bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differentiation of African trypanosomes is controlled by a density sensing mechanism which signals cell cycle arrest via the cAMP pathway
Authors: Vassella Erik , Reuner Birgit , Yutzy Barbara , Boshart Michael 
Published: April 2021

ABSTRACT
Differentiation of African trypanosomes from replicating slender bloodstream forms to nondividing stumpy forms limits the parasite population size, allowing survival of the mammalian host and establishment of a stable host-parasite relationship. Using a novel in vitro culture system we have shown that slender to stumpy differentiation is induced by parasite density alone and thus is independent of host cues. Here we investigate the density sensing mechanism and show that trypanosomes release a soluble activity of low relative molecular mass, termed stumpy induction factor (SIF), which accumulates in conditioned medium. SIF activity triggers cell cycle arrest in G1/G0 phase and induces differentiation with high efficiency and rapid kinetics. Membrane-permeable derivates of cAMP or the phosphodiesterase inhibitor etazolate perfectly mimic SIF activity. Furthermore, SIF activity elicits an immediate two-to threefold elevation of intracellular cAMP content upon addition to slender forms. We conclude that SIF and hence density sensing operate through the cAMP signalling pathway. Temporal correlation of markers indicates that cell cycle arrest invariably precedes differentiation. Thus, our results indicate that the cell cycle regulation of blood-stream forms is under dominant control of cAMP signalling. Irreversible commitment to the quiescent state is elicited by a cAMP agonist within a period shorter than one complete cell cycle.
Dual action of sonic hedgehog on chondrocyte hypertrophy: retrovirus mediated ectopic sonic hedgehog expression in limb bud micromass culture induces novel cartilage nodules that are positive for alkaline phosphatase and type X collagen
Authors: Stott N. Susan , Chuong Cheng-Ming 
Published: April 2021

ABSTRACT
Members of the vertebrate hedgehog gene family (HH) are involved in patterning and modulation of differentiation. Recently it has been shown that ectopic expression of HH gene family members in vivo blocks chondrocyte maturation through activation of a parathyroid hormone related peptide (PTHrP) dependent negative regulatory loop in the perichondrium. However, the direct effect of HH on chondrocyte maturation has not been tested. Here, we studied the effect of retroviral overexpression of the chicken sonic hedgehog gene (Shh) on the growth and maturation of limb bud cells in micromass cultures. Shh is neither expressed nor required for the initiation of cellular condensation in normal micromass cultures. With Shh overexpression, micromass cultures developed novel tightly whorled nodules in addition to the normal Alcian Blue positive cartilage nodules. We characterized the new nodules and showed that they are strongly positive for alkaline phosphatase, enriched in type X collagen and weakly positive for Alcian Blue staining. Shh overexpression also increased cell proliferation, but this cannot account for the formation of the new nodules. This current study shows that misexpression of Shh in in vitro chondrogenic cultures promotes characteristics of hypertrophic chondrocytes. Thus HH has two complementary functions; a direct positive effect on chondrocyte hypertrophy in the absence of PTHrP pathway, and an indirect negative feedback loop through PTHrP to prevent other less differentiated chondrocytes from becoming hypertrophic. These two complementary actions of HH coordinate the progression of cartilage maturation.
Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract
Authors: Lu Zhi Hong , Sittman Donald B. , Brown David T. , Munshi Rajan , Leno Gregory H. 
Published: April 2021

ABSTRACT
We investigated the effects of histone H1s on DNA replication using Xenopus egg extract. Mouse variants H1c and H10 were assembled onto Xenopus sperm chromatin by the extract during the remodeling that accompanies nuclear decondensation. The association of H1 with chromatin was rapid and concentration dependent. H1-associated chromatin displayed a typical nucleosomal repeat pattern indicating that linker histones are properly positioned along the DNA. The presence of H1 on sperm chromatin reduced both the rate and extent of DNA replication in egg extract. This reduction in rate is due, in part, to a delay in initiation of replication within individual nuclei. Initiation in extract is dependent upon nuclear assembly. Analysis of the assembly process revealed that H1 does not inhibit nuclear membrane formation or the import of nuclear protein, however, it does slow the rate of nuclear lamina formation. This H1-induced delay in lamina assembly is responsible for the delay in initiation as pre-assembled H1-containing nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete suggesting that H1 also affects replication directly. These data indicate that H1 modulates DNA replication through multiple pathways in egg extract.
αβ-crystallin interacts with intermediate filaments in response to stress
Authors: Djabali Karima , Néchaud Béatrice de , Landon Françoise , Portier Marie-Madeleine 
Published: April 2021

ABSTRACT
The small heat shock protein αβ-crystallin interacts with intermediate filament proteins. Using a co-sedimentation assay, we showed that in vitro binding of αβ-crystallin to peripherin and vimentin was temperature-dependent. Specifically, a synthetic peptide representing the first ten residues of αβ-crystallin was involved in this interaction. When cells were submitted to different stress conditions such as serum starvation, hypertonic stress, or heat shock, we observed a dynamic reorganisation of the intermediate filament network, and concomitant recruitment of αB-crystallins on intermediate filament proteins. Under normal conditions αβ-crystallin was extracted from cells by detergent. In stressed cells, αβ-crystallin colocalised with intermediate filament proteins, and became resistant to detergent extraction. The intracellular state of αB-crystallin seemed to correlate directly with the remodelling of the intermediate filament network in response to stress. This suggested that αβ-crystallin functions as a molecular chaperone for intermediate filament proteins.
Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture
Authors: Sandig Martin , Negrou Ella , Rogers Kem A. 
Published: April 2021

ABSTRACT
To determine changes in the spatial and temporal distribution of cell-cell adhesion molecules during transendo-thelial migration of monocytes, we examined an in vitro model system of diapedesis using high resolution laser scanning confocal microscopy. Human arterial endothelial cells were cultured to confluence on coverslips coated with Matrigel and activated with IL-1β before the addition of monocytic THP-1 cells. Seventy per cent of monocytes transmigrated through the endothelium within one hour. Diapedesis, but not adhesion and spreading, was inhibited 8-fold in cocultures that contained endothelial cell conditioned medium, suggesting the release of an endothelial derived inhibitor. Double immunofluorescence labeling with antibodies to LFA-1, α- and β-catenin, VE-cadherin and with Texas Red phalloidin, identified a circular transmigration passage in endothelial cell-cell contact regions. This passage was formed by an LFA-1-containing pseudopodium that penetrated between endothelial cells. Apical to the transmigration passage, monocytes remained round in shape, while underneath the endothelium, they spread along the Matrigel. The margins of the transmigration passage contained high levels of LFA-1 and F-actin, suggesting a major role of these molecules during the transmigration process itself. Endothelial adherens junctions, as judged by the presence of VE-cadherin and α-catenin adjacent to the passage, remained intact during diapedesis. The presence of catenins at heterotypic contact regions between monocytes and endothelial cells during diapedesis suggested cadherin-mediated interactions between the two cell types. These results reveal dynamic changes in the distribution of adhesion molecules and the actin cytoskeleton during monocyte transendothelial migration in culture.
Genetic analysis of β1 integrin function: confirmed, new and revised roles for a crucial family of cell adhesion molecules
Authors: Brakebusch Cord , Hirsch Emilio , Potocnik Alexandre , Fässler Reinhard 
Published: April 2021

ABSTRACT
Integrins are heterodimeric cell adhesion proteins connecting the extracellular matrix to the cytoskeleton and transmitting signals in both directions. These integrins are suggested to be involved in many different biological processes such as growth, differentiation, migration, and cell death. Of more than 20 known integrins, 10 contain the nearly ubiquitously expressed β1 integrin subunit. Disruption of the β1 integrin gene by homologous recombination allows us to assess the supposed functions of β1 containing integrins in vivo in a new way. This review will present and discuss recent findings derived from such studies concerning the biological roles of β1 integrins in early development, differentiation and migration, hematopoiesis, tumorigenesis, and supramolecular assembly of extracellular matrix proteins. While several former results were confirmed, others were contradicted and new functions found, significantly changing the previous view of β1 integrin function in vivo.
In situ inhibition of vesicle transport and protein processing in the dominant negative Rab1 mutant of Drosophila
Authors: Satoh Akiko K. , Tokunaga Fumio , Kawamura Satoru , Ozaki oichi 
Published: April 2021

ABSTRACT
Rab proteins play an essential role in vesicle transport. In particular, RAB1 is thought to participate in the transport of most membrane and secretory proteins. To investigate the role of RAB1 in developing or functioning cells in situ, we constructed transgenic, dominant-negative Rab1 mutants of Drosophila, and examined the protein transport and cellular and subcellular structures of mutant photoreceptor cells. In the transgenic fly, the expression of mutant RAB1 was induced by Gal4 protein, whose expression was triggered by heat treatment (37°C) of the fly. Within several hours after the heat induction, the lumens of the rough endoplasmic reticulum (rER) became swollen, and Golgi bodies were disassembled into vesicle clusters. Corresponding to these changes in cell structure, rhodopsin transport was blocked between the rER and the Golgi body, as indicated by the accumulation of immature rhodopsin carrying a large high-mannose-type oligosaccharide chain. Long-term expression of mutant RAB1 caused the degradation of photoreceptive microvilli and the accumulation of numerous swollen rERs, whereas no distinct changes were found in the axonal regions. These results indicate that, in Drosophila photoreceptor cells, RAB1 contributes to the maintenance of local cell structure by mediating vesicle transport between the rER and Golgi body.
Localization of cardiac α-myosin heavy chain mRNA is regulated by its 3′ untranslated region via mechanical activity and translational block
Authors: Goldspink Paul H. , Sharp W. W. , Russell Brenda 
Published: April 2021

ABSTRACT
We have altered the spontaneous contractile activity of neonatal cardiac myocytes in culture to investigate the relationship between mechanical forces, myofibril assembly, and the localization and translation of α-myosin heavy chain mRNA. Immunofluorescence and in situ hybridization techniques revealed that contracting myocytes display well aligned myofibrils and a diffuse distribution of α-myosin heavy chain mRNA. Inhibition of contractile activity with the calcium channel blocker verapamil (10 μM) resulted in myofibril disassembly and a perinuclear mRNA distribution within six hours. There was a significant decrease (P&lt;0.05) of mRNA levels, 5 to 15 μm away from the nucleus following 6 hours of verapamil treatment compared with control cells. Inhibition of protein synthesis with cycloheximide (10 μM) also resulted in perinuclear mRNA localization despite having little effect on contractile activity or myofibril assembly. To determine if the 3′ untranslated region of α-myosin heavy chain mRNA was sufficient for localizing the entire message, a chimeric construct composed of β-galactosidase coding region followed by α-myosin heavy chain 3′ untranslated region sequences was made as a reporter plasmid and transfected into cultured myocytes. A perinuclear accumulation of β-galactosidase was exhibited in many of the contractile arrested cells (48.3±2.4%, n=7). In contrast, significantly fewer (P&lt;0.05) contracting control (29.1±3.3%, n=7) and strongly contracting, isoproterenol-treated cells (27.2±6.1%, n=3) exhibited a perinuclear localization of protein. The distribution of the reporter protein was not affected by the contractile state in cells transfected with a constitutively translated 3′UTR. We propose that mechanical activity of neonatal cardiac myocytes regulates the intracellular localization of α-myosin heavy chain mRNA via the 3′ untranslated region mediated by an initial block in translation.
The AMF-R tubule is a smooth ilimaquinone-sensitive subdomain of the endoplasmic reticulum
Authors: Wang Hui-Jun , Benlimame Naciba , Nabi Ivan R. 
Published: April 2021

ABSTRACT
Autocrine motility factor receptor (AMF-R) is a marker for a distinct smooth membranous tubule. Ilimaquinone (IQ) is a sea sponge metabolite which induces the complete vesiculation of the Golgi apparatus and we show here that the addition of IQ to MDCK cells also results in the disruption of the AMF-R tubule. By immunofluorescence microscopy, the resultant punctate AMF-R label resembles the products of IQ-mediated vesiculation of the trans-Golgi network, however, the two labels can be distinguished by confocal microscopy. AMF-R tubule fragmentation occurs after nocodazole or taxol treatment of the cells demonstrating that the action of IQ on AMF-R tubules is not related to the ability of IQ to depolymerize microtubules. IQ activity is therefore not Golgi-specific. Electron microscopy of IQ-treated cells reveals that AMF-R is distributed to fenestrated networks of narrow interconnected tubules which are distinguishable from the uniform Golgi-derived vesicles and morphologically equivalent to smooth ER. Distinct fenestrations are visible in incompletely fragmented tubules which may represent intermediates in the fragmentation process. Smooth AMF-R labeled tubules exhibit continuity with rough ER cisternae and IQ selectively targets smooth and not rough ER. AMF-R tubules can be distinguished from the intermediate compartment labeled for ERGIC-53 by confocal microscopy and thus constitute a distinct IQ-sensitive subdomain of the smooth ER.
A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte
Authors: Gross Stefan D. , Simerly Calvin , Schatten Gerald , Anderson Richard A. 
Published: April 2021

ABSTRACT
Casein kinase I is a family of serine/threonine protein kinases common to all eukaryotes. In yeast, casein kinase I homologues have been linked to the regulation of growth, DNA repair and cell division. In addition, their subcellular localization to membraneous structures and the nucleus is essential for function. In higher eukaryotes, there exist seven genetically distinct isoforms: α, β, γ1, γ2, γ3, δ and ε. Casein kinase Iα exhibits a cell cycle-dependent subcellular localization including an association with cytosolic vesicular structures and the nucleus during interphase, and the spindle during mitosis. casein kinase I has also been shown to modulate critical regulators of growth and DNA synthesis/repair in mammalian cells such as SV40 large T antigen and p53. These results suggest that casein kinase I may be involved in processes similar to those ascribed to the yeast casein kinase I homologues. To define a role for casein kinase Iα in cell cycle regulation, the mouse oocyte was utilized because of its well-defined cell cycle and ease of micromanipulation. Immunofluorescence studies from meiosis I of maturation to the first zygotic cleavage demon-strated that the kinase was associated with structures similar to those previously reported. Microinjection of casein kinase Iα antibodies at metaphase II-arrest and G2 phase, had no effect on the completion of second meiosis or first division. However, microinjection of these antibodies during the early pronucleate phase prior to S-phase onset blocked uptake of the kinase into pronuclei and interfered with proper and timely cell cycle progression to first cleavage. These results suggest that the kinase regulates the progression from interphase to mitosis during the first cell cycle.
Rapid, microtubule-dependent fluctuations of the cell margin
Authors: Dunn Graham A. , Zicha Daniel , Fraylich Paul E. 
Published: April 2021

ABSTRACTUsing data automatically acquired by microinterferometry from large numbers of chick fibroblasts, we have detected rapid microfluctuations in the rates of protrusion and retraction of the cell margin which were strongly sup-pressed by colcemid, nocodazole and taxol. Fluctuations in the rate of retraction of the margin were about twice as powerful as fluctuations in the rate of protrusion. High-frequency fluctuations were also apparent in the cell track and in measures of cell spreading, shape and speed. These rapid fluctuations were also all suppressed by colcemid and nocodazole, sometimes by doses insufficient to disrupt the majority of microtubules. Taxol on the other hand did not suppress fluctuations in direction of the cell track nor fluctuations in the spreading of the cells but it was very effective at suppressing variations in protrusion and retraction and in cell speed and shape.We discovered that much slower, larger-scale variations in protrusion, retraction, spreading, shape and speed resulted from the accumulation of these rapid, microtubule-dependent fluctuations of the cell margin. These large-scale variations in cell behaviour were also suppressed by the same drug treatments that were effective in suppressing the corresponding high-frequency fluctuations. We speculate that a function of microtubules is to enhance the fibroblast’s responses to its environment by causing microfluctuations of the cell’s margin which give rise to large-scale variations in cell behaviour.
The combination of epidermal growth factor and transforming growth factor-β induces novel phenotypic changes in mouse liver stem cell lines
Authors: Isfort Robert J. , Cody David B. , Stuard Sharon B. , Randall Catherine J. , Miller Catherine , Ridder Gregg M. , Doersen Claus-Jens , Richards William G. , Yoder Bradley K. , Wilkinson J. Erby , Woychik Richard P. 
Published: April 2021

ABSTRACT
Mouse liver stem cell (oval cell) lines were investigated in order to determine the role which two families of growth and differentiation factors (GDFs), epidermal growth factor (EGF) family and transforming growth factor beta (TGF-β) family, play in liver regeneration. EGF family members, including EGF, amphiregulin, betacellulin, heparin-binding epidermal growth factor, and TGF-alpha, were mitogenic for oval cell lines while TGF-β family members, including TGF-β1, TGF-β2 and TGF-β3, inhibited mitogenesis and induced apoptosis in oval cell lines. Surprisingly, the combination of EGF family members and TGF-β family members resulted in neither proliferation nor apoptosis but instead in a novel cellular response, cellular scattering in tissue culture and morphological differentiation in Matrigel. Analysis of the signal transduction pathways activated by exposure of oval cell lines to either EGF, EGF+TGF-β, or TGF-β indicated that novel combinations of intracellular signals result following stimulation of the cells with the combination of EGF+TGF-β. These data reveal that the dynamics of synergistic GDF action following tissue injury and regeneration results in a new level of complexity not obvious from the study of indi-vidual GDFs.
Centrosome-microtubule nucleation
Authors: Pereira Gislene , Schiebel Elmar 
Published: April 2021

ABSTRACT
In many cell types the formation of microtubules from tubulin subunits is initiated at defined nucleation sites at the centrosome. Thesse sites contain the conserved γ-tubulin which is in association with additional not very well characterised proteins, identified as components of a γ-tubulin ring complex from Xenopus egg extracts or from suppressor screens in the yeast Saccharomyces cerevisiae. In this review we discuss two recently proposed models of how the γ-tubulin complex assists in the assembly of tubulin to form microtubules. These models propose different roles for γ-tubulin and the other proteins in the complex in tubulin assembly. While the structure and composition of a microtubule nucleation site is becoming clearer, it is still unknown how the cell-cycle dependent regulation of microtubule nucleation sites is achieved and whether they disassemble after microtubule formation in order to allow microtubule fluxes towards the centrosome which have been observed in mitotic cells.
E-cadherin mediates adhesion and suppresses cell motility via distinct mechanisms
Authors: Chen Haiyan , Paradies Nancy E. , Fedor-Chaiken Mary , Brackenbury Robert 
Published: April 2021

ABSTRACT
Expression of the calcium-dependent adhesion molecule E-cadherin suppresses the invasion of cells in vitro, but the mechanism of this effect is unknown. To investigate this mechanism, we analyzed the effects of expressing E-cadherin in mouse L-cells and rat astrocyte-like WC5 cells. Increased cellular adhesion mediated by E-cadherin reduced invasion in WC5 cells and in some L-cells, but not in others. In all cases, suppression of invasion was correlated with decreased cell movement as assessed in an in vitro wound-filling assay and a transwell motility assay. To define the relationship between adhesion mediated by E-cadherin and suppression of motility, we analyzed the effects of deleting different regions of the E-cadherin cytoplasmic domain. E-cadherin lacking the entire cytoplasmic domain did not mediate calcium-dependent adhesion and did not reduce cell motility when expressed in WC5 cells. E-cadherin lacking a portion of the catenin-binding domain did not associate with the cytoskeleton and did not promote adhesion, yet still suppressed the motility of WC5 cells. In addition, E-cadherin that retains an intact catenin-binding domain, but lacks a juxtamembrane portion of the cytoplasmic domain, mediated effective adhesion, but did not suppress motility. These results indicate E-cadherin mediates adhesion and suppresses cell motility via distinct mechanisms, and suggest that the juxtamembrane domain of E-cadherin plays a key role in suppressing motility.