P shows strong sequence homology with a murine p120 protein, which is a substrate of protein tyrosine kinase receptors and of p60v-rc. P120 and B6P show amino-terminal domains differing distinctly in length and sequence. These are followed in both proteins by 460 residues that display a series of imperfect repeats corresponding to the repeats in the cadherin binding proteins armadillo, plakoglobin and P-catenin. Over this repeat region B6P and p120 share 33% sequence identity (54% similarity). These sequence characteristics define B6P as a novel member of the armadillo multigene family and raise the question of whether the structural proteins B6P, plako-globin, β-catenin and armadillo share some function. Since armadillo, plakoglobin, β-catenin and p120 seem involved in signal transduction this may also hold for B6P. The amino-terminal region of B6P (residues 1 to 263) shows no significant homology to any known protein sequence. It may therefore be involved in unique functions of B6P.
Differential calmodulin binding to three myosin-1 isoforms from liver
Authors: Coluccio Lynne M.
Published: April 2021

We have previously purified and characterized two myosin-1 isoforms from rat liver (molecular masses 130 kDa and 110 kDa; L. M. Coluccio and C. Conaty (1993) Cell Motil. Cytoskel. 24, 189–199). Here, we describe the purification and characterization from liver of a third myosin-1 (molecular mass 105 kDa) and determine the number of calmodulin molecules associated with each of these three myosin-1 isoforms. The 105 kDa polypeptide, solubilized from liver homogenates with the addition of ATP, co-sediments with F-actin, co-purifies with calmod-ulin, and binds calmodulin in the presence of EGTA. Anti-bodies directed against chicken intestinal brush border myosin-1 cross-react with the 105 kDa polypeptide on immunoblots. Partial peptide sequence analysis indicates that the polypeptide corresponds with an MM1γ gene product that represents a myosin-1 isoform cloned from mouse brain (Sherr et al. (1993)J. Cell Biol. 120, 1405-1416). A comparison of calmodulin binding to the now three isolated forms of myosin-1 in liver shows that in solution the 105 kDa and 110 kDa polypeptides bind two molecules of calmodulin each whereas the 130 kDa binds six molecules of calmodulin.
Ultraviolet radiation-induced melanogenesis in human melanocytes Effects of modulating protein kinase C
Authors: Carsberg C. J. , Warenius H. M. , Friedmann P. S.
Published: April 2021

The mechanism by which ultraviolet radiation induces melanogenesis in epidermal melanocytes is unknown. Previous observations that in cultured human melanocytes 1-oleoyl-2-acetylglycerol augmented both basal and ultra-violet radiation-induced melanogenesis, suggested that the responses were mediated via protein kinase C. However, paradoxically the phorbol ester TPA was without effect. Therefore, the present study has examined the involvement of protein kinase C in melanogenesis. Analysis of the isozyme profile of human melanocytes revealed the presence of protein kinase C α, βI, ε and ζ but not the isozyme η. Following exposure to 500 nM TPA for 24 hours, isozymes α, βI and ε were downregulated, but ζ was unaffected. Similar isozyme profiles were observed in S91 and SKMEL3 melanoma cells. The melanogenic responses to 1-oleoyl-2-acetylglycerol and ultraviolet radiation were unaffected by inhibition of protein kinase C with Ro31-8220, or ablation by downregulation with 500 nM TPA, in human melanocytes and melanoma cells. 1-Oleoyl-2-acetyl-glycerol had no effect on protein kinase C activity in human melanocytes, as measured by rapid phosphorylation of the 80 kDa protein myristoylated alanine-rich C kinase substrate (MARCKS). Ultraviolet radiation induced a small increase in MARCKS protein phosphorylation but this effect was inhibited by pretreatment for 24 hours with 500 nM TPA, which had no effect on ultraviolet-induced melanogenesis. Overall, these findings indicate that 1-oleoyl-2-acetylglycerol and ultraviolet radiation activate melanogenesis via protein kinase C-independent pathways.
The heparin-binding domain of heparin-binding EGF-like growth factor can target Pseudomonas exotoxin to kill cells exclusively through heparan sulfate proteoglycans
Authors: Mesri Enrique A. , Ono Minoru , Kreitman Robert J. , Klagsbrun Michael , Pastan Ira
Published: April 2021

Heparin-binding EGF-like growth factor (HB-EGF) is a smooth muscle cell mitogen composed of both EGF receptor and heparin-binding domains. To better understand the function of its domains, intact HB-EGF or its heparin-binding (HB) domain (amino acids 1-45) were fused to a mutant Pseudomonas exotoxin with an inactivated cell-binding domain. The resulting chimeric toxins, HB-EGF-PE* and HB-PE*, were tested on tumor cells, proliferating smooth muscle cells and a mutant Chinese hamster ovary cell line deficient in heparan sulfate proteoglycans (HSPGs). Two targets were found for HB-EGF-PE*. Cells were killed mainly through EGF receptors, but the HB domain was responsible for killing via HSPGs. HB-PE* did not bind to the EGF receptor and thus was cytotoxic by interacting exclusively with HSPGs. We conclude that the HB domain of HB-EGF is able to mediate internalization through HSPGs, without requiring the EGF receptor.
Yeast homolog of mammalian mitogen-activated protein kinase, FUS3/DAC2 kinase, is required both for cell fusion and for G1 arrest of the cell cycle and morphological changes by the CDC37 mutation
Authors: Fujimura Hiro-aki
Published: April 2021

Saccharomyces cerevisiae FUS3/DAC2 protein kinase, a homolog of mammalian mitogen-activated protein (MAP) kinase, inactivates a G1 cyclin encoded by the CLN3 gene to arrest cell division in the G1 phase and activates a tran-scriptional factor STE12 in response to mating pheromone during sexual conjugation. To elucidate the role of the FUS3/DAC2 gene product in the mating process, I con-structed and characterized dac2 cln3 double mutants. Here, I show that FUS3/DAC2 is required for completion of cell fusion even in the dac2 cln3 double mutants in which the pheromone response is restored, suggesting that FUS3/DAC2 plays a positive role in cell fusion during con-jugation. In addition, the cdc dac2 and cdc37 ste double mutants were constructed and investigated for their phe-notypes to clarify the relationship between FUS3/DAC2, STE7 or STE11 and CDC gene products (CDC28, 36, 37 and 39). The results indicate that FUS3/DAC2 may act upstream of CDC28 and provide evidence that the G1 arrest and morphological changes conferred by the cdc37 mutation may require FUS3/DAC2 (MAP kinase), STE7(MEK) and STE11 (MEK kinase).
The regulated degradation of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase reporter construct occurs in the endoplasmic reticulum
Authors: Lecureux Lloyd W. , Wattenberg Binks W.
Published: April 2021

The rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase, is regulated at a number of levels. One important mechanism is regulation of the half-life of the protein by a controlled proteolytic system. This comes about in response to downstream products of the sterol biosynthetic pathway. Little is known about this system, including where in the cell this regulated degradation occurs. HMG CoA reductase resides in the endoplasmic reticulum. To localize the site of regulated degradation of HMG CoA reductase, we used a construct that fuses the N-terminal membrane-anchoring domain of HMG CoA reductase in-frame with β-galactosidase as a reporter domain (HM-Gal). HM-Gal has previously been shown to reproduce faithfully the degradative properties of native HMG CoA reductase (Chun et al. (1990)J. Biol. Chem. 265, 22004-22010). CHO cells transfected with DNA encoding HM-Gal were exposed to mevalonic acid, which enhances the rate of HMG CoA reductase degradation several fold, and leads to the reduction of the steady state levels of HM-Gal by 80-90%. To accumulate HMG CoA reductase at the site of degradation, cells were simultaneously treated with N-acetyl-leucyl-leucyl-norleucinal (ALLN), which inhibits the protease responsible for reductase degradation. HM-Gal was localized morphologically by immunofluorescence and biochemically by measuring β-galactosidase activity in Percoll gradients of cellular homogenates. Using either technique HM-Gal localization was indistinguishable from that of ER markers in both control cells and in cells treated to accumulate HMG CoA reductase at the site of degradation. We conclude that the regulated degradation of HMG CoA reductase occurs in the ER or an ER-like compartment.
Telomere length and proliferation potential of hematopoietic stem cells
Authors: Lansdorp Peter M.
Published: April 2021

Hematopoietic stem cells have typically been defined as pluripotent cells with self-renewal capacity. Recent studies have shown striking differences in the mean length of telomeric repeat sequences at the end of chromosomes from human hematopoietic cells at different stages of development. The most likely explanation for these observations is that hematopoietic stem cells, like all other somatic cells studied to date, lose telomeric DNA upon each cell division. In this review, limitations in the replicative potential of hematopoietic stem cells are discussed in the context of possible clinical use of such cells for transplantation and gene therapy.
Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures
Authors: Chen Ping , Vukicevic Slobodan , Sampath T. Kuber , Luyten Frank P.
Published: April 2021

We examined the effect of recombinant human osteogenic protein-1 (OP-1, or bone morphogenetic protein-7), a member of the bone morphogenetic protein family, on growth and maturation of day 11, 15 and 17 chick sternal chondrocytes in high density monolayers, suspension and agarose cultures for up to 5 weeks. OP-1 dose-dependently (10-50 ng/ml) promoted chondrocyte maturation associated with enhanced alkaline phosphatase activity, and increased mRNA levels and protein synthesis of type X collagen in both the presence and absence of serum. In serum-free conditions, OP-1 promoted cell proliferation and chondrocyte maturation, without requiring either thyroid hormone or insulin, agents known to support chick chondrocyte differentiation in vitro. When grown in agarose under the same conditions, TGF-β1 and retinoic acid neither initiated nor promoted chondrocyte differentiation. The results demonstrate that OP-1, as the sole medium supplement, supports the maturation of embryonic chick sternal chondrocytes in vitro.
Three-dimensional co-location of RNA polymerase I and DNA during interphase and mitosis by confocal microscopy
Authors: Gilbert N. , Lucas L. , Klein C. , Menager M. , Bonnet N. , Ploton D.
Published: April 2021

The relative three-dimensional co-location of RNA polymerase I (RPI) and DNA was studied using confocal laser scanning microscopy during interphase and all the steps of mitosis in human cancerous cells.

For each step of the cell cycle, immunolabeled RPI molecules and DNA specifically stained with chromomycin A3 were simultaneously imaged at high resolution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic representations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuclei, in which DNA is organized as a regular 3-D network, RPI was present within numerous irregular spheres arranged as several twisted necklaces. During metaphase, RPI labeling was segregated into pairs of spheres and typical crescent-shaped structures; both were centrally located within the set of chromosomes. During anaphase and telophase, a typical central and symmetric arrangement of labeled structures was systematically seen among the decondensing chromosomes, arranged as a regular cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the non-random organization of the genome during interphase and mitosis.