bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cytoskeletal interactions with post-mitotic migrating nuclei in the oyster mushroom fungus, Pleurotus ostreatus: evidence against a force-generating role for astral microtubules
Authors: Kaminskyj S. G. W. , Yoon K. S. , Heath I. B. 
Published: April 2021

ABSTRACT
Nuclei of Pleurotus ostreatus migrate in a highly predictable manner foflowing conjugate mitosis at clamp connections. Parameters were determined by observations in living hyphae and these data were used to predict the living behaviour of freeze-substituted nuclei. Three of four classes of nucleus migrate immediately after telophase and move at similar speeds. Freeze-substitution electron microscopy shows that these nuclei have prominent nucleus-associated organelles (NAOs) with large astral microtubule (MT) arrays. Although the NAOs do not have a consistent position with respect to the nuclear motion, they are preferentially located near the hyphal axis. The fourth class of nucleus remains in the developing clamp until it fuses with the main hypha, whereupon it migrates to its interphase position at a rate much faster than the other classes. This class of nucleus has a small NAO and no astral MTs. Treatment of hyphae with a MT-disrupting drug, MBC, reduced the astral arrays in the first three classes but did not slow their rate of movement. Moreover, serial section analysis of drug-treated nuclei whose migration rate at the time of fixation was known showed no relationship, positive or negative, between astral MT number and rate of movement. These data suggest that astral MTs neither generate nor transduce force for post-mitotic nuclear migration in P. ostreatus. The rofe of astral MTs and possible mechanisms of postmitotic nuclear migration are discussed.
Translocation of precursor proteins into the mitochondrial matrix occurs through an environment accessible to aqueous perturbants
Authors: Sztul Elizabeth S. , Chu Tom W. , Strauss Arnold W. , Rosenberg Leon E. 
Published: April 2021

ABSTRACT
We have identified translocational intermediates generated during import of precursor proteins into the mitochondrial matrix and have characterized their association with mitochondrial membranes. Partially translocated forms of mitochondrial malate dehydrogenase (MDH) and ornithine transcarbamylase (OTC) were generated during import of the corresponding precursors (pMDH and pOTC) into mitochondria at 2°C. Import at this temperature results in the formation of intermediate-sized MDH (iMDH) and OTC (iOTC) produced by the removal of a portion of the leader peptide, and in the production of mature-sized MDH. All of these forms contain NHz termini located within the mitochondrial matrix, although the majority of theirpofypeptide chains remain extramitochondrial. All three are strongly associated with mitochondrial membranes, but can be extracted by protein denaturants such as urea. These translocational intermediates appear to be hydrophilic proteins, on the basis of their partitioning properties during extraction with the nonionic detergent Triton X-114. The data indicate that the translocation of pofypeptide chains into mitochondria occurs in a microenvironment that is aqueous in nature and is mediated by integral membrane proteins.
The early expression of myofibrillar proteins in round postmitotic myoblasts of embryonic skeletal muscle
Authors: Colley Nansi Jo , Tokuyasu K. T. , Singer S. J. 
Published: April 2021

ABSTRACT
Previous reports on skeletal muscle myogenesis have shown that postmitotic spindle-shaped myoblasts express muscle-specific proteins, some of which are organized into nascent myofibrils. However, we show that, in skeletal muscle cultures derived from 12-day chick embryos, by 6h after plating the predominant mononucleated cell type that expresses muscle-specific proteins is a round cell. These round myoblasts appear to precede spindle-shaped myoblasts in development, since the latter are more abundant in later cultures and contain larger amounts of muscle proteins and more highly organized myofibrils. By double immunofluorescence microscopy using antibodies specific for the muscle proteins titin, myosin heavy chain (MHC) and zeugmatin we find that 18 h after plating approximately 20% of the round myoblasts that are titin-positive are negative for myofibrillar MHC and zeugmatin. On the other hand, all spindle-shaped myocytes that are positive for titin are also positive for myofibrillar MHC and zeugmatin. These results suggest that titin expression precedes that of myofibrillar MHC and zeugmatin in the non-synchronized round myoblasts, and is consistent with earlier suggestions that titin may function as an initial organizer of myofibrillar proteins during myogenesis. Immunofluorescence data indicate that the earliest localization of the myofibrillar proteins titin, MHC, zeugmatin and a-actinin in the round myoblasts is surrounding the nucleus with no. immunofluorescent labeling of the cytoplasm or near the plasma membrane. Furthermore, pairwise double immunofluorescence experiments show that these four myofibrillar proteins are all co-localized, at the light-microscopic level of resolution, in irregular patterns that may appear in either a punctate or a basket-like distribution. These labeling patterns around the nucleus are resistant to extraction with Triton X-100, suggesting that the proteins are associated in a stable array. These Triton X-100-resistant assemblies in round myoblasts appear to be composed solely of structural myofibrillar proteins, since the non-structural myofibrillar protein creatine kinase (CK) does not colocalize with the other myofibrillar proteins. These results indicate that in early myoblasts myofibrillar proteins form stable pre-myofibrillar assemblies surrounding the nucleus, and raise the possibility that these initial assemblies may play an organizing role during subsequent early stages of myofibrillogenesis.
Monoclonal antibodies to the adhesive cell coat secreted by Pythium aphanidermatum zoospores recognise 200×103Mr glycoproteins stored within large peripheral vesicles
Authors: Estrada-Garcia M. Teresa , Callow James A. , Green Jonathan R. 
Published: April 2021

ABSTRACT
During encystment the motile zoospores of the plant pathogen Pythium aphanidermatum secrete an adhesive cell coat that is involved in their attachment to roots. Previous ultrastructural studies have indicated that the adhesive material is pre-packaged within large peripheral vesicles underlying the zoospore plasma membrane. In the present study, four monoclonal antibodies (MAbs) designated PAЗ-6, which were raised against zoospores and cysts of P. aphanidermatum, were used to re-examine the formation of the adhesive cell coat and to study its molecular nature. Immunogold labelling of zoospores and cysts shows that all the antibodies recognise material contained within the large peripheral vesicles of zoospores. These structures are morphologically distinct from the microbodies, which also underly the plasma membrane, and the latter are not labelled by the antibodies. During encystment the material recognised by the MAbs is secreted to form a cell coat around the zoospores and cysts and this can be seen to be separated from the cyst plasma membrane by a distinct layer (cyst wall). The MAbs also label material within vesicles that are located towards the centre of the cyst cytoplasm. Western blotting and antigen-modification techniques have shown that all the MAbs bind to carbohydrate epitopes of a set of high molecular weight glycoproteins (&gt;200×103Mr). One of the antibodies, PA6, also binds to several lower molecular weight components. Overall, the results show that the adhesive material secreted by P. aphanidermatum zoospores is stored within large peripheral vesicles and is composed of several glycoproteins. The results are discussed in the context of studies on the secretion of adhesive material by zoospores of related oomycete fungi.
Modulation of type X collagen gene expression by calcium β-glycerophosphate and levamisole: implications for a possible role for type X collagen in endochondral bone formation
Authors: Thomas J. Terrig , Boot-Handford Raymond P. , Grant Michael E. 
Published: April 2021

ABSTRACT
Chondrocytes from the cephalic region of 18-day chick embryo sterna were cultured within type I collagen gels in the presence of increasing concentrations of calcium β-glycerophosphate (Ca PGP) (2.5 mM, 5mM and 10 mM) or 2mM levamisole. Addition of Ca β-GP produced an increase in type X collagen synthesis and when 10 mM Ca β-GP was used, mineral deposits were observed in the matrix elaborated by the chondrocytes. Measurement of mRNA levels of type II and type X collagen μg−1 total RNA revealed that the level of αl (II) mRNA decreased whereas the level of αl(X) mRNA remained relatively constant in the presence of Ca β-GP. Thus, Ca β-GP appears to increase type X collagen synthesis by enhancing the rate of collagen type X mRNA translation. In levamisole-treated cultures the synthesis of type X collagen and its deposition within the matrix were inhibited. Measurement of mRNA levels μg−1 total RNA revealed that the level of al (II) mRNA increased whereas the level of al(X) mRNA was markedly decreased in the presence of levamisole. Consequently, it must be concluded that levamisole inhibits type X collagen synthesis by inhibiting type X collagen gene transcription.
These results add support for type X collagen having an important role in the mineralization process and have demonstrated that type X collagen synthesis is probably regulated at the mRNA translational stage during chondrocyte hypertrophy.
The α6β1 (VLA-6) and α6β4 protein complexes: tissue distribution and biochemical properties
Authors: Sonnenberg A. , Linders C. J. T. , Daams J. H. , Kennel S. J. 
Published: April 2021

ABSTRACT
A member of the integrin family, the α6β4 complex was previously identified on human and mouse carcinoma cell lines by using a rat monoclonal antibody to α6. Here we describe two monoclonal antibodies that recognize epitopes on the β1subunit of the human and mouse α6β4 complexes. The monoclonal antibodies against β1 were able to preclear α6β4, but not α 6β4from cell line extracts. A substantial fraction of the total β4 subunits present on the cell surface was not associated with α6, as it could not be removed by anti-α6 antibodies, but remained precipitable with anti-β4 antibodies. There was no evidence for novel a subunits associated with pi.
The α6 subunit consists of disulfide-linked heavy and light chains. The variability in size of these two chains from different cell types is largely due to differences in modifications of N-linked glycans. Additional heterogeneity may be caused by differential proteolytic cleavage of the α6 precursor.
Immunoperoxidase staining of tissue sections of neonatal and adult mice revealed that pi expression is limited to epithelial tissues and peripheral nerves. The α6 subunit has a wider distribution that includes all tissues and cells stained by antibodies against β4 Cells and tissue that are positive for α6, but negative for β4, may express the α6β4 complex.
Total internal reflection fluorescence (TIRF) microscopy I. Modelling cell contact region fluorescence
Authors: Reichert W. M. , Truskey G. A. 
Published: April 2021

ABSTRACT
Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1 % for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10 % as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1 %.
Phosphorylation of algal centrin is rapidly responsive to changes in the external milieu
Authors: Martindale Valerie E. , Salisbury Jeffrey L. 
Published: April 2021

ABSTRACTCentrin, a calcium-sensitive contractile phosphoprotein, is the major component of the striated flagellar roots of the flagellate green alga, Tetraselmis striata. Flagellar roots contract in response to elevated calcium levels. Data presented here indicate that the level of centrin phosphorylation is rapidly responsive to changes in the cell’s external environment. Centrin is dephosphorylated in response to elevated calcium or to heat shock. An increase in centrin phosphorylation accompanies pH shock or ethanol treatment. These changes are compared with flagellar excision, flagellar root contraction, and protein synthesis under the same treatments. We conclude that under certain conditions phosphorylation/dephosphorylation of centrin and extension/contraction of the flagellar root are uncoupled. Possible role(s) of centrin phosphorylation independent of force generation in the flagellar root are discussed.
Cytoplasmic pool of histone H1 in mammalian cells
Authors: Zlatanova J. S. , Srebreva L. N. , Banchev T. B. , Tasheva B. T. , Tsanev R. G. 
Published: April 2021

ABSTRACT
Two types of cell populations, nondividing mouse liver cells and exponentially growing Friend erythroleukemia cells, were studied for the presence of a histone H1 pool in the cytoplasm. Purified cytoplasmic fractions were extracted with 5% perchloric acid and the resulting protein preparation was characterized by two types of electrophoresis, gel filtration, peptide mapping, ELISA and immunoblotting. The occurrence of significant quantities of H1 in isolated cytoplasmic fractions was confirmed by indirect immunofluorescence on whole cells. The existence of a cytoplasmic pool of H1 contrasts with the lack of detectable amounts of core histones in the cytoplasm. This indicates that the observed H1 pool is not just a reflection of its cytoplasmic synthesis but probably has some functional significance.
Ultrastructural localization of the regulatory (RII) subunit of cyclic AMP-dependent protein kinase to subcellular compartments active in endocytosis and recycling of membrane receptors
Authors: Griffiths Gareth , Hollinshead Ruth , Hemmings Brian A. , Nigg Erich A. 
Published: April 2021

ABSTRACT
The subcellular distribution of the regulatory (RII) subunit of cyclic AMP-dependent protein kinase (PK-A) was analyzed at the electron-microscopical level using thawed cryo-sections of Madin Darby Bovine Kidney (MDBK) cells. The highest density of labelling for RII was found on membranes of the prelysosomal compartment (PLC; marked with the cation-independent mannose 6-phosphate receptor, MPR) and the trans-Golgi network, TGN (at 20°C, marked with the G protein of vesicular stomatitis virus, VSV), as well as in coated buds on the latter. Significant labelling was also localized to the cytoplasmic surface of the plasma membrane, including clathrin-coated pits and microvilli, and to early endosomes (identified using internalized HRP). In contrast, no significant label was seen over the Golgi compartments proximal to the TGN, the endoplasmic reticulum (ER) or over lysosomes. From these results we conclude that PK-A type II is associated with the membranes of precisely those subcellular compartments that are active in endocytosis and recycling of cell surface receptors. We believe these findings to be related to the well-established role of cyclic AMP in signal transduction. In particular, we propose that activation of PK-A in endocytic compartments may contribute to regulation (via phosphorylation) of the subcellular distribution of internalized surface receptors or their functional coupling to effector systems involved in signal propagation.
Use of conjugates made between a cytolytic T cell clone and target cells to study the redistribution of membrane molecules in cell contact areas
Authors: André Pascale , Benoliel Anne-Marie , Capo Christian , Foa Colette , Buferne Michel , Boyer Claude , Schmitt-Verhulst Anne-Marie , Bongrand Pierre 
Published: April 2021

ABSTRACT
In many models of cell-cell adhesion, it was reported that some cell membrane molecules might be redistributed into contact areas. However, this phenomenon was not subjected to precise quantification. In the present work, fluorescence microscopy, immunolabelling and digital image processing were combined to analyse quantitatively the spatial organization of specific or nonspecific conjugates made with a cytolytic T (CTL) lymphocyte clone (BM3.3) and target cells (EL4 or RDM4). Binding was achieved under calcium-free conditions to study the earliest steps of cell interaction, preceding CTL activation. Fluorescent antibodies were used to label class I histocompatibility molecules on both killer and target cells, and T cell receptor, CD3, CDS and LFA-1 (CD18/CDlla) on the killer cells. Membrane bilayers were stained with a fluorescent phospholipid, glycoconjugates were labelled with periodic oxidation and Lucifer Yellow uptake, and polymerized actin was revealed with a fluorescent phallacidin derivative. Also, the fine geometry of killer–target interaction area was studied with electron microscopy and computer-assisted contour analysis.
It is concluded that: (1) qualitative examination of fluorescence photomicrographs cannot permit accurate comparison between different fluorescence densities. (2) The cell–cell contact area was about fourfold higher in specific conjugates than in nonspecific ones. (3) The surface density of adhesion molecules exhibited similar increases (between 30 and 80%) in the contact areas of both specific and nonspecific conjugates. (4) However, the amount of redistributed surface molecules was higher when cell-cell interaction was enhanced either by specific immunological recognition (in specific conjugates) or periodate oxidation. (5) Since redistribution did not require extracellular calcium and it was detected on nonspecific conjugates, this did not require full lymphocyte activation.
Spatial reorganization of cell surface molecules may thus be a general consequence of adhesion, cell surface mobility and intermolecular forces.
Selective internalization of the apical plasma membrane and rapid redistribution of lysosomal enzymes and mannose 6-phosphate receptors during osteoclast inactivation by calcitonin
Authors: Baron Roland , Neff Lynn , Brown William , Louvard Daniel , Courtoy Pierre J. 
Published: April 2021

ABSTRACT
The effects of inhibition of bone resorption by the peptide hormone calcitonin have been studied at the level of the osteoclast. Although not epithelial, the osteoclast is polarized with the secretion of newly synthesized lysosomal enzymes and of acid occurring specifically at the apical pole, facing the bone compartment. The membranes composing the apical (ruffled-border) and basolateral domains contain topologically restricted antigens, a 100×103Mr lysosomal membrane protein and the Na+,K+-ATPase, respectively. It was found that calcitonin induces a rapid (15–60 min) redistribution of the apical marker as well as of markers of the secretory compartment of the osteoclast (arylsulfatase and cation-independent mannose 6-phosphate (Man6P) receptors). The apical plasma membrane, in contrast to the basolateral membrane, is selectively internalized. This internalization leads to the disappearance of the ruffled border. The vesicular translocation of apical membranes is reminiscent of the events occurring ingastric oxyntic cells and in kidney tubule intercalated cells during the regulation of acid secretion. In parallel, the synthesis of both the lysosomal enzyme arylsulfatase and Man6P receptors is arrested. The products that were already present in the secretory pathway seem to be rerouted to intracellular vacuoles instead of being targeted to the plasma membrane, leading to marked accumulation of enzymes in the inhibited cells. These results suggest that the rapid inhibition of bone resorption by calcitonin involves the vesicular translocation of the apical membranes and the rapid arrest in the synthesis and secretion of lysosomal enzymes in osteoclasts.
Centriole and centrosome cycle in the early Drosophila embryo
Authors: Callaini Giuliano , Riparbelli Maria Giovanna 
Published: April 2021

ABSTRACT
Centriole and centrosome cycles were examined by indirect immunofluorescence and electron microscopy techniques in the early Drosophila embryo. The centrosomes, which are already divided at interphase, appear as compact spheres during prophase and metaphase, expand and flatten from anaphase to telophase and split into two units in late telophase. Centriole separation starts in late metaphase, becomes evident in anaphase and increases during telophase. Procentrioles appear during the following interphase.
Localization of two fibronectin-binding glycoproteins in rat liver and primary hepatocytes: Co-distribution in vitro of integrin (α5β1) and non-integrin (AGp110) receptors in cell-substratum adhesion sites
Authors: Stamatoglou Stamatis C. , Sullivan Kate H. , Johansson Staff An , Bayley Peter M. , Burdett Ian D. J. , Hughes R. Colin 
Published: April 2021

ABSTRACT
We have compared the localization of integrin α5β 1 and AGp100 (apical glycoprotein of Mr 110×103) in rat liver parenchyma and in primary hepatocyte cultures. Integrin α5β 1 is a heterodimeric fibronectin receptor. AGp110 is a newly described monomeric glycoprotein of the apical (bile canalicular) membrane domain of liver parenchyma that binds in an RGD-independent manner to fibronectin and mediates spreading of hepatocytes onto fibronectin-coated substrata. Using Western blotting of fractionated liver membranes and immunocytochemistry of liver sections at light- and electron-microscope levels, we have confirmed that AGp110 is a canalicular glycoprotein and have established that integrin is located in approximately equal proportions in the sinusoidal, lateral and canalicular membrane domains. In the canalicular surface domain both glycoproteins are associated with microvilli. Examination of immunolabelled primary hepatocytes spread on fibronectin-coated substrata by light and laser scanning confocal microscopy revealed colocalization of AGp110, integrin, actin and vinculin in substratum-attached microextensions at the periphery of the basal cell surface. Actin filaments that terminated at these cell processes originated from circular sub-cortical actin fibres. Interference reflection microscopy revealed focal adhesive contacts at the edge of the basal cell periphery at the same location where AGp110 and integrin were observed by immunofluorescence. In vitro, a proportion of the primary hepatocytes seeded onto fibronectin-coated substrata aggregated into colonies of several cells with intercellular contacts between neighbouring cells. Cell-substratum contacts containing integrin, AGp110, actin and vinculin followed the contours of these colonies in the same manner as they delineated the basal periphery of single, substratum-attached cells. We conclude that both integrin and AGp110 contribute to hepatocyte-fibronectin adhesive interactions and that intercellular adhesion and cooperation among hepatocytes in their response to fibronectin matrices leads to colony formation and morphological differentiation of parenchymal cell monolayers in vitro.