bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of cyclic gmp in regulating myosin during chemotaxis of Dictyostelium: evidence from a mutant lacking the normal cyclic gmp response to cyclic amp
Authors: Liu Gang , Kuwayama Hidekazu , Ishida Shuji , Newell Peter C. 
Published: April 2021

ABSTRACT
Evidence has previously been reported that, during chemotaxis of the cellular slime mould Dictyostelium dis - coideum, cyclic GMP regulates the association of myosin II with the cytoskeleton and that this regulation is effected by inhibiting myosin II heavy chain phospho-rylation (Liu and Newell, J.Cell Sci., 90, 123-129, 1988; 98, 483-490, 1991). Here we provide further evidence in support of this hypothesis using a mutant (KI-10) that is defective in chemotaxis and lacks the normal cyclic AMP-induced cyclic GMP response. We found that the cyclic AMP-induced cytoskeletal actin response was similar to that of the parental strain in this mutant (although showing a slight displacement in the dose-response curve) but the cytoskeletal myosin II heavy chain response was abolished. Moreover, the mutant showed no phosphorylation of myosin II heavy chain in response to cyclic AMP.
Compared to the parental strain XP55, the mutant cells contained approximately 40% more protein and their doubling time was 30% longer. These differences could be due to differences in the efficiency of cell divi-sion, a process in which the proper regulation of myosin function is essential and in which cyclic GMP may there-fore play a role.
Dual function of tenascin: simultaneous promotion of neurite growth and inhibition of glial migration
Authors: Wehrle-Haller Bernhard , Chiquet Matthias 
Published: April 2021

ABSTRACT
The extracellular matrix molecule tenascin is expressed within the developing peripheral nervous system, first by migrating neural crest cells and later by satellite (Schwann precursor) cells at the growing tips of periph-eral nerves. Here we found that the neurite promoting activity of tenascin for sensory neurons is developmentally regulated: very young sensory ganglia of stage 23 (4 days old) embryos grew neurites on tenascin as fast as on laminin and fibronectin. The growth response of older (day 7 and 9) ganglia on laminin and fibronectin was similar to that of 4-day-old ganglia, while on tenascin neurite growth occured only after a lag phase and at a slower rate. Neurite growth on tenascin was inhibited by antibodies to 1 integrin and by heparin. While tenascin promotes neurite outgrowth of periph-eral neurons, we found that it does not allow satellite cell migration when it is present on the substratum, and it inhibits migration of satellite cells on fibronectin when added in soluble form. In contrast, soluble tenascin did not significantly alter the rate of neurite growth on tenascin, fibronectin or laminin substrata, although neu-rites were straighter and less attached. When isolated satellite cells were added to neurites grown on tenascin, they preferentially adhered to and elongated along neu-rite surfaces. Using patterned substrata of tenascin versus fibronectin or laminin confirmed that tenascin borders allow neurites to pass but act as barriers to migrating satellite cells. We postulate that tenascin or related molecules with dual functions in cell adhesion are important for peripheral nerve morphogenesis. Tenascin allows axonal growth, but may restrict random satellite cell migration into the fibronectin-rich mes-enchyme, thereby inducing the compaction of nerve fascicles.
Tumor spheroid-induced vesicle formation on endothelial cells is associated with procoagulant properties
Authors: Lichtenbeld Hera H. C. , Muller Annemarie D. , Dam-Mieras Maria C. E. van , Blijham Geert H. 
Published: April 2021

ABSTRACT
Fibrin deposits in tumor beds are an intriguing phenomenon. It has been suggested that fibrin plays a role as a provisional matrix in which the tumor grows and induces development of a vascular network. On the other hand fibrin possibly protects the tumor nodule from host defense mechanisms. Wetherefore investigate whether tumor cells can induce a procoagulant response in endothelial cells leading to fibrin formation. For our studies we employed a modification of the matrix model of Montesano in which sprouting of endothelial cell aggregates can be followed. This system allows us to study in vitro the involvement of coagulation in tumor growth and angiogenesis.
Cocultures of endothelial cell aggregates and avascular tumor spheroids in collagen type I gels results in the appearance of extracellular vesicle-like structures on the endothelial sprouts. The vesicles formed on endothelial cell sprouts upon coculturing with tumor cells exhibit an increased amidolytic activity, suggestive of factor X/Xa activity, not dependent on tissue factor exposure. Experiments using HgCl2 and Iodoacetamide point to the importance of SH groups in the factor X/Xa activity on endothelial cell sprouts.
Osmoregulation in Paramecium: the locus of fluid segregation in the contractile vacuole complex
Authors: Ishida Masaki , Aihara Marilynn S. , Allen Richard D. , Fok Agnes K. 
Published: April 2021

ABSTRACT
In a previous study, monoclonal antibody DS-1 was found to specifically label the decorated spongiome along the radial arms of the contractile vacuole complexes in Paramecium multimicronucleatum. Fluorescein isothiocyanate-conjugated DS-1, when injected into cells, labels the radial arms initially, but with increasing postinjection time both the intensity of fluorescence and the number of fluorescently labeled radial arms were reduced. When these cells were fixed after 45 minutes and probed indirectly using a second fluorochrome, little new label was seen on the already fluorescein-labeled radial arms. Thin sections showed that the amount of decorated tubules along some collecting canals decreased from the proximal to the distal end and vesicles, which were never seen in control cells, appeared next to the decorated spongiome. These results suggested that the decorated spongiome was undergoing disassembly and sequestration into one region of the cell. The injected DS-1 also reduced the expulsion frequency of the contractile vacuoles in a dose-, time- and site-dependent manner. The contractile vacuole complexes near the injection site were affected more than those farther from the site, but the sizes of both contractile vacuoles were only transiently affected so that fluid output per cell was reduced by ∼60%. Beyond 45 minutes postinjection, both the expulsion frequency and total fluid output began to recover as the DS-1 was sequestered into one part of the cell. This region persisted in cells up to 18 hours but disappeared by 24 hours, which coincided with the full recovery of the expulsion frequency and of decorated spongiome along the radial arms. The contractile vacuole, the collecting canals and the smooth spongiome were morphologically unaffected. These results indicate that when the decorated spongiome is dissociated from the contractile vacuole complex, the complex’s function is strongly inhibited, showing the decorated spongiome to be the site of fluid segregation.
Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells
Authors: Schweitzer Erik S. 
Published: April 2021

ABSTRACT
PC12 cells secrete the enzyme acetylcholinesterase (AChE) while at rest, and increase the overall rate of this secretion 2-fold upon depolarization. This behavior is different from the release of other markers by the constitutive or regulated secretory pathways in PC12 cells. Both the resting and stimulated release of AChE are unchanged after treatment with a membrane-imper-meable esterase inhibitor, demonstrating that it repre-sents true secretion and not shedding from the cell sur-face. The stimulated release of AChE is Ca2+-dependent, while the unstimulated release is not. Analysis of the molecular forms of AChE secreted by PC12 cells indicates that the release of AChE actually involves two con-current but independent secretory processes, and that the G4 form of the enzyme is secreted constitutively, while both the G2 and G4 forms are secreted in a reg-ulated manner, presumably from regulated secretory vesicles. Compared with other regulated secretory proteins, a much smaller fraction of cellular AChE is secreted, and the intracellular localization of this enzyme differs from that of other regulated secretory proteins. The demonstration that a cell line that exhibits regulated secretion of acetylcholine (ACh) is also capable of regulated secretion of AChE provides additional evidence for the existence of multiple regulated secretory pathways within a single cell. Moreover, there appears to be a selective packaging of different molecular forms of AChE into the regulated versus the constitutive secretory pathway. Both the specificity of sorting of AChE and the regulation of its secretion suggest that AChE may play a more dynamic role in synaptic function than has been recognized previously.
Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations
Authors: Lesuffleur Thécla , Porchet Nicole , Aubert Jean-Pierre , Swallow Dallas , Gum James R. , Kim Young S. , Real Francisco X. 
Published: April 2021

ABSTRACT
Mucin expression was analysed, in relation to cell growth, in parental HT-29 cells and in two populations of mucus-secreting HT-29 cells selected by adaptation to methotrexate (HT29-MTX) or 5-fluorouracil (HT29-FU). These two populations express mature mucins that differ in their immunoreactivity to antibodies against gastric (HT29-MTX) or colonic mucins (HT29-FU). In the parental population, at late confluency, only very few cells produce mucins or the MUC1 glycoprotein, this being consistent with the low level of expression of the mRNAs corresponding to the MUC1 to MUC5C mucin genes. In the HT29-MTX and HT29-FU populations, the appearance of mucus droplets, as shown by histochemistry and immunofluorescence, starts a few days after confluency, progressively involving a greater proportion of cells and reaching a steady state at late confluency. The MUC1 glycoprotein appears earlier, already being detectable in preconfluent cells. Its distribution is restricted to the apical surface of the cells and is distinct from that of the mucus droplets. In both populations the growth-related levels of MUC1 mRNA are concordant with the apparent levels of expression of the MUC1 glycoprotein. The levels of MUC2, MUC3, MUC4 and MUC5C mRNAs differ from one population to another and, within each population, according to the stage of the culture. The highest levels of MUC2 and MUC4 mRNAs are found in the HT29-FU cells, whereas the highest levels of MUC3 and MUC5C are found in the HT29-MTX cells, suggesting that the differences observed in the mature mucins expressed by either population may be related to which MUC genes are expressed. In both populations significant or even high levels of MUC mRNAs are already present in early cul-tures, i.e. at a stage when the mature mucins are not yet detectable, suggesting that mucin maturation is a later event.
The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail
Authors: Pieters Jean , Bakke Oddmund , Dobberstein Bernhard 
Published: April 2021

ABSTRACT
The oligomeric complex formed by major histocompat-ibility complex (MHC) class II and chains and invariant chain (Ii) assembles in the endoplasmic retic-ulum and is then transported via the Golgi complex to compartments of the endocytic pathway. When Ii alone is expressed in CV1 cells it is sorted to endosomes. The Ii cytoplasmic tail has been found to be essential for tar-geting to these compartments. In order to characterize further the signals responsible for endosomal targeting, we have deleted various segments of the cytoplasmic tail. The Ii mutants were transiently expressed and the cel-lular location of the proteins was analyzed biochemi-cally and morphologically. The cytoplasmic tail of Ii was found to contain two endosomal targeting sequences within its cytoplasmic tail; one targeting sequence was present within amino acid residues 12-29 and deletion of this segment revealed the presence of a second endosomal targeting sequence, located within the first 11 amino acid residues. The presence of a leucine-isoleucine pair at positions 7 and 8 within this sequence was found to be essential for endosomal targeting.
In addition, the presence of this L-I motif lead to accumulation of Ii molecules in large endosomal vacuoles containing lysosomal marker proteins. Both wild type Ii and Ii mutant molecules containing only one endosomal targeting sequence were rapidly internalized from the plasma membrane.
When the Ii cytoplasmic tail was fused to the mem-brane-spanning region of neuraminidase, a resident plasma membrane protein, the resulting chimera (INA) was found in endocytic compartments containing lyso-somal marker proteins. Thus the cytoplasmic tail of Ii is sufficient for targeting to the endocytic/lysosomal pathway.
Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and high-pressure frozen/freeze-substituted protoplasts of white spruce (Picea glauca)
Authors: Galway M. E. , Rennie P. J. , Fowke L. C. 
Published: April 2021

ABSTRACT
An ultrastructural study of endocytosis has been made for the first time in protoplasts of a gymnosperm, white spruce (Picea glauca), fixed by high-pressure freezing and freeze substitution. Protoplasts derived from the WS1 line of suspension-cultured embryogenic white spruce were labelled with cationized ferritin, a non-specific marker of the plasma membrane. The timing of cationized ferritin uptake and its subcellular distribu-tion were determined by fixing protoplasts at various intervals after labelling. To address concerns about using chemical fixation to study the membrane-bound transport of cationized ferritin, protoplasts were fixed both by conventional glutaraldehyde fixation and by rapid freezing in a Balzers high-pressure freezing appa-ratus (followed by freeze substitution). Cationized fer-ritin appeared rapidly in coated pits and coated vesicles after labelling. Later it was present in uncoated vesicles, and in Golgi bodies, trans-Golgi membranes and par-tially coated reticula, then subsequently in multivesicular bodies, which may ultimately fuse with and deliver their contents to lytic vacuoles. The results show that the time course and pathway of cationized ferritin uptake in the gymnosperm white spruce is very similar to the time course and pathway elucidated for cation-ized ferritin uptake in the angiosperm soybean. High-pressure freezing yielded much better preservation of intracellular membranes and organelles, although plasma membranes appeared ruffled. Protoplasts fixed by both methods possessed numerous smooth vesicles in the cortex and smooth invaginations of the plasma mem-brane. These became labelled with cationized ferritin, but apparently did not contribute directly to the inter-nalization of cationized ferritin, except via the forma-tion of coated pits and vesicles from their surfaces.
In vitro assembly properties of vimentin mutagenized at the-site tail motif
Authors: Kouklis Panos D. , Hatzfeld Mechthild , Brunkener Mariann , Weber Klaus , Georgatos Spyros D. 
Published: April 2021

ABSTRACT
The intermediate filament (IF) proteins vimentin, desmin and peripherin share a 9-residue sequence motif (β-site) located near the end of their COOH-terminal tail domain. Peptide inhibition experiments have previously suggested that the-site is involved in interactions that limit the lateral growth of IFs and prevent inappropriate filament-filament associations. To investigate this question further, we have constructed and expressed, in Escherichia coli, hamster vimentin bearing different mutations in the β-site. We show here that a single exchange of glycine 450 with a valine residue, or an internal deletion of amino acids 444-452, strongly interferes with the normal assembly of IFs under in vitro conditions. These mutants polymerize into irregular fibrils that have a strong tendency to anastomose and laterally aggregate under isotonic conditions. In contrast, a non-conservative substitution of arginine 448 for glutamic acid does not significantly interfere with filament structure and yields subunits that polymerize into long, smooth filaments that show a slight aberration in thickness. All mutant proteins are soluble in low salt and form oligomers similar to the ones formed by wild-type vimentin. On the basis of these findings and on related observations, we propose that the tail domain of type III IF proteins contains important structural elements involved in lateral protofilament-protofilament interactions.
Subcellular distribution of Ro ribonucleoprotein complexes and their constituents
Authors: Peek Ron , Pruijn Ger J. M. , Kemp Annemiete J. W. van der , Venrooij Walther J. van 
Published: April 2021

ABSTRACT
Ro ribonucleoprotein particles (Ro RNPs) are complexes of several proteins with a small RNA polymerase III-transcribed Ro RNA. Despite their relative abundance and evolutionary conservation no function has as yet been ascribed to these complexes. Also their subcellular distribution is still largely unknown as immuno-fluorescence studies concerning their localization have produced conflicting data. We have used cell enucleation to fractionate cells into cytoplasmic and nuclear fractions. Analysis of these fractions revealed an exclusively cytoplasmic localization for the Ro RNPs. The majority of the Ro RNAs are shown to be stably associated with all three known Ro RNP proteins. Although no Ro RNAs could be detected in the nuclear fraction, the Ro RNP-specific proteins were abundantly present. These nuclear non-Ro RNA-associated proteins are shown to be capable of binding Ro RNAs.
Regulation of vesicle transport in CV-1 cells and extracts
Authors: Hamm-Alvarez Sarah F. , Kim Preston Y. , Sheetz Michael P. 
Published: April 2021

ABSTRACT
To investigate the regulation of microtubule-dependent vesicle motility, we have studied the effects of pharmacological agents on the frequency and velocity of small vesicle movements in intact CV-1 cells. Nocodazole, but not cytochalasin B or D, abolished vesicle movements, indicating that these movements were microtubule and not actin-dependent. The frequency of vesicle movements was stimulated maximally sixfold by okadaic acid from a resting value of 1.6 movements/min per m2 in serum-starved CV-1 cells. Other activators in decreasing order of effectiveness are fetal calf serum, dibutyryl cAMP, cholera toxin, genistein, A23187, and trental. On the other side, taxol inhibited vesicle movements by a factor of four. The activators, okadaic acid, fetal calf serum, and dibutyryl cAMP, also increased vesicle velocity and run length, while taxol decreased vesicle velocity. Although modulation of the frequency of vesicle movements over a &gt;20-fold range was observed, under all conditions the fraction of vesicles moving inward versus outward did not significantly change. Only in the case of taxol was the distribution of microtubules altered within this same time period. Both inward and outward microtubule-dependent vesicle movements therefore appear to be coordinately regulated. The enhanced vesicle motility elicited by fetal calf serum in intact cells correlated with in vitro measurements of vesicle motility and velocity on purified microtubules using microtubule affinity-purified motors and carbonate-washed vesicles from cells treated with fetal calf serum. This suggests that the amount of vesicular intracellular membrane traffic is coordinately regulated with microtubule-dependent motor activity.
Identification of a 102 kDa protein (cytocentrin) immunologically related to keratin 19, which is a cytoplasmically derived component of the mitotic spindle pole
Authors: Paul Eileen C. A. , Quaroni Andrea 
Published: April 2021

ABSTRACT
The mAb RK7, previously shown to recognize keratin 19, was also found to cross-react with a biologically unrelated 102 kDa protein, which becomes associated with the poles of the mitotic apparatus. This newly identified protein, called cytocentrin, is a stable cellular component, may be at least in part phosphorylated, and displays a cell cycle-dependent cellular localization. In interphase cells, it is diffusely distributed in the cytosol and shows no affinity for cytoplasmic microtubules. It becomes localized to the centrosome in early prophase, prior to nuclear envelope breakdown, separation of replicated centrosomes, and nucleation of mitotic apparatus microtubules. During metaphase, cytocentrin is located predominately at the mitotic poles, often appearing as an aggregate of small globular sub-components; it also associates with some polar microtubules. In late anaphase/early telophase cytocentrin dissociates entirely from the mitotic apparatus and becomes temporarily localized with microtubules in the midbody, from which it disappears by late telophase. In taxol-treated cells cytocentrin was associated with the center of the miniasters but also showed affinity for some cytoplasmic microtubules. Studies employing G2-synchronized cells and nocodazole demonstrated that cytocentrin can become associated with mitotic centrosomes independently of tubulin polymerization and that microtubules regrow from antigen-containing foci. We interpret these results to suggest that cytocentrin is a cytoplasmic protein that becomes specifically activated or modified at the onset of mitosis so that it can affiliate with the mitotic poles where it may provide a link between the pericentriolar material and other components of the mitotic apparatus.
Gene deletion suggests a role for Trypanosoma Cruzi surface glycoprotein gp72 in the insect and mammalian stages of the life cycle
Authors: Jesus Amelia Ribeiro de , Cooper Robin , Espinosa Martha , Gomes José Eugenio P. L. , Garcia Eloi S. , Paul Simon , Cross George A. M. 
Published: April 2021

ABSTRACT
We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene dele-tion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the aquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular popula-tion consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector.