bluuebunny/crossref_metadata_2025_split huggingface dataset:
Df31 is a novel nuclear protein involved in chromatin structure in Drosophila melanogaster
Authors: Crevel Gilles , Huikeshoven Hella , Cotterill* Sue 
Published: April 2021

ABSTRACT
We originally isolated the Df31 protein from Drosophila embryo extracts as a factor which could decondense Xenopus sperm, by removing the sperm specific proteins and interacting with histones to facilitate their loading onto DNA. We now believe that this protein has a more general function in cellular DNA metabolism.
The Df31 gene encodes a very hydrophilic protein with a predicted molecular mass of 18.5 kDa. Immunostaining showed that Df31 was present in a wide range of cell types throughout differentiation and in both dividing and non- dividing cells. In all cases the protein is present in large amounts, comparable with the level of nucleosomes. Injection of antisense oligonucleotides to lower the level of Df31 in embryos caused severe disruption of the nuclear structure. Large irregular clumps of DNA were formed, and in most cases the amount of DNA associated with each clump was more than that found in a normal nucleus. Immunofluorescence, cell fractionation, and formaldehyde cross-linking show that Df31 is associated with chromatin and that a significant fraction of it binds very tightly. It also shows the same binding characteristics when loaded onto chromatin in vitro. Chromatin fractionation shows that Df31 is tightly associated with nucleosomes, preferentially with oligonucleosomes. Despite this no differences were observed in the properties of nucleosomes loaded in the in vitro system in the presence and absence of Df31.
These results suggest that Df31 has a role in chromosomal structure, most likely acting as a structural protein at levels of folding higher than that of nucleosomes.
Two type V myosins with non-overlapping functions in the fission yeast Schizosaccharomyces pombe: Myo52 is concerned with growth polarity and cytokinesis, Myo51 is a component of the cytokinetic actin ring
Authors: Win Thein Z. , Gachet Yannick , Mulvihill Daniel P. , May Karen M. , Hyams Jeremy S. 
Published: April 2021

ABSTRACT
The fission yeast genome project has identified five myosin genes: one type I myosin, myo1+, two type II myosins, myo2+ and myp2+, and two type V myosins, myo51+ and myo52+. Cells deleted for myo51+ show normal morphology and growth rates whereas deletion of myo52+ results in a partial loss of cell polarity, slow growth and cytokinetic defects. Combining both deletions in a single strain is phenotypically non-additive, myo52Δ being epistatic to myo51Δ. Overproduction of Myo51 gives rise to elongated cells which fail to form functional septa whereas overproduction of Myo52 results in branched cells with aberrant septa that fail to cleave. Myo52 localises to the poles of growing cells but during cell division it relocalises to the cell equator as a bar that is bisected by the cytokinetic septum. Myo51 shows no obvious localisation during interphase but at cytokinesis it is associated with the contractile cytokinetic actin ring (CAR). Both myosins are dependent upon an intact actin cytoskeleton for localisation. Myo52 partially colocalises with the α-glucan synthase Mok1 at the cell tips and to a lesser extent at the septum. Mok1 is delocalised and upregulated in myo52Δ and myo52Δ cell walls are resistant to digestion by the cell wall degrading enzyme zymolyase. Thus myo52+ appears to be involved in the local delivery or positioning of vesicles containing cell wall precursors at the cell tips and has a role in the maturation or cleavage of the septum. Myo51 has a non-essential role in cytokinesis as a component of the cytokinetic actin ring.
A nuclear tale of two yeasts: The Yeast Nucleus
Authors: Fantes P. , Beggs J. 
Published: April 2021

ABSTRACT
Without question, numerous studies in yeast and mammals have revealed a striking commonality of underlying mechanisms that govern basic biological operations. Perhaps the most famous example from recent years has been the recognition that genes required for maintaining the yeast genome play a critical role in preventing cancer in humans. However, examining the molecular differences -the variations on a common theme, so to speak -can also be useful for understanding core biological processes. These ideas are the foundation for The Yeast Nucleus, a valuable contribution to Oxford University Press’s ‘Frontiers in Molecular Biology’ series. The textbook compares and contrasts various nuclear processes in budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), pointing out the similarities - and differences -that make these two somewhat unrelated yeasts the dominant model systems for studying fundamental eukaryotic processes.
Leukotriene D4 affects localisation of vinculin in intestinal epithelial cells via distinct tyrosine kinase and protein kinase C controlled events
Authors: Massoumi Ramin , Sjölander Anita 
Published: April 2021

ABSTRACT
Local inflammatory reactions affect the integrity of intestinal epithelial cells, such as E-cadherin-mediated cell-cell interactions. To elucidate this event, we investigated the effects of an inflammatory mediator, leukotriene D4 (LTD4), on the phosphorylation status and properties of vinculin, a multi-binding protein known to interact with both the E-cadherin-catenin complex and the cytoskeleton. Treatment of an intestinal epithelial cell line with LTD4 induced rapid tyrosine phosphorylation of vinculin, which was blocked by the Src family tyrosine kinase inhibitor PP1. Simultaneously, LTD4 caused an increased association between vinculin and actin, and that association was decreased by PP1. LTD4 also induced dissociation of vinculin from α-catenin without affecting the catenin complex itself. This dissociation was not blocked by PP1 but was mimicked by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Also, the PKC inhibitor GF109203X abolished both the LTD4- and the TPA-induced dissociation of vinculin from α-catenin. Furthermore, LTD4 caused a colocalisation of vinculin with PKC-α in focal adhesions. This accumulation of vinculin was blocked by transfection with a dominant negative inhibitor of PKC (PKC regulatory domain) and also by preincubation with either GF109203X or PP1. Thus, various LTD4-induced phosphorylations of vinculin affect the release of this protein from catenin complexes and its association with actin, two events that are necessary for accumulation of vinculin in focal adhesions. Functionally this LTD4-induced redistribution of vinculin was accompanied by a PKC-dependent upregulation of active β1 integrins on the cell surface and an enhanced β1 integrin-dependent adhesion of the cells to collagen IV.
Collagen IV synthesis is restricted to the enteroendocrine pathway during multilineage differentiation of human colorectal epithelial stem cells
Authors: Kirkland Susan C. , Henderson Karen 
Published: April 2021

The human large intestine is lined by a rapidly renewing epithelial monolayer where cell loss is precisely balanced with cell production. The continuous supply of new cells is produced by undifferentiated multipotent stem cells via a coordinated program of proliferation and differentiation yielding three epithelial lineages: absorptive, goblet and enteroendocrine. Cell-matrix interactions have been suggested to be regulators of the multilineage differentiation program of the colorectal crypt but the expression of matrix proteins or their receptors does not appear to have the subtlety expected for this task.We have developed an in vitro model system of intestinal epithelial stem cells to facilitate the direct analysis of stem cells undergoing lineage commitment and differentiation. Using this culture system, we can now directly investigate the role of cell-matrix signalling in stem-cell decisions. In this study, collagen-IV synthesis has been followed in monolayers of multipotent cells that have been induced to differentiate into absorptive, goblet and enteroendocrine cells. Our experiments demonstrate that commitment to the enteroendocrine lineage is specifically accompanied by the expression of type-IV collagen that remains enteroendocrine-cell associated. Undifferentiated cells, absorptive cells and goblet cells do not express collagen IV. To confirm that the differential lineage-specific expression of collagen IV observed in the model system was representative of the in vivo situation, collagen-IV synthesis was analysed in isolated human colorectal crypts and tissue sections using immunocytochemistry and in situ hybridisation. These studies confirmed the in vitro findings, in that implementation of the enteroendocrine differentiation program involves synthesis and accumulation of a collagen-IV matrix. Thus, human colorectal enteroendocrine cells are unique in the colorectal crypt in that they assemble a cell-associated collagen-IV-rich matrix not observed on other colorectal epithelial cells.This study provides the first evidence for differential matrix synthesis between colorectal epithelial lineages in human colorectal epithelium. The specialised pericellular environment of the enteroendocrine cells might explain some of the unique phenotypic characteristics of this cell lineage. Furthermore, these findings suggest a potential mechanism whereby individual epithelial cells could modulate their cell-matrix signalling even while rapidly migrating in heterogeneous sheets over a shared basement membrane.
Myosin II-dependent cylindrical protrusions induced by quinine inDictyostelium: antagonizing effects of actin polymerization at the leading edge
Authors: Yoshida Kunito , Inouye Kei 
Published: April 2021

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc− cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general.Movies available on-line
Functional interaction between the SSeCKS scaffolding protein and the cytoplasmic domain of β1,4-galactosyltransferase
Authors: Wassler Michael J. , Foote Cynthia I. , Gelman Irwin H. , Shur Barry D. 
Published: April 2021

The β1,4-galactosyltransferase family contains at least seven unique gene products, of which β1,4-galactosyltransferase I (GalT) is the most exhaustively studied. GalT exists in the Golgi complex, similar to many other glycosyltransferases, as well as on the cell surface, where it functions as a signaling receptor for extracellular glycoside ligands. When expressed on the surface, GalT associates with the cytoskeleton and, upon ligand-induced aggregation, induces cell-type specific intracellular signal cascades. In an effort to define the mechanisms by which surface GalT exerts these intracellular effects, we used the yeast two-hybrid system to identify proteins that specifically interact with the GalT cytoplasmic domain.The yeast two-hybrid screen identified two distinct clones (1.12 and 2.52) that showed identity to portions of SSeCKS (Src Suppressed CKinase Substrate). SSeCKS is a previously defined kinase and cytoskeleton scaffolding protein whose subcellular distribution and functions are remarkably similar to those attributed to GalT. Both SSeCKS and GalT have been localized to the perinuclear/Golgi region as well as to filopodia/lamellipodia. SSeCKS and GalT have been implicated in regulating cell growth, actin filament dynamics, and cell spreading. Interestingly, 1.12 and 2.52-GFP constructs were localized to subcellular domains that correlated with the two purported subcellular distributions for GalT; 2.52 being confined to the Golgi, whereas 1.12 localized primarily to filopodia. Coimmunoprecipitation assays demonstrate stable binding between the GalT cytoplasmic domain and the 1.12 and 2.52 domains of SSeCKS in appropriately transfected cells. Similar assays demonstrate binding between the endogenous GalT and SSeCKS proteins also. Coimmunoprecipitation assays were performed in both directions and produced similar results (i.e. using either anti-GalT domain or anti-SSeCKS domain antibodies as the precipitating reagent). A functional interaction between the GalT cytoplasmic domain and SSeCKS was illustrated by the ability of either the 1.12 or 2.52 SSeCKS domain to restore a normal adhesive phenotype in cells overexpressing the TL-GFP dominant negative construct. TL-GFP is composed of the GalT cytoplasmic and transmembrane domains fused to GFP, and leads to a loss of cell adhesion on laminin by displacing the endogenous GalT from its cytoskeleton binding sites. This is the first reported interaction between a glycosyltransferase and a scaffolding protein, and suggests that SSeCKS serve to integrate the various functions ascribed to the GalT cytoplasmic domain.
Search, capture and signal: games microtubules and centrosomes play
Authors: Schuyler Scott C. , Pellman David 
Published: April 2021

ABSTRACT
Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression. Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation. Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules. Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin. In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved. Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.
The interplay between cyclin-B–Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes
Authors: Abrieu Ariane , Dorée Marcel , Fisher Daniel 
Published: April 2021

ABSTRACT
Throughout oocyte maturation, and subsequently during the first mitotic cell cycle, the MAP kinase cascade and cyclin-B–Cdc2 kinase are associated with the control of cell cycle progression. Many roles have been directly or indirectly attributed to MAP kinase and its influence on cyclin-B–Cdc2 kinase in different model systems; yet a principle theme does not emerge from the published literature, some of which is apparently contradictory. Interplay between these two kinases affects the major events of meiotic maturation throughout the animal kingdom, including the suppression of DNA replication, the segregation of meiotic chromosomes, and the prevention of parthenogenetic activation. Central to many of these events appears to be the control by MAP kinase of cyclin translation and degradation.
Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues
Authors: Kabore Albert F. , Wang Wen-Jing , Russo Scott J. , Beers Michael F. 
Published: April 2021

ABSTRACT
Surfactant protein C (SP-C) is a lung-specific secreted protein, which is synthesized as a 21-kDa propeptide (SP-C21) and then proteolytically processed as a bitopic transmembrane protein in subcellular compartments distal to the medial Golgi to produce a 3.7 kDa mature form. We have shown that initial processing of SP-C21 involves two endoproteolytic cleavages of the C terminus and that truncation of nine amino acids from the C-flanking peptide resulted in retention of mutant protein in proximal compartments. Because these truncations involved removal of a conserved cysteine residue (Cys186), we hypothesized that intralumenal disulfide-mediated folding of the C terminus of SP-C21 is required for intracellular trafficking. To test this, cDNA constructs encoding heterologous fusion proteins consisting of enhanced green fluorescent protein (EGFP) attached to the N terminus of wild-type rat proSP-C (EGFP/SP-C1-194), C-terminally deleted proSP-C (EGFP/SP-C1-185; EGFP/SP-C1-191) or point mutations of conserved cysteine residues (EGFP/SP-CC122G; EGFP/SP-CC186G; or EGFP/SP-CC122/186G) were transfected into A549 cells. Fluorescence microscopy revealed that transfected EGFP/SP-C1-194 and EGFP/SP-C1-191 were expressed in a punctate pattern within CD-63 positive, EEA-1 negative cytoplasmic vesicles. In contrast, EGFP/SP-C1-185, EGFP/SP-CC122G, EGFP/SP-CC186G and EGFP/SP-CC122/186G were expressed but retained in a juxtanuclear compartment that stained for ubiquitin and that contained γ-tubulin and vimentin, consistent with expression in aggresomes. Treatment of cells transfected with mutant proSP-C with the proteasome inhibitor lactacysteine enhanced aggresome formation, which could be blocked by coincubation with nocodazole. Western blots using a GFP antibody detected a single form in lysates of cells transfected with EGFP/SP-C cysteine mutants, without evidence of smaller degradation fragments. We conclude that residues Cys122 and Cys186 of proSP-C are required for proper post-translational trafficking. Mutation or deletion of one or both of these residues results in misfolding with mistargeting of unprocessed mutant protein, leading to formation of stable aggregates within aggresomes.
A calcineurin- and NFAT-dependent pathway regulates Myf5 gene expression in skeletal muscle reserve cells
Authors: Friday Bret B. , Pavlath Grace K. 
Published: April 2021

ABSTRACT
Myf5 is a member of the muscle regulatory factor family of transcription factors and plays an important role in the determination, development, and differentiation of skeletal muscle. However, factors that regulate the expression and activity of Myf5 itself are not well understood. Recently, a role for the calcium-dependent phosphatase calcineurin was suggested in three distinct pathways in skeletal muscle: differentiation, hypertrophy, and fiber-type determination. We propose that one downstream target of calcineurin and the calcineurin substrate NFAT in skeletal muscle is regulation of Myf5 gene expression. For these studies, we used myotube cultures that contain both multinucleated myotubes and quiescent, mononucleated cells termed ‘reserve’ cells, which share many characteristics with satellite cells. Treatment of such myotube cultures with the calcium ionophore ionomycin results in an ≈4-fold increase in Myf5 mRNA levels, but similar effects are not observed in proliferating myoblast cultures indicating that Myf5 is regulated by different pathways in different cell populations. The increase in Myf5 mRNA levels in myotube cultures requires the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. We used immunohistochemical analyses and fractionation of the cell populations to demonstrate that the calcium regulated expression of Myf5 occurs in the mononucleated reserve cells. We conclude that Myf5 gene expression is regulated by a calcineurin- and NFAT-dependent pathway in the reserve cell population of myotube cultures. These results may provide important insights into the molecular mechanisms responsible for satellite cell activation and/or the renewal of the satellite cell pool following activation and proliferation.
Chondroitin sulfate is involved in lysosomal transport of lysozyme in U937 cells
Authors: Lemansky Peter , Hasilik Andrej 
Published: April 2021

ABSTRACT
Human promonocytes U937 synthesize lysozyme and retain approximately one third of it within lysosomes. Lysozyme is not glycosylated; thus, it cannot be subject to mannose-6-phosphate-dependent targeting to lysosomes. It is a basic protein with a pI of 10.5 and is known to interact with negatively charged macromolecules like proteoglycans. Therefore, we examined whether the latter are involved in the lysosomal targeting of lysozyme in U937 cells. We partially diminished the electronegative charge of newly synthesized proteoglycans by inhibiting their sulfation with chlorate. This increased the rate of secretion of lysozyme. Upon treatment of U937 cells with phorbol esters, the rate of secretion of lysozyme was increased to more than 90%. This coincided with an almost complete redistribution of a [35S]sulfate bearing proteoglycan to the secretory pathway. After a brief pulse with [35S]sulfate in the control, 80% of the [35S]sulfate-bearing proteoglycan was retained within the cells, whereas in the treated cells this proportion was decreased to 13%. The secreted proteoglycan was sensitive to chondroitinase ABC and bound to immobilized lysozyme. This interaction was disrupted by 50-300 mM NaCl. The intracellularly retained proteoglycan was degraded with a half-life of 50-60 minutes and seemed to be directed to lysosomes because in the presence of NH4Cl the degradation was strongly inhibited. Our results suggest that the proteoglycan is involved in lysosomal targeting of lysozyme in U937 cells.