bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential expression of plasminogen activators and their inhibitors in an organotypic skin coculture system
Authors: Chen Chih-Shan , Lyons-Giordano Bernadette , Lazarus Gerald S. , Jensen Pamela J.
Published: April 2021

Using immunohistochemistry and in situ hybridization, we have characterized the expression and localization of components of the plasminogen activator proteolytic cascade in an organotypic coculture system which consists of a “dermal” portion (human dermal fibroblasts throughout a collagen matrix) and a stratified, well-differentiated epidermal portion. Specifically, the following components were examined: the enzymes urokinasetype plasminogen activator and tissue-type plasminogen activator and their type 1 and type 2 inhibitors. Urokinase plasminogen activator mRNA and antigen were found predominantly in the least differentiated, basal keratinocytes; in some fields there was also faint deposition of antigen beneath the basal cells. The distribution of plasminogen activator inhibitor type 1 was similar to that of urokinase, except that inhibitor type 1 antigen deposition beneath the basal cells appeared more intense and uniform. In contrast to the results with urokinase plasminogen activator and inhibitor type 1, tissue plasminogen activator mRNA and antigen were localized focally in the suprabasal, i.e. more differentiated, keratinocytes. Plasminogen activator inhibitor type 2 mRNA and antigen were detected in most epidermal layers, but were more intense suprabasally and often spared the basal layer. These studies demonstrate that the same type of cell, i.e. the keratinocyte, can express different components of the plasminogen activator cascade depending on its state of differentiation. The change in expression of plasminogen activator cascade components with keratinocyte differentiation suggests distinct epidermal functions for these components, related to cell-matrix interaction and epidermal differentiation.
Phorbol ester-induced scattering of HT-29 human intestinal cancer cells is associated with down-modulation of E-cadherin
Authors: Fabre Myriam , García de Herreros Antonio
Published: April 2021

The effects of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth characteristics of the colon cancer cell line HT-29 M6 were studied. TPA induced the scattering of proliferative HT-29 M6 cells: in the presence of the phorbol ester, HT-29 M6 colonies scattered and the cells acquired a flatter aspect with diminished cell-cell contacts. This effect of TPA required a persistent activation of PK-C and was acompanied by a slight decrease (30%) in the growth rate. Modifications by TPA of two scattering associated properties of these cells were also detected: TPA decreased cell-to-cell aggregation and enhanced the cellular attachment to matrix substrata (collagen, laminin). The decrease in cell-to-cell adhesion was correlated with a loss of cellular E-cadherin as evidenced by immunofluorescence or immunoblotting with a specific monoclonal antibody. Cell scattering was dependent on the extra-cellular concentration of Ca2+; an increase from 1.6 to 10 mM in the concentration of this ion completely blocked the morphological effects of TPA as well as its action on cell aggregation. This high concentration of Ca2+ also prevented the down modulation of E-cadherin as determined by immunofluorescence. However, the TPA-induced increase in cell attachment to the matrix was not affected by high calcium. These findings support the importance of altered cell-cell adhesion in the process of scattering and provide a good system for the study of down modulation of E-cadherin, a protein involved in the control of cell growth, differentiation and invasion of epithelial cells.
Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora Crassa are mediated by cytoplasmic microtubules
Authors: Steinberg Gero , Schliwa Manfred
Published: April 2021

The cellular basis of organelle transport in filamentous fungi is still unresolved. Here we have studied the intra-cellular movement of mitochondria and other organelles in the fungus Neurospora crassa. Four different model systems were employed: hyphae, protoplasts, a cell wallless mutant, and experimentally generated small, flattened cell fragments of the mutant cells. Organelle movements were visualized by DIC optics and computer-enhanced video microscopy. In all cell models the transport of organelles was vectorial and saltatory in nature. The mean velocities for mitochondria, particles and nuclei were 1.4, 2.0, and 0.9 m/s, respectively. Treatment with 10 M nocodazole for 30 minutes caused a complete disappearance of microtubules and reversibly blocked directed transport of virtually all organelles, whereas cytochalasin D up to 20 M was without effect. Correlative video and immunofluorescence microscopy of small fragments of wall-less mutant cells revealed a clear match between microtubule distribution and the tracks of moving organelles. We conclude that organelle movement in the filamentous fungus Neurospora crassa is a microtubule-dependent process.
α7β1 integrin is a component of the myotendinous junction on skeletal muscle
Authors: Bao Z. Z. , Lakonishok M. , Kaufman S. , Horwitz A. F.
Published: April 2021

Immunization against a 70 kDa band that co-purifies with skeletal muscle integrins has resulted in an anti-body directed against the avian 7 integrin subunit. The specificity of the antibody was established by patterns of tissue staining and cross-reactivity with antibodies directed against the cytoplasmic domain of the rat 7 cytoplasmic domain. On sections of adult skeletal muscle the 7 integrin was enriched in the myotendinous junc-tion (MTJ). This localization was unique as neither the 1, 3, 5, 6 and v subunit localizes in the myotendi-nous junction. The distribution of the 7 subunit in the MTJ was examined during embryonic development. 7 expression in the junction is first apparent around embryo day 14 and is almost exclusively at the devel-oping MTJ at this stage. 3 is expressed with distinctive punctate staining around the junctional area in earlier embryos (11-day). The time of appearance of the 7 sub-unit in the MTJ correlates with the insertion of myofib-rils into subsarcolemmal densities and folding of the junctional membrane, suggesting a role of the 7 inte-grin in this process. Vinculin is present throughout development of the myotendinous junction, suggesting that the 7 integrin recognizes a preformed cytoskele-tal structure. The presence of the 7 subunit in the myotendinous junction and the 5 subunit in the adhe-sion plaque demonstrates a molecular difference between these two adherens junctions. It also points to possible origins of junctional specificity on muscle. Dif-ferences between these two junctions were developed further using an antibody against phosphotyrosine (PY20). Phosphotyrosine is thought to participate in the organization and stabilization of adhesions. The focal adhesion and the neuromuscular junction, but not the MTJ, contained proteins phosphorylated on tyrosine.
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

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

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

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

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

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

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

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

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

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

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

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 >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

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

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.
Autoantibodies to a novel cell cycle-regulated protein that accumulates in the nuclear matrix during S phase and is localized in the kinetochores and spindle midzone during mitosis
Authors: Casiano Carlos A. , Landberg Göran , Ochs Robert L. , Tan Eng M.
Published: April 2021

We have employed human autoantibodies to character-ize a novel cell cycle-regulated nuclear protein, provi-sionally designated p330d (doublet polypeptide of 330 kDa). The expression and intracellular distribution of this protein was followed throughout the cell cycle using immunofluorescence microscopy, laser confocal microscopy, immunoelectron microscopy and flow cytometry. p330d was expressed only in proliferating cells and began accumulating in the nucleus during early S phase. The protein reached maximum expression levels during G2/M. In situ extractions with detergent, salt and nucleases failed to abolish the nuclear staining of interphase cells, suggesting a tight binding of p330d to the nuclear matrix during interphase. p330d was con-centrated in the kinetochores during prophase but was relocated to the spindle midzone at the onset of anaphase. By late telophase, it was localized predomi-nantly in the intercellular bridge regions flanking the midbody and disappeared gradually as the daughter cells separated. Immunoblotting analysis showed that the autoimmune sera recognized a doublet of 330 kDa, and affinity-purified antibodies from this doublet repro-duced the fluorescence staining pattern of the whole serum. We propose that p330d is a novel member of the class of ‘chromosomal passenger’ proteins, which are associated transiently with centromeres during early mitosis and are then redistributed to other sites of the mitotic apparatus after the metaphase/anaphase transi-tion. Possible in vivo functions for p330d and related proteins might include roles in centromere/kinetochore maturation and assembly, chromosome segregation, central spindle stabilization and cytokinesis.
Numerical analysis of a comprehensive model of M-phase control in Xenopus oocyte extracts and intact embryos
Authors: Novak Bela , Tyson John J.
Published: April 2021

To contribute to a deeper understanding of M-phase control in eukaryotic cells, we have contructed a model based on the biochemistry of M-phase promoting factor (MPF) in Xenopus oocyte extracts, where there is evi-dence for two positive feedback loops (MPF stimulates its own production by activating Cdc25 and inhibiting Wee1) and a negative feedback loop (MPF stimulates its own destruction by indirectly activating the ubiquitin pathway that degrades its cyclin subunit). To uncover the full dynamical possibilities of the control system, we translate the regulatory network into a set of differential equations and study these equations by graphical techniques and computer simulation. The positive feed-back loops in the model account for thresholds and time lags in cyclin-induced and MPF-induced activation of MPF, and the model can be fitted quantitatively to these experimental observations. The negative feedback loop is consistent with observed time lags in MPF-induced cyclin degradation. Furthermore, our model indicates that there are two possible mechanisms for autonomous oscillations. One is driven by the positive feedback loops, resulting in phosphorylation and abrupt dephosphorylation of the Cdc2 subunit at an inhibitory tyrosine residue. These oscillations are typical of oocyte extracts. The other type is driven by the negative feedback loop, involving rapid cyclin turnover and negligible phosphorylation of the tyrosine residue of Cdc2. The early mitotic cycles of intact embryos exhibit such character-istics. In addition, by assuming that unreplicated DNA interferes with M-phase initiation by activating the phosphatases that oppose MPF in the positive feedback loops, we can simulate the effect of addition of sperm nuclei to oocyte extracts, and the lengthening of cycle times at the mid-blastula transition of intact embryos.
Abnormal cytoskeletal and chromosome distribution in po, ms4 and ms6; mutant alleles of polymitotic that disrupt the cell cycle progression from meiosis to mitosis in maize
Authors: Liu Qinqin , Golubovskaya Inna , Cande W. Zacheus
Published: April 2021

The maize cell cycle regulation mutant polymitotic (po) progresses through abnormal cell cycles, characterized by premature cell divisions without chromosome dupli-cation of the daughter cells produced by meiosis during microsporogenesis and macrosporogenesis. There are three recessive alleles of the Po gene; po, ms4, and ms6. A new method of permeabilizing cells based on freeze-fracture technology was used to study the distribution of microtubules in wild-type and mutant microspores. Here we show that an abnormal distribution of micro-tubules is correlated with changes in chromosome mor-phology in a cell cycle-dependent manner in po, ms4 and ms6 mutant alleles. After meiosis II, the cell cycle is com-plete and becomes progressively less synchronous in po homozygotes compared with wild-type cells. During microsporogenesis, the distribution of microtubules is abnormal, and chromosome morphology is altered in both po, ms4 and ms6 mutants. However, more chromosome fragments or micronuclei associated with mini-spindles are present in ms6 than po and ms4. After microspores are released from the tetrads, disruptions in structure and organization of chromosomes and microtubules continues in subsequent abnormal cell cycles. However, these cell cycles are incomplete since phragmoplasts are not formed. During these incomplete cell cycles, abnormal spindles and microtubule arrays are induced and extra microtubule arrays are associated with irregularly distributed chromosome fragments. States corresponding to interphase, prophase, metaphase and anaphase can be recognized in the mutant microspores. Abnormal cell cycles also occur after female meiosis during ms4 macrospore development. Since only the cell that normally undergoes embryo sac development (the chazal-most cell) under-goes supernumerary divisions this suggests that the po phenotype can be characterized as premature haploid divisions rather than repetition of meiosis II.
A novel method for measuring protein expression at the cell surface
Authors: Stoorvogel Willem , Oorschot Viola , Neve Bernadette
Published: April 2021

All methods described in the literature that allow quantitative measurements of protein expression at the cell surface are applicable to subsets of surface-exposed proteins only. We developed a new method, involving 3,3-diaminobenzidine (DAB) cytochemistry, which allowed determination of cell-surface expression of all plasma membrane proteins measured, in at least three different cell lines. Adherent cells were first brought into sus-pension by proteinase K and EDTA treatment at 0°C removing many, but not all, surface-exposed proteins. Subsequently, horseradish peroxidase (HRP) was linked by means of its glycosyl residues to specific cell-surface-exposed sugar moieties using the multivalent lectin con-canavalin A (ConA). The suspended cells were encap-sulated by polymerized DAB, a process that was catalysed by plasma membrane-bound HRP. After cell lysis, and removal of nuclei and most of the DAB polymer by centrifugation, proteins were analysed by SDS-PAGE. Surface proteins encapsulated by non-pelleted DAB polymer were retained on top of the stacking gel. After 125I-labelling the cell surface, protease-resistant125I-labelled proteins could be quantitatively coupled to DAB polymer. This process was completely dependent on the presence of ConA, HRP, DAB and H2O2. Sur-face 125I-labelled-Na+,K+-ATPase was resistant to proteinase K but could be completely removed using DAB cytochemistry. Intracellular ConA binding proteins were not affected. Other intracellular proteins, includ-ing endosomal asialoglycoprotein receptor and cation-independent mannose 6-phosphate/insulin-like growth factor II receptor were also not affected. Metabolically [35S]methionine-labelled ‘high-mannose’ glycosylated-Na+,K+-ATPase was not touched by DAB cytochemistry whereas complex-glycosylated surface-exposed 35S--Na+,K+-ATPase was removed by the procedure. The results show that the method can be used to measure both endocytic uptake and biosynthetic arrival at the plasma membrane of membrane-associated proteins.
Stacking of Golgi cisternae in Schizosaccharomyces pombe requires intact microtubules
Authors: Ayscough Kathryn , Hajibagheri Nasser M. A. , Watson Rose , Warren Graham
Published: April 2021

Fission yeast was treated with the anti-microtubule agent, thiabendazole. Cytoplasmic microtubules broke down with a half-time of less than 10 minutes followed closely by the unstacking of Golgi cisternae. The final product appeared to be single Golgi cisternae. No other organelle seemed to be affected by this treatment, which was completely reversible. The nda3 mutant strain has an alteredtubulin and its cytoplasmic microtubules are resistant to thiabendazole. The Golgi in this cold-sensitive mutant was unaffected by treatment at the per-missive temperature but unstacked at the non-permissive temperature even in the absence of thiabendazole. Taken together these data show that disruption of the microtubular network can cause dissociation of Golgi cisternae. Newly synthesised acid phosphatase was transported and secreted to the same extent and with the same kinetics whether or not the Golgi was unstacked. The possible role of microtubules in Golgi stacking and the lack of effect on secretion are discussed.
Transient expression of the intermediate filament nestin during skeletal muscle development
Authors: Sejersen Thomas , Lendahl Urban
Published: April 2021

It has previously been established that skeletal muscle development is accompanied by changes in the compo-sition of intermediate filaments: vimentin is expressed predominantly in myoblasts and desmin in adult myotubes. We show that the intermediate filament tran-sitions during muscle development are more complex, and involve a transient expression of the recently dis-covered intermediate filament nestin. Nestin RNA is expressed predominantly early, in a biphasic pattern, and is markedly downregulated in adult rat muscle, whereas desmin RNA becomes more abundant through-out development. Nestin protein was found up to the postnatal myotube stage, where it colocalized with desmin in Z bands. The intracellular distribution of nestin, vimentin and desmin was analysed in the human myogenic cell line G6 before and after in vitro differ-entiation. Despite its more distant evolutionary and structural relationship to the other two intermediate fil-aments, nestin formed a cytoplasmic filamentous net-work indistinguishable from that of desmin and vimentin, both in undifferentiated myoblasts and after differentiation to multinuclear myotubes. In conclusion, our data suggest that nestin is an integrated component of the dynamic intermediate filament network during muscle development and that nestin copolymerizes with desmin and vimentin at stages of coexpression.
Contribution of melanogenic proteins to the heterogeneous pigmentation of human melanocytes
Authors: Abdel-Malek Zalfa , Swope Viki , Collins Cynthia , Boissy Raymond , Zhao Huiquan , Nordlund James
Published: April 2021

Human melanocytes from individuals with different skin types, as well as from the skin of the same indi-vidual, are heterogeneous in their melanin content. This heterogeneity may be attributed to differences in the activity and expression of the three melanogenic proteins: tyrosinase and tyrosinase-related proteins 1 and 2 (gp75 and DOPAchrome tautomerase, respectively), which in turn are affected by certain regulatory factors. Established melanocyte strains that exhibited intrinsic melanogenic heterogeneity could be separated into sub-populations according to density and melanin content by Percoll density gradient centrifugation. The least melanotic subpopulation consisted of melanocytes that contained an active tyrosinase enzyme and a low amount of melanin. Tyrosinase activity and the quantities of tyrosinase enzyme, tyrosinase-related protein-1 and DOPAchrome tautomerase gradually increased with increased melanin content and Percoll density of the isolated melanocyte subpopulations. We have found a direct correlation between melanin content, tyrosinase activity and the expression of the three melanogenic pro-teins in melanocyte strains established from different skin types. Addition of the two epidermal cytokines, tumor necrosis factor- or interleukin-1, to cultures of human melanocytes from different skin types caused decreased proliferation, tyrosinase activity and expression of tyrosinase, tyrosinase-related protein-1 and DOPAchrome tautomerase. Similar results were obtained when Percoll-derived melanocyte subpopula-tions were treated with tumor necrosis factor- and interleukin-1. These results indicate that the variation in melanin content in human melanocytes is due to differences in the activity and expression of the melanogenic proteins, which are influenced by autocrine and paracrine factors.
Ultrastructural rRNA localization in plant cell nucleoli RNA/RNA in situ hybridization, autoradiography and cytochemistry
Authors: Olmedilla A. , Testillano P. S. , Vicente O. , Delseny M. , Risueño M. C.
Published: April 2021

The distribution of ribosomal transcripts in the plant nucleolus has been studied by non-isotopic in situ hybridization in ultrathin Lowicryl K4M sections and by high-resolution autoradiography after labelling with tritiated uridine. In parallel, cytochemical techniques were applied to localize RNA on different plant nucle-olar components of Allium cepa L. root meristematic cells and Capsicum annuum L. pollen grains.

For RNA/RNA in situ hybridization, several biotiny-lated single-stranded ribosomal RNA probes were used for mapping different fragments of the 18 S and the 25 S rRNA gene transcribed regions. Ribosomal RNAs (from pre-rRNAs to mature 18 and 25 S RNAs) were found in the nucleolus, in the dense fibrillar (DFC) and granular components (GC). Hybridization signal was found at the periphery of some fibrillar centres (FCs) with probes recognizing both 18 and 25 S rRNA sequences. A quantitative study was performed to analyze the significance of this labelling.

Incorporation of tritiated uridine into roots was car-ried out and, later, after a long time-exposure, autora-diography revealed the presence of newly synthesized RNA mainly in the DFC and at the periphery of the FCs. The presence of RNA in these areas was also con-firmed by the cytochemical techniques used in this study.

Taken together, these data favour the hypothesis that transcription can begin at the periphery of the FCs, although we cannot exclude the possibility that the DFC plays a role in this process.
Intracellular distribution of β-actin mrna is polarized in embryonic corneal epithelia
Authors: Yeh Berhan , Svoboda Kathy Kay Hartford
Published: April 2021

The intracellular distribution of filamentous actin (F-actin), all actin isoforms and β-actin mRNA were analyzed in whole-mount preparations of freshly isolated corneal epithelia. Filamentous actin distribution was analyzed with fluorescently tagged phalloidin. An antibody that recognizes an epitope on both globular (G-actin) and F-actin was used in an immunohistochemical analysis of actin protein distribution. Whole-mount epithelial tissues were examined with a confocal laser scanning microscope (CLSM). Biotinylated oligonucleotide probes specific for the β-actin mRNA were used, and visualized with avidinFITC. The intracellular localization of the β-actin mRNA was similar to the F-actin protein distribution. In the most apical optical sections of embryonic cornea, actin staining delineated the cell borders and microvilli of the periderm cells. The actin is also detected as an organized network at the interface between the basal and periderm cells. At the level of the basal cell nucleus, F-actin is sparse, associating only with the lateral cell membranes. However, at the optical plane below the nuclei, the actin forms an elaborate actin cortical mat. Actin mRNA staining was visualized as discrete punctate areas. The β-actin mRNA was positive at the optical plane just below the periderm cell apical membrane surface, similar to actin in microvilli. These cells also contained punctate staining near the cell membranes and in the periderm-basal cell junction area. At the level of the basal cell nucleus the actin mRNA was present in a punctate pattern along the cell membranes. Below the basal cell nuclei the actin mRNA staining increased at the level of the actin cortical mat. These experiments are the first demonstration that actin mRNA is polarized in embryonic corneal epithelia and co-localized with actin protein in an intact tissue.