bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribosome crystals in necrotizing cells from the posterior necrotic zone of the developing chick limb
Authors: Mottet N. K. , Hammar S. P.
Published: April 2021

Ribosome and ‘protein crystals’ were observed in electron micrographs of degenerating cells from the posterior necrotic zone (PNZ) of developing chick limbs, stages 22 through 24.

Ribosome crystals were made up of basic units of 4 ribosomes arranged in a square tetramer. They were monomorphic with the surface lattice being of the p4 plane group. The 1-ribosome thick sheets of crystals were often seen stacked upon one another forming a 3-dimensional P422 configuration. The percentage of crystallized ribosomes within degenerating PNZ cells is nearly directly proportional to the degree of degeneration of the cell. Crystals identical to this have been observed in numerous types of embryonic chick cells subjected to hypothermia.

The ‘protein crystals’ were composed of straight filamentous structures separated by a distance of 20–30 nm. They were always closely associated with the ribosome crystals. These crystals are similar to those seen in cells treated with high doses of vinblastine and vincristine. The pathogenesis of the ribosome and protein crystals occurring in degenerating PNZ cells is unknown. They are not associated in any way with hypothermia or vinblastine-vincristine treatment.
Intermediate cells of the pancreas: I. ultrastructural characterization
Authors: Melmed R. N. , Benitez Carol J. , Holt S. J.
Published: April 2021

The normal existence of cells in the pancreas with a structure intermediate between those of exocrine and endocrine cell types has long been a matter of dispute. The present study shows that, based upon morphological criteria, such intermediate cells are present in both the endocrine and exocrine tissues of the normal pancreas of the rat, guinea-pig, rhesus monkey, goat, chicken and frog. There is a tendency for intermediate cells to occur most frequently in the frog, where exocrine and endocrine cells are intermingled, and least frequently in higher species such as the rat, guinea-pig, monkey and goat where the endocrine cells are localized in discrete islets. Their occurrence in the chick appears to lie between these 2 extremes. The existence of intermediate cell types has been attributed to a ‘transformation’ of one form of specialized cell in the pancreas into another in response to a metabolic demand. However, the widespread occurrence of inter mediate cells in the normal pancreas suggests that they represent, ab initio, a distinct category of cell, the existence of which poses interesting questions concerning the genetic control of their specialized functions and of developmental processes in the pancreas. Moreover, intermediate cells, such as acinar cells containing endocrine β-granules might serve as a source of insulin additional or alternative to that provided by cells that are wholly endocrine in character.
Absence of centrioles in the first and second meiotic spindles of mouse oocytes
Authors: Szollosi D. , Calarco Patricia , Donahue R. P.
Published: April 2021

Centrioles are present in oogonia and oocytes of hamsters, mice, rats, gerbils and man up to the pachytene stage. In subsequent meiotic stages, centrioles are absent. At the time of germinal vesicle breakdown in the mouse several foci composed of electron-dense fibrillar material from which microtubules radiate appear near the nucleus. These fibrillar aggregates, referred to as microtubule foci, form small asters and collectively perform the role of the spindle pole during the two meiotic divisions. There are multiple centres at each pole and their number may vary. Thus, centrioles are not obligatory organelles of the spindle apparatus.
The classes of kappa in Paramecium aurelia
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Authors: Preer L. B. , Jurand A. , Preer J. R. , Rudman B. M.
Published: April 2021

Kappas (bacterial symbionts containing R bodies) have been studied in 16 strains of Para mecium aurelia, syngens 2 and 4. All produce toxins capable of killing sensitive paramecia. The first major class, the 51 group (consisting of the kappas of strains 51, 116, and 298), has R bodies which, when the pH is lowered below 6·5, unwind reversibly from the inside to form a tight tube; the outside end of the R-body ribbon forms an acute angle; no sheath surrounds the R body. The phage-like structures of the 51 group are helical; no capsomere-like structures are present; isolated R bodies do not have any killing activity. The second major class, the 7 group (strains 7, 576, BI 166-1, 249, 1041, 310, 1039), has R bodies which unwind irreversibly from the outside to form a loose twisted ribbon whose outside tip is blunt, irregular or finger-like; a single membraneous sheath covers the R body. Phage-like structures in the 7 group are spherical; capsomere-like structures are present; isolated R bodies show killing activity. The third major class, the 562 group (strains 562, 517, 511), is similar to the 7 group except that there is no sheath, no capsomeres, and free R bodies are almost or completely inactive. The phage-like structures are spherical, and unlike those of the 7 group, do not stick to the R bodies. In addition to these 3 major classes there are 3 strains which show important differences from the major classes and also differ among themselves. 1038 is like 7 in all respects except that the phage-like structures are helical and isolated R bodies are not very active. 51ml, presumably a mutant of 51, has spherical phage-like structures and 7 type-R bodies which are inactive when isolated. 570 is the only known mate-killer with R bodies; it has spherical phage-like structures.

It is noted that strain 1039 of the 7 group has very few phage-like structures, virtually all of which are empty. Since free 1039 R bodies are highly active, it is likely that intact phage-like structures are not essential for the toxic action on sensitives. The spherical and helical phage-like structures are probably very closely related because 51, which has helical structures, apparently gave rise to 51ml, which has spherical structures. Likewise the kappa symbionts of 1038 with helical phage-like structures are virtually identical to the kappas of the 7 group with spherical phage-like structures. The presence of phage-like structures in all strains with R bodies suggests that R bodies may be a product of phage activity. Strain 570, a mate-killer whose symbionts contain R bodies, provides a link between kappas and mus, the mate-killer symbionts. The symbionts of 570 are the only ones containing R bodies which are non-toxic when encountered by sensitives in the medium in which the paramecia are cultured.
Evidence that the synchronized production of new basal bodies is not associated with dna synthesis in Stentor coeruleus
Authors: Younger K. B. , Banerjee S. , Kelleher J. K. , Winston M. , Margulis Lynn
Published: April 2021

Stentors were induced to produce synchronously thousands of new ciliated oral membranellar band basal bodies in less than 3 h. DNA synthesis does not accompany this process, as determined by [3H]thymidine incorporation into isolated bands and by sensitivity to DNA synthesis inhibitors (mitomycin C, ethidium bromide, cytosine arabinoside and hydroxyurea). Yet DNA could be detected in the cortex and the band at basal body sites by autoradiography. Since [3H]thymidine incorporation into membranellar band was eliminated in concentrations of ethidium bromide that had no effect on basal body formation, the previous reports of ciliate kinetosomal (basal body) DNA are interpreted as due to mitochondrial contamination. Specific cortical patterns of DNA that could have been easily misinterpreted as basal body-related were especially apparent in autoradiographs using [3H]actinomycin D as a ‘stain’.

In no experiment involving induced basal body regeneration could evidence be found for a correlation between new basal body production and DNA synthesis; RNA and protein synthesis correlated with basal body and cilia regeneration were, however, easily detected by the same techniques. We concluded that there is no evidence that basal body DNA synthesis is required for new basal body production.
Limpet Haemocytes I. Studies on Aggregation and Spike Formation
Authors: Davies P. S. , Partridge T.
Published: April 2021

In this paper a promising new spontaneously aggregating system of amoebocytes from the blood of limpets is described. The system has the advantage of being readily available without cell culture and of being operational at room temperature.

The blood of the limpet Patella contains 2 types of haemocytes, amoebocytes and macrophages, the latter accounting for less than 1 % of the cell population. The concentration of cells in suspension varies with the body temperature of the animal, from about 1 ×108 cells/ml at 5 °C to 9 × 108 cells/ml at 25 °C.

Following blood withdrawal the amoebocytes are approximately spherical in shape with superficial convoluted lamellae, but over a period of 5 min develop stout marginal spikes. This development is temperature dependent and independent of divalent cations in the medium.

Most interestingly, and apparently associated with the stimulus of blood withdrawal, the cells become spontaneously adhesive and commence an extremely rapid aggregation. With a shaking speed of 700 strokes/min the half time of aggregation varies from 15 s at an initial cell density of 4·7 × 106 cells/ml to 60 s at a density of 1·4 × 106 cells/ml. The aggregates so formed are very resistant to mechanical dispersion and tight junctions have been observed between aggregated cells in electron micrographs. Complete aggregation requires the presence of divalent cations and another unidentified plasma component. Aggregation will occur at 0°C but the process is slower and less complete than at 20 °C. It is suggested that aggregation involves the triggering or activation of a pre-synthesized adhesive system and that the adhesiveness per unit area increases with increase in temperature.
Embryoglycans regulate FGF-2-mediated mesoderm induction in the rabbit embryo
Authors: Dvořák P. , Flechon J.-E. , Thompson E. M. , Horák V. , Adenot P. , Renard J. P.
Published: April 2021

Several peptide growth factors, including members of the fibroblast growth factor (FGF) superfamily, are potential inducers of mesoderm in vertebrates. Receptor binding of basic FGF (FGF-2) is promoted by cell surface or extracellular matrix proteoglycans. The substantial biosynthesis of proteoglycans by embryonic cells (called embryoglycans) and their potential role as ligands for growth factor receptors led us to examine the role of embryoglycans that carry the developmentally regulated oligosaccharide epitope TEC 1, in the binding of FGF-2 to cultured rabbit inner cell masses (ICMs).

Culture of isolated ICMs in the presence of FGF-2 gave rise to well delimited colonies with migrating cells at the periphery. In these cells, TEC 1 staining shifts from a punctate pattern over the entire membrane, to an apical, finely granular distribution with some internalization. This shift occurs after 96 hours in culture.

Here we show that: (1) migrating cells are mesoderm-like in phenotype; (2) antibodies against TEC 1 blocked FGF-2 mediated differentiation in vitro; (3) antibodies against TEC 1 selectively blocked binding of FGF-2 to ectodermal receptors and, vice versa, the binding of TEC 1-specific antibodies to ectodermal cells can be competed by excess FGF-2; (4) the same switch in TEC 1 staining patterns was observed in vivo, between the day 7 and the day 9 rabbit embryo.

These data suggest the involvement of defined species of embryonic cell surface epitopes in the regulation of FGF-2 receptor binding. Moreover, this proposed binding activity is temporally restricted to ectodermal cells and disappears early during differentiation. Thus, the apical TEC 1 redistribution can be considered as the earliest indicator of mesoderm formation.
Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments
Authors: Toivola Diana M. , Goldman Robert D. , Garrod David R. , Eriksson John E.
Published: April 2021

The importance of protein phosphatases in the maintenance of cytoskeletal structure is supported by the serious liver injury caused by microcystin-LR, a hepatotoxic inhibitor of type-1 and type-2A serine/threonine protein phosphatases. We used the microcystin-LR-induced cell injury as a model to study the roles of protein dephosphorylation in maintaining cytoskeletal structure and cellular interactions in primary rat hepatocyte cultures. Confocal microscopy revealed that the first visible effect of micro-cystin-LR is disruption of desmoplakin organization at the cell surface, indicating dissociation of desmosomes. This effect is followed by a dramatic reorganization of both the intermediate filament (keratins 8 and 18) and microfilament networks, resulting in a merged structure in which the intermediate filaments are organized around a condensed actin core. Keratin 8, keratin 18 and desmo-plakin I/II are the major cytoskeleton-associated targets for microcystin-LR-induced phosphorylation. Hyperphosphorylation of keratin 8 and 18 is accompanied by an increased keratin solubility, which correlates with the observed mor-phological effects. Phosphopeptide mapping shows that four specific tryptic phosphopeptides are highly phosphorylated predominantly in the soluble pool of keratin 18, whereas keratin 8 shows no indications of such assembly state-specific sites. Phosphopeptide maps of keratins phosphorylated in vivo and in vitro indicate that Ca2+/calmod-ulin-dependent kinase may be involved in regulating the serine-specific phosphorylation of both keratin 8 and keratin 18, while cAMP-dependent protein kinase does not seem to play a major role in this context. Taken together, our results show that the interactions between keratin intermediate filaments and desmosomes as well as the assembly states of their main constituent proteins, are directly regulated by serine/threonine kinase/phosphatase equilibria.
ERD2 proteins mediate ER retention of the HNEL signal of LRP’s receptor-associated protein (RAP)
Authors: Bu Guojun , Rennke Stephanie , Geuze Hans J.
Published: April 2021

The 39 kDa receptor-associated protein (RAP) is a receptor antagonist that interacts with several members of the low density lipoprotein (LDL) receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. Our recent studies have demonstrated that RAP is an endoplasmic reticulum (ER) resident protein and an intracellular chaperone for the LDL receptor-related protein (LRP). The HNEL sequence at the carboxyl terminus of RAP represents a novel ER retention signal that shares homology with the well-characterized KDEL signal. In the present study, using immunoelectron microscopy we demonstrate that cells stably transfected with human growth hormone (GH) tagged with either KDEL (GH+KDEL) or HNEL (GH+HNEL) signals exhibit ER and cis-Golgi localization typical of ER-retained proteins. Overexpression of not only GH+HNEL but also GH+KDEL cDNA in transfected cells results in saturation of ER retention receptors and secretion of endogenous RAP indicating that the two signals interact with the same ER retention receptor(s). The role of RAP in the maturation of LRP is further supported by the observation that functional LRP is reduced about 60% as a result of decreased intracellular RAP. Pulse-chase labeling and immunolocalization studies of ERD2.1 and ERD2.2 proteins in transfected cells demonstrate a long half-life and Golgi localization for both receptors. Finally, overexpression of either ERD2.1 or ERD2.2 proteins significantly increases the capacity of cells to retain both KDEL and HNEL-contain-ing proteins. Taken together, our results thus demonstrate that ERD2 proteins are capable of retaining the novel ER retention signal associated with RAP.
Translocation of a UV-damaged DNA binding protein into a tight association with chromatin after treatment of mammalian cells with UV light
Authors: Rapić Otrin Vesna , McLenigan Mary , Takao Masashi , Levine Arthur S. , Protić Miroslava
Published: April 2021

A UV-damaged DNA binding protein (UV-DDB) is the major source of UV-damaged DNA binding activity in mammalian cell extracts. This activity is defective in at least some xeroderma pigmentosum group E (XP-E) patients; microinjection of the UV-DDB protein into their fibroblasts corrects nucleotide excision repair (NER). In an in vitro reconstituted NER system, small amounts of UV-DDB stimulate repair synthesis a few fold. After exposure to UV, mammalian cells show an early dose-dependent inhibition of the extractable UV-DDB activity; this inhibition may reflect a tight association of the binding protein with UV-damaged genomic DNA. To investigate the dynamics and location of UV-DDB with respect to damaged chromatin in vivo, we utilized nuclear fractionation and specific antibodies and detected translocation of the p127 component of UV-DDB from a loose to a tight association with chromatinized DNA immediately after UV treatment.

A similar redistribution was found for other NER proteins, i.e. XPA, RP-A and PCNA, suggesting their tighter association with genomic DNA after UV. These studies revealed a specific protein-protein interaction between UV-DDB/p127 and RP-A that appears to enhance binding of both proteins to UV-damaged DNA in vitro, providing evidence for the involvement of UV-DDB in the damage-recognition step of NER. Moreover, the kinetics of the reappearance of extractable UV-DDB activity after UV treatment of human cells with differing repair capacities positively correlate with the cell’s capacity to repair 6-4 pyrimidine dimers (6-4 PD) in the whole genome, a result consistent with an in vivo role for UV-DDB in recognizing this type of UV lesion.
Cytomechanics of neurite outgrowth from chick brain neurons
Authors: Chada Sandeep , Lamoureux Phillip , Buxbaum Robert E. , Heidemann Steven R.
Published: April 2021

Mechanical tension is a direct and immediate stimulus for neurite initiation and elongation from peripheral neurons. We report here that the relationship between tension and neurite outgrowth is equally initimate for embryonic chick forebrain neurons. Culture of forebrain neurons was unusually simple and reliable, and some of these cells undergo early events of axonal-dendritic polarity. Neurite outgrowth can be initiated de novo by experimental application of tension to the cell margin of forebrain neurons placed into culture 8-12 hours earlier, prior to spontaneous neurite outgrowth. Experimentally induced neurite elongation from these neurons shows the same robust linear relationship between elongation rate and magnitude of applied tension as peripheral neurons, i.e. both show a fluid-like growth response to tension. Although forebrain and sensory neurons manifest a similar distribution of growth sensitivity to tension (growth rate/unit tension), chick forebrain neurons initiated and elongated neurites at substantially lower net tensions than peripheral neurons. This is because, unlike peripheral neurons, there is no minimum threshold tension required for elongation in forebrain neurons; all positive tensions stimulate neurite outgrowth. Consistent with this observation, chick forebrain neurons showed weak retractile behavior in response to slackening compared to sensory neurons. Neurites that were slackened showed only transient elastic behavior and never actively produced tension, as do chick sensory neurons after slackening. We conclude that tension is an important regulator of both peripheral and central neuronal growth, but that elastic behavior is much weaker for forebrain neurons than peripheral neurons from the same developing organism. These data have significance for the understanding of the morphogenetic events of brain development.
Molecular cloning, over-expression, developmental regulation and immunolocalization of fragminP, a gelsolin-related actin-binding protein from Physarum polycephalum plasmodia
Authors: T’Jampens Davy , Meerschaert Kris , Constantin Bruno , Bailey Juliet , Cook Lynnette J. , Corte Veerle De , Mol Hans De , Goethals Mark , Damme José Van , Vandekerckhove Joël , Gettemans Jan
Published: April 2021

FragminP is a Ca2+-dependent actin-binding and micro-filament regulatory protein of the gelsolin family. We screened a Physarum polycephalum cDNA library with polyclonal fragminP antibodies and isolated a cDNA clone of 1,104 bp encoding 368 amino acids of fragminP, revealing two consensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the central part of the protein. The first methionine is modified by an acetyl group, and three amino acids were missing from the protein coded for by the cDNA clone. Full-length recombinant fragminP was generated by PCR, purified after over-expression from Escherichia coli and displayed identical properties to native Physarum fragminP. Northern blot analysis against RNA, isolated from cultures at various stages of development, indicated that fragminP is absent from amoebae and that expression is initiated at an early stage during apogamic development, in a similar way to that observed for the profilin genes. In situ immunolocalization of fragminP in Physarum microplasmodia revealed that the protein is localized predominantly at the plasma membrane, suggesting a role in the regulation of the subcortical actin meshwork. Our data indicate that we have isolated the plasmodium-specific fragminP cDNA (frgP) and suggest that, in each of its two vegetative cell types, P. polycephalum uses a different fragmin isoform that performs different functions.
Mesenchyme-mediated effects of retinoic acid during rat intestinal development
Authors: Plateroti Michelina , Freund Jean-Noël , Leberquier Cathy , Kedinger Michèle
Published: April 2021

In previous experiments we showed that intestinal development was dependent upon epithelial-mesenchymal cell interactions. The aim of this study was to investigate the possible role of retinoic acid (RA), a morphogenetic and differentiating agent, on the gut epithelial-mesenchymal unit. For this purpose we first analyzed the effects of a physiological dose of RA on 14-day fetal rat intestine using short-term organ culture experiments, or long-term grafts under the skin of nude mice. In these conditions, RA accelerated villus outgrowth and epithelial cell differentiation as assessed by the onset of lactase expression, and it also stimulated muscle and crypt formation. In order to analyze potential effects of RA mediated by mesenchymal cells, we isolated and characterized gut mucosa mesenchyme-derived cell cultures (mesenchyme-derived intestinal cell lines, MIC). These cells were shown to express mRNAs for retinoid binding proteins similar to those expressed in situ in the intestinal mesenchyme. MIC cells co-cultured with 14-day intestinal endoderms promoted endodermal cell adhesion and growth, and the addition of exogeneous RA enhanced epithelial cell polarization and differentiation assessed by cytokeratin and lactase immunostaining. Such a differentiating effect of RA was not observed on endo-dermal cells when cultured without a mesenchymal feeder layer or maintained in conditioned medium from RA-treated MIC cells. In the co-cultures, immunostaining of laminin and collagen IV with polyclonal antibodies, as well as α1 and β1 laminin chains mRNAs (analyzed by RT-PCR) increased concurrently with the RA-enhanced differentiation of epithelial cells. It is worth noting that this stimulation by RA was also obvious on the mesenchymal cells cultured alone. These results show that RA plays a role in intestinal morphogenesis and differentiation. In addition, they indicate that RA acts on the mesenchymal cell phenotype and suggest that RA may modify the mes-enchymal-epithelial cell interactions during intestinal development.
Pathogen-induced programmed cell death in tobacco
Authors: Mittler Ron , Simon Lee , Lam Eric
Published: April 2021

Sacrificing an infected cell or cells in order to prevent systemic spread of a pathogen appears to be a conserved strategy in both plants and animals. We studied some of the morphological and biochemical events that accompany programmed cell death during the hypersensitive response of tobacco plants infected with tobacco mosaic virus. Certain aspects of this cell death process appeared to be similar to those that take place during apoptosis in animal cells. These included condensation and vacuolization of the cytoplasm and cleavage of nuclear DNA to 50 kb fragments. In contrast, internucleosomal fragmentation, condensation of chromatin at the nuclear periphery and apoptotic bodies were not observed in tobacco plants during tobacco mosaic virus-induced hypersensitive response. A unique aspect of programmed cell death during the hypersensitive response of tobacco to tobacco mosaic virus involved an increase in the amount of monomeric chloroplast DNA. Morphological changes to the chloroplast and cytosol of tobacco cells and increase in monomeric chloroplast DNA occurred prior to gross changes in nuclear morphology and significant chromatin cleavage. Our findings suggest that certain aspects of programmed cell death may have been conserved during the evolution of plants and animals.
Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent protein kinase
Authors: Goretzki Lothar , Mueller Barbara M.
Published: April 2021

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/α2-macroglobulin (α2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RIα of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RIα protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RIα antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated α2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.
Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C
Authors: Chung Chang Y. , Erickson Harold P.
Published: April 2021

We have investigated the role of glycosaminoglycans in fibronectin matrix assembly and the incorporation of tenascin-C into matrix fibrils. Chinese hamster ovary cell mutants with a total block in heparan and chondroitin sulfate production failed to assemble a fibronectin matrix, and incorporated no tenascin-C. Another mutant with reduced heparan sulfate produced a normal fibronectin matrix but failed to incorporate tenascin-C. Excess soluble glycosaminoglycans inhibited the binding of tenascin-C to purified fibronectin in ELISA, and completely blocked incorporation into matrix fibrils. Treating cultured cells with xyloside, which interferes with glycosaminoglycan attachment to proteoglycans, also completely blocked their ability to incorporate tenascin-C into matrix fibrils. We conclude that proteoglycans bound to fibronectin fibrils play a major role in binding tenascin-C to these fibrils. We examined more closely the large heparan sulfate proteoglycan, perlecan, and found that it co-localizes with tenascin-C and fibronectin in the matrix. The perlecan binding site in tenascin-C was mapped to the fibronectin type III domains 3-5, but this binding was strongly enhanced for the small splice variant, which is the major form incorporated into the matrix. Apparently when the alternative splice segment is inserted after domain 5 it inhibits perlecan binding. Thus heparan sulfate glycosaminoglycans, and perlecan in particular, may play a role in incorporation of the small splice variant of tenascin-C into fibronectin matrix fibrils.
Molecular chaperones and the cytoskeleton
Authors: Liang P. , MacRae T. H.
Published: April 2021

Heat shock proteins, first observed because they are preferentially synthesized by organisms exposed to heat or other physiological stress, are also synthesized constitutively. These proteins are divided into several families, namely, HSP100, 90, 70, 60 (chaperonin), and the small heat shock/α-crystallin proteins. They enjoy a wide phylogenetic distribution and are important because they function as molecular chaperones, able to mediate many cellular processes through an influence on higher order protein structure. For example, molecular chaperones assist in the transport of proteins into mitochondria and chloroplasts, as well as influencing clathrin lattice dynamics, viral replication and transcriptional activation. Under conditions of stress, some molecular chaperones prevent denaturation of proteins while others may dissociate protein aggregates, refolding monomers derived therefrom or directing their proteolytic destruction. We present in this review an analysis of the emerging literature on the relationship between molecular chaperones and the cytoskeleton, a collection of polymeric structures consisting of microtubules, microfilaments and intermediate filaments. A recent development in this field is identification of the TCP-1 complex as the eukaryotic cytoplasmic chaperonin which directs folding of cytoskeletal proteins such as α/β/γ-tubulin, actin and centractin. Moreover, the TCP-1 complex is a centrosomal component, apparently involved in the nucleation of microtubules. Other molecular chaperones recognize one or more cytoskeletal elements and in most cases they modulate the assembly of and/or provide protection for their constituent proteins. For example, HSP70 protects the centrosome and perhaps intermediate filaments during heat shock, and like HSP90, it binds to microtubules. Small heat shock proteins interact with microfilaments and intermediate filaments, affect their polymerization and guard them from heat shock by a phosphorylation-dependent mechanism. We conclude that molecular chaperones have different but cooperative roles in the formation and function of the eukaryotic cell cytoskeleton.
Association of the γ12 subunit of G proteins with actin filaments
Authors: Ueda Hiroshi , Saga Shinsuke , Shinohara Haruo , Morishita Rika , Kato Kanefusa , Asano Tomiko
Published: April 2021

Recent studies have suggested an association between heterotrimeric G proteins, which play a major role in transmembrane signal transduction, and intracellular components. We therefore examined the subcellular localization of isoforms of G protein γ subunits in Swiss 3T3 and C6 glioma cells, mainly containing the γ5 and γ12 subunits. Immunocytochemical double staining with phalloidin showed co-localization of the γ12 subunit with actin filaments (F-actin), while the γ5 co-localized with vinculin, suggesting an association with focal adhesion. Pretreatment of cells with Triton X-100 eliminated the γ5 but not the γ12 staining. Co-localization of γ12 and F-actin was preserved when F-actin was disorganized with cytochalasin D or reorganized using fetal calf serum. Large amounts of γ12 were recovered in the vimentinand tubulin-free F-actin-rich fraction prepared from crude cytoskeleton preparations by double depolymerization-repolymerization. Co-localization of Gi2α, β and γ12 in the F-actin-rich fraction suggested the existence of γ12 as a βγ or heterotrimeric complex. Furthermore, purified βγ12 was found to associate with F-actin in vitro more tightly than βγ5. These results strongly suggest that the γ12 subunit associates with F-actin in cells. The observed differential distribution of γ12 and γ5 implies functional differences for the two γ subunits.
A programmed oxyradical burst causes hatching of mouse blastocysts
Authors: Thomas M. , Jain S. , Kumar G. P. , Laloraya M.
Published: April 2021

The emergence of the mammalian blastocysts from their thick glycoprotein investment known as the zona pellucida is an important, but poorly understood, event in embryogenesis. In this paper, we demonstrate that peri-hatching blastocysts generate a considerably high quantum of an active oxyradical species for an extremely short period of time when compared to the pre-hatching (unhatched) and post-hatching (hatched) blastocysts. Hatching could be induced in pre-hatching blastocysts by exposing them to superoxide artificially generated to match the observed peri-implantation stage specific levels of superoxide, without impairing their viability. These observations suggest the operation of a superoxide-dependent hatching initiation in developing mammalian embryos.
NNF1 is an essential yeast gene required for proper spindle orientation, nucleolar and nuclear envelope structure and mRNA export
Authors: Shan Xiaoyin , Xue Zhixiong , Euskirchen Ghia , Mélèse Teri
Published: April 2021

The nuclear envelope is central to nuclear structure and function. It plays a role in maintaining nuclear shape, allowing the exchange of macromolecules between the nucleus and the cytoplasm (via the nuclear pore complexes), and providing attachment sites for microtubules during chromosome segregation and nuclear migration (via the spindle pole body). We have isolated an essential yeast gene, NNF1 that is required for a number of nuclear functions. Cells depleted of Nnf1p or containing a temperature-sensitive nnf1 mutation have elongated microtubules and become bi- and multinucleate. They also have a fragmented nucleolous and accumulate poly(A) * RNA inside the nucleus. A similar constellation of phenotypes has been reported in cells carrying mutations in a number of nuclear pore proteins, components of the Ran GTPase cycle, and the nuclear localization sequence receptor protein. Our results suggest that Nnf1p plays a role in a number of nuclear functions.
Arachidonic acid stimulates protein kinase C-ε redistribution in heart cells
Authors: Huang Xu Pei , Pi YeQing , Lokuta Andrew J. , Greaser Marion L. , Walker Jeffery W.
Published: April 2021

Arachidonic acid is elevated in a variety of cell types in response to extracellular stimuli, and has been hypothesized to exert at least some of its intracellular actions via activation of protein kinase C. Here we show that arachidonic acid stimulates a unique pattern of translocation of the μ-isoform of protein kinase C in isolated adult rat cardiac myocytes. Using western blot analysis, the majority of ε-protein kinase C was found in a cytosolic fraction in unstimulated cells. Treatment with 50 μM arachidonic acid caused a transient increase of δ-protein kinase C in a membrane fraction within 1 minute, then after 5-20 minutes most was found in a filament/nuclear fraction. Immunofluorescence and confocal microscopy of the filament fraction revealed a striated staining pattern with ε-protein kinase C localized near the Z-line where actin filaments are anchored and where transverse tubules are closely apposed to the myofilaments. δ-Protein kinase C, another isoform highly expressed in these cells, did not redistribute significantly in response to arachidonic acid, but in response to phorbol ester displayed a predominantly nuclear localization. Arachidonic acid also stimulated phosphorylation of the thin filament protein, troponin I, consistent with a filament localization for activated PKC. The physiological relevance of these findings was supported by the observation that 50 μM arachidonic acid promoted a 2.3-fold enhancement of myocyte twitch amplitude, an effect that was significantly blocked by the protein kinase C antagonist chelerythrine. Moreover, the onset of this physiological response correlated in time with translocation of ε-protein kinase C to the filaments. The results suggest that arachidonic acid initiates a redistribution of ε-protein kinase C to myofilament structures at or near the Z-line where this isozyme would be strategically located to regulate myofilament function and excitation-contraction coupling.
Cytosolic phospholipase A2 is activated by the hepatocyte growth factor receptor-kinase in Madin Darby canine kidney cells
Authors: Skouteris George G. , Schröder Claus H.
Published: April 2021

The hepatocyte growth factor/scatter factor (HGF/SF) receptor which is a transmembrane protein encoded by the Met oncogene, possesses intrinsic tyrosine kinase activity which transduces the mitogenic, morphogenic and the scattering effect of HGF/SF. The pluripotent signal of HGF/SF is transduced through association of the Met receptor with various intracellular adaptors. Phosphorylation of cytosolic phospholipase A2 (cPLA2) is associated with activation of this molecule which in turn leads to arachidonic acid production followed by release of prostaglandins and related compounds exerting their roles onto cell proliferation, chemotaxis and vascular motility. Arachidonic acid and its metabolites were shown to be involved in processes like liver regeneration where growth factor receptors possessing tyrosine kinase activity are implicated. In this study we examined whether stimulation of the HGF/SF-receptor’s tyrosine kinase activity would involve changes in the phosphorylation state and the activity of cPLA2 in MDCK cells, where HGF/SF is known to induce scattering responses rather than mitogenesis. The activated p145βMET was shown to associate with and to phosphorylate cPLA2on tyrosine residues, this leading to subsequent release of arachidonic acid. cPLA2 was also phosphorylated in serine residues and such a role has been so far assigned to the mitogen activated protein (MAP) kinase. Our data have also shown that MAP kinase is associated and phosphorylated on tyrosine by the activated p145βMET Immunodepletion of MAP kinase via electroporation of an anti-MAP kinase antibody, did not significantly decrease arachidonic acid release in HGF/SF-stimulated MDCK cells. It is therefore emerging that phosphorylation of cPLA2 on tyrosine by the HGF/SF receptor kinase is capable of triggering arachidonic acid release and that MAP kinase is contributing to full, but does not drive, the activity of cPLA2. The release of arachidonic acid by MDCK cells following HGF/SF stimulation is establishing this fatty acid and its metabolites as major components involved in the transduction of MET-driven signals and at the same time in the amplification of such signals.
A specific domain in α-catenin mediates binding to β-catenin or plakoglobin
Authors: Huber Otmar , Krohn Michael , Kemler Rolf
Published: April 2021

The E-cadherin-catenin adhesion complex has been the subject of many structural and functional studies because of its importance in development, normal tissue function and carcinogenesis. It is well established that the cytoplasmic domain of E-cadherin binds either β-catenin or plakoglobin, which both can assemble α-catenin into the complex. Recently we have identified an α-catenin binding site in β-catenin and plakoglobin and postulated, based on sequence analysis, that these protein-protein interactions are mediated by a hydrophobic interaction mechanism. Here we have now identified the reciprocal complementary binding site in α-catenin which mediates its interaction with β-catenin and plakoglobin. Using in vitro association assays with C-terminal truncations of α-catenin expressed as recombinant fusion proteins, we found that the Nterminal 146 amino acids are required for this interaction. We then identified a peptide of 27 amino acids within this sequence (amino acid positions 117-143) which is necessary and sufficient to bind β-catenin or plakoglobin. As shown by mutational analysis, hydrophobic amino acids within this binding site are important for the interaction. The results described here, together with our previous work, give strong support for the idea that these proteins associate by hydrophobic interactions of two α-helices.
Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II
Authors: Grande Marjolein A. , Kraan Ineke van der , Jong Luitzen de , Driel Roel van
Published: April 2021

We have investigated the spatial relationship between sites containing newly synthesized RNA and domains containing proteins involved in transcription, such as RNA polymerase II and the transcription factors TFIIH, Oct1, BRG1, E2F-1 and glucocorticoid receptors, using dual immunofluorescence labelling followed by confocal microscopy on cultured cells. As expected, a high degree of colocalisation between the RNA polymerase II and sites containing newly synthesised RNA was observed. Like the newly synthesised RNA and the RNA polymerase II, we found that all the transcription factors that we studied are distributed more or less homogenously throughout the nucleoplasm, occupying numerous small domains. In addition to these small domains, TFIIH was found concentrated in coiled bodies and Oct1 in a single large domain of about 1.5 μm in 30% of the cells in an asynchronous HeLa cell culture. Remarkably, we found little or no relationship between the spatial distribution of the glucocorticoid receptor, Oct1 and E2F-1 on the one hand and RNA polymerase II and transcription sites on the other hand. In contrast, a significant but incomplete overlap was observed between the spatial distributions of transcription sites and BRG1 and TFIIH. These results indicate that many of the transcription factor-rich nuclear domains are not actively involved in transcription. They may represent incomplete transcription initiation complexes, inhibitory complexes, or storage sites.
Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast
Authors: Yamada Hiroshi , Kumada Kazuki , Yanagida Mitsuhiro
Published: April 2021

We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction. The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa. The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast. The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase. Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase. The truncated forms of Cut9 block entry into mitosis, however. The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcn1, similar to budding yeast Cdc26. Furthermore, deletion of protein kinase PKA (Pka1) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9. In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9. The Cut9 subunit is likely to be a target for regulating APC/cyclosome function through protein-protein interactions and phosphorylation.
Transient increase of free cytosolic calcium during neutrophil motility responses
Authors: Alteraifi Abdullatif M. , Zhelev Doncho V.
Published: April 2021

The release of free cytosolic calcium is a secondary messenger for many cell functions. Here we study the coupling between the release of intracellular calcium and motility responses of the human neutrophil. Two groups of motility responses are studied: motility responses in the presence of adhesion, such as cell crawling and phagocytosis, and motility responses ‘in suspension’, such as pseudopod formation. The motility responses are stimulated by the chemoattractant N-formyl-methionyl-leucylphenylalanine (fMLP) and the release of calcium is monitored by measuring the fluorescence from fluo-3. fMLP induces a single release of free cytosolic calcium both in suspended cells and in crawling cells. Calcium release is a threshold process where the number of cells releasing calcium is dependent on the chemoattractant concentration while the amount of released calcium is not. For suspended cells the threshold fMLP concentration for calcium release is in the order of 10−7 M, while for crawling cells it is in the order of 5×10−9 M. The smaller value of the threshold fMLP concentration for crawling cells compared to that for suspended cells suggests that bound adhesion receptors are involved in the calcium release. The threshold fMLP concentration for suspended cells is also larger than the minimum fMLP concentration (in the order of 10−10 M) for initiating pseudopod formation. So, there is a range of fMLP concentrations where pseudopod formation occurs without calcium release. To explore this relationship further, pseudopod extension and calcium release are stimulated many times in a single cell by using fMLP concentrations above the threshold. The result is that calcium release is desensitized by fMLP while pseudopod extension is not. All the results taken together suggest that the release of free cytosolic calcium and the rearrangement of the F-actin network during motility follow different signaling pathways.
Cell-specific expression and heat-shock induction of Hsps during spermatogenesis in Drosophila melanogaster
Authors: Michaud Sébastien , Marin Raquel , Westwood J. Timothy , Tanguay Robert M.
Published: April 2021

The developmental and heat-shock-induced expression of two small heat-shock proteins (Hsp23 and Hsp27) was investigated during spermatogenesis in Drosophila melanogaster. Both of these Hsps were expressed in unstressed and stressed male gonads as shown by immunoblotting. Immunostaining of whole-mount organs and thin sections of testes showed that an anti-Hsp23 antibody specifically decorated cells of the somatic lineage, such as the cyst cells and the epithelial cells of the testis and of the seminal vesicle. Hsp27 was expressed in some somatic cells (cyst cells and epithelial cells of the accessory glands) and, in addition, was also visible in the maturing spermatocytes of the germline. The same cell-specific pattern of expression was observed after heat shock, and cells which did not express Hsp23 and Hsp27 in the absence of stress were similarly unable to mount a heat shock response for these s-Hsps. However other Hsps such as Hsp70 and Hsp22 were induced under heat-shock conditions in testes. Actinomycin D prevented the heat-induced accumulation of these Hsps indicating that the induction of Hsps was regulated at the transcriptional level. The heat shock transcriptional factor of Drosophila (DmHSF), present in significantly lower amount in testes when compared to other tissues such as the head, was shown to be required for the heat activation of Hsp22 and Hsp70. Immunostaining revealed that HSF expression was restricted to specific cells such as cyst cells, epithelial pigment cells, spermatogonia and spermatids but not the primary spermatocytes. These data show that the expression and induction of the different small Hsps is regulated in a cell-specific manner under both normal and heat shock conditions and suggest that factors other than the DmHSF are involved in this regulation in male gonads.