bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment
Authors: Jahraus Andrea , Storrie Brian , Griffiths Gareth , Desjardins Michel
Published: April 2021

We have investigated the interactions occuring between the prelysosomal compartment, PLC/late endosome, and terminal lysosomes using an approach that allowed us to internalize and deliver specific tracers to these compartments, and look for evidence of their meeting. After internalization of sucrose, the lysosomes (sucrosomes), but not the PLC/late endosomes, became significantly swollen and visible in the light microscope. If invertase is then added to the medium it reaches the lysosomes where it cleaves sucrose into fructose and glucose. These sugars, unlike sucrose, can be transported into the cytosol, resulting in the disappearance of the sucrosomes. We previously showed that phagocytosed latex beads are delivered specifically to, and reside in, the PLC/late endosome, a stage earlier than the lysosomes in the endocytic pathway (Rabinowitz et al. (1992)J. Cell Biol. 116, 95-112). In the present study, we demonstrate that invertase conjugated to the latex beads, and thus immobilized in late endosomes, has access to the sucrose present in the more distal lysosomes. Experiments using nocodazole indicate that this retrograde fusion event requires the presence of an intact microtubule network. The simplest interpretation of our results is that the two compartments fuse, allowing for a retrograde transport of sucrose from the lysosomes to the PLC/late endosomes.
Merosin is synthesized by thyroid cells in primary culture irrespective of cellular organization
Authors: Andre Frédéric , Filippi Pierre , Feracci Hélène
Published: April 2021

The in vitro synthesis and deposition of laminin family gly-coproteins were studied using primary porcine thyroid cells cultured as monolayers or in follicles. The latter organization mimics the in vivo state of these polarized epithelial cells. In both cell systems a trimeric molecule was immunoprecipitated by using polyclonal antibodies against EHS-laminin. When the cells were fully polarized the protein was found at the basal pole of cells, irrespective of their organization. However, this molecule was different from laminin purified from a traditional source, the murine Engelbreth-Holm-Swarm (EHS) tumor. Thyroid cell laminin was composed of two light chains, analogous to EHS B1 and B2, and a disulfide-bonded heavy chain not found in EHS-laminin. The heavy chain was first synthe-sized as a 380 kDa polypeptide, then rapidly cleaved to a doublet of 350-380 kDa, which was subsequently found in both cell extracts and conditioned culture media. This thyroid laminin variant was compared with merosin, another variant found in the basement membranes of tro-phoblast, Schwann cells, striated muscle and liver. The heavy chain (M) of merosin shows homology to EHS-laminin heavy chain at the C-terminal domain, and is usually found as two polypeptides of 80 kDa and 300 kDa (Ehrig K., Leivo I., Argraves W. S., Ruoslahti E. and Engvall E. Proc. Nat. Acad. Sci. 87, 3264-3268, 1990). mRNA of the M chain was identified by RT-PCR in freshly isolated thyrocytes as well as in 6-day-old cultured thyroid cells. Furthermore, both the classical laminin heavy chain and the 350 kDa variant were detected by immunoblotting and immunofluorescence in the thyroid gland in vivo. All these results suggest strongly that merosin is a basement membrane component of thyroid cells in vivo and in vitro.
An amino-terminal domain of the growth-associated protein gap-43 mediates its effects on filopodial formation and cell spreading
Authors: Strittmatter Stephen M. , Valenzuela Dario , Fishman Mark C.
Published: April 2021

GAP-43 is a neuronal protein that is believed to be important to neuronal growth and nerve terminal plastic-ity. It is enriched on the inner surface of growth cone membranes, a localization that may depend upon palmi-toylation of Cys3 and Cys4. It is a major substrate for protein kinase C, which phosphorylates Ser41. Isolated GAP-43 can bind to actin and to calmodulin, and can activate the heterotrimeric GTP-binding proteins, Go and Gi. A peptide consisting of the GAP-43 sequence 39-55 binds calmodulin, and an amino-terminal GAP-43 (1-10) peptide activates Go, suggesting that these stretches may be functional domains of the intact protein.

When expressed in non-neuronal cells, GAP-43 enhances filopodial extension and has effects upon cell spreading. We have examined the effects of various GAP-43 domains upon this assay, by expression of GAP-43, GAP-43 mutant proteins, and GAP-43-CAT fusion proteins in COS-7 cells. We find that the amino terminus (Met-Leu-Cys-Cys-Met-Arg-Arg-Thr-Lys-Gln) is an important contributor to these effects on cell shape. A GAP-43 protein mutant in Cys3 and Cys4 does not bind to the membrane, and is inactive. Mutants in Arg6 or Lys9 also are inactive, although they remain localized to particulate fractions; Arg7 mutants are active. A chimeric gene consisting of GAP-43 (1-10) fused to chloramphenicol acetyl transferase (CAT) also causes cell shape changes. As for GAP-43, the effects of this fusion protein are abolished by mutations of Cys3, Cys4, Arg6 or Lys9, but not by mutation of Arg7. Therefore, the cell surface activity of transfected GAP-43 depends upon its amino terminus, although other domains may regulate it in this regard.

Since the amino-terminal domain includes the peptide stretch known to be capable of activating Go and Gi, we examined the effect of GAP-43 on a Gi-regulated second messenger system, the inhibition of cAMP production in A431 cells. A431 cells stably transfected with GAP-43 spread less well than do controls. In addition, they evidence decreased levels of forskolin-stimulated cAMP, consistent with chronic stimulation of Gi. Stimulation of adenylate cyclase by isoproterenol reverses the GAP-43-induced changes in cell shape. This suggests that G protein stimu-lation is involved in GAP-43 effects upon cell shape.
A human nuclear protein with sequence homology to a family of early s phase proteins is required for entry into s phase and for cell division
Authors: Todorov Ivan T. , Pepperkok Rainer , Philipova Radka N. , Kearsey Stephen E. , Ansorge Wilhelm , Werner Dieter
Published: April 2021

Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccha-romyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2.

Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bro-modeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.
Histone h1 kinase activity, germinal vesicle breakdown and m phase entry in mouse oocytes
Authors: Gavin Anne-Claude , Cavadore Jean-Claude , Schorderet-Slatkine Sabine
Published: April 2021

Meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphase I, beginning with germinal vesicle breakdown and ending with spindle formation. It is accompanied by a cascade of protein kinases and phos-phatases increasing protein phosphorylation. The activa-tion of histone H1 kinase and that of the mitogen-activated protein kinase p42 have been compared during sponta-neous or okadaic acid-induced meiotic reinitiation.

In spontaneously maturing oocytes, histone H1 kinase activity increases before germinal vesicle breakdown (2-fold), in a protein synthesis-independent manner. It is asso-ciated with the disappearance of the upper migrating form of p34cdc2, which, in our system, seems to represent the tyrosine phosphorylated form. Following germinal vesicle breakdown, histone H1 kinase activity culminates (8-fold) in metaphase I and requires protein synthesis. Activation by phosphorylation of p42MAPK is observed as a permanent shift upward-migrating form and by its myelin basic protein kinase activity. It occurs after germinal vesicle breakdown and depends on protein synthesis.

In contrast, no increase of histone H1 kinase is detectable in oocytes induced to reinitiate meiosis by a transient inhi-bition of okadaic acid-sensitive phosphatase(s), either before germinal vesicle breakdown or during the following 7 hours of culture. A slight increase is nevertheless evident after 17 hours, when oocytes are arrested with an abnormal metaphase I spindle. The upper migrating form of p34cdc2 is present for 8 hours. The activation of p42MAPK begins before germinal vesicle breakdown. Furthermore, when okadaic acid is applied to oocytes that have had meiosis reinitiation prevented by p13suc1 microinjection, neither increase of histone H1 kinase activity nor p34cdc2 dephos-phorylation is observed although germinal vesicle breakdown is induced; p42MAPK is activated.

Altogether these results suggest that germinal vesicle breakdown may or may not be associated with a detectable activation of histone H1 kinase, depending on the experi-mental conditions. Activation of p34cdc2 and p42MAPK are separable events. The effect of okadaic acid on mouse oocyte maturation is positive and negative. It induces meiotic events (germinal vesicle breakdown) independently of histone H1 kinase activation, perhaps through premature activation of p42MAPK. It interferes negatively with the meiotic post-germinal vesicle breakdown events, most probably through mechanisms inhibiting protein synthesis and preventing cell cycle progression. We propose that, in the mouse oocyte, okadaic acid might be able to activate an alternative pathway leading to germinal vesicle breakdown that is cdc2-independent and that involves p42MAPK activation ensuing maturation promoting factor-independent phosphorylations.
Characterization of the novel brown adipocyte cell line hib 1b adrenergic pathways involved in regulation of uncoupling protein gene expression
Authors: Klaus Susanne , Choy Lisa , Champigny Odette , Cassard-Doulcier Anne-Marie , Ross Susan , Spiegelman Bruce , Ricquier Daniel
Published: April 2021

The HIB 1B cell line, derived from a brown fat tumor of a transgenic mouse, is the first established brown adipocyte cell line capable of expressing the brown fat-specific mitochondrial uncoupling protein (UCP). UCP gene expression, which was virtually undetectable under basic conditions, was stimulated by acute catecholamine or cyclic AMP treatment to levels comparable to primary cultures of brown adipocytes. Elevation of UCP mRNA levels following stimulation was very rapid but transient, decreasing after about 4 hours with a half-life between 9 and 13 hours. Immunoblotting showed the presence of UCP in HIB 1B mitochondria, but expression was much lower than observed in BAT or primary cultures of brown adipocytes. Upon transfection of HIB 1B cells with a reporter gene containing the UCP promoter, the activity of the transgene was regulatable by cAMP and norepineph-rine. Investigation of the possible adrenergic receptors involved in UCP stimulation showed that specific β3-adren-ergic agonists were much less effective than nonspecific β-adrenergic agonists and that mRNA levels of the atypical, fat-specific β3-adrenoceptor were lower than those observed in brown adipocytes differentiated in primary culture. From pharmacological evidence we conclude that β3-adrenergic receptors account for approximately 30-40% of catecholamine induced UCP gene stimulation, whereas about 60-70% is stimulated via the classical β1/2 adrenergic pathway. We conclude that HIB 1B cells represent a functional system for the study of mechanisms related to brown adipose thermogenesis.
Protein binding and cell adhesion properties of two laminin isoforms (amb1eb2e, amb1sb2e) from human placenta
Authors: Brown Judith C. , Wiedemann Hanna , Timpl Rupert
Published: April 2021

Two isoforms of laminin were extracted from human placenta by neutral buffer containing EDTA, copurified through several steps and finally separated by Mono Q anion exchange chromatography. One variant consisted of disulphide-linked 340, 230 and 190 kDa subunits, which were identified by immunoblotting as Am, B1e and B2e chains. In the other variant, the B1e chain was replaced by B1s of 180 kDa. After rotary shadowing, both variants showed a similar cross-shaped structure. The nidogen content of these laminins was substoichiometric and variable (3-70%), indicating loss by endogenous proteolysis. Yet both human isoforms were able to bind mouse nidogen with an affinity (Kd∼0.5 nM) comparable to that of AeB1eB2e laminin from a mouse tumour. Since the binding site is known to be contributed by a single EGF-like motif of the B2e chain, this demonstrates that activity of this site is independent of chain assembly. Binding activity of both isoforms to collagen IV and the heparan sulphate proteo-glycan perlecan was correlated to the nidogen content and could be enhanced by adding nidogen. Binding to heparin was only partial and heparin did not inhibit perlecan binding. This indicated a crucial role for nidogen in mediating the integration of these laminin isoforms into basement membranes. Variant AmB1sB2e showed calcium-dependent binding to fibulin-1, while only a little activity was found for AmB1eB2e. Both isoforms promoted adhesion and spreading of several cell lines. Adhesion could be completely inhibited by antibodies to the integrin β1 subunit but not, or only weakly, by antibodies against β3,α2, α3, α5 and α6 subunits. No inhibition was observed with an Arg-Gly-Asp-containing peptide.
Characterisation of articular and growth plate cartilage collagens in porcine osteochondrosis
Authors: Wardale R. John , Duance Victor C.
Published: April 2021

The articular and growth plate cartilages of osteochon-drotic pigs were examined and compared with those from clinically normal animals. Both types of osteochondrotic cartilage showed considerable localised thickening appar-ently due to a lack of ossification. Histological examination of cartilage lesions demonstrated a breakdown in the normal pattern of chondrocyte maturation. Articular cartilage lesions lacked mature clones of chondrocytes in the calcifying region. Growth plate cartilage showed an accumulation of disorganised hypertrophic chondrocytes rather than the well-defined columns seen in normal tissue.

The overall percentages of collagen in osteochondrotic lesions from both articular and growth plate cartilage were significantly reduced compared with levels in unaffected cartilage. There were substantial increases in the propor-tion of type I collagen in lesions from both osteochondrotic articular and growth plate cartilages and a reduction in the proportion of type II collagen. Type X collagen was detected in osteochondrotic but not normal articular cartilage. The proportion of type X collagen was unchanged in osteochondrotic growth plate cartilage.

The levels of the collagen cross-links, hydroxylysyl-pyridinoline, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine were radically reduced in samples from osteochondrotic growth-plate cartilage lesions when compared with normal tissue. Less dramatic changes were observed in articular cartilage although there was a significant decrease in the level of hydroxylysyl-ketonorleucine in osteochondrotic lesions.

Immunofluorescence examination of osteochondrotic lesions showed a considerable disruption of the organisa-tion of the collagenous components within both articular and growth-plate cartilages. Normal patterns of staining of types I and VI collagen seen at the articular surface in unaf-fected tissue were replaced by a disorganised, uneven stain in osteochondrotic articular cartilage lesions. Incomplete removal of cartilage at the ossification front of osteochon-drotic growth plate was demonstrated by immunofluores-cence staining of type IX collagen. Type X collagen was produced in the matrix of the calcifying region of osteo-chondrotic articular cartilage by small groups of hyper-trophic chondrocytes, but was not detected in normal articular cartilage. The distribution of type X collagen was unchanged in osteochondrotic growth plate cartilage.
A gradient method for the quantitative analysis of cell movement and tissue flow and its application to the analysis of multicellular Dictyostelium development
Authors: Siegert Florian , Weijer Cornelis J. , Nomura Atsushi , Miike Hidetoshi
Published: April 2021

We describe the application of a novel image processing method, which allows quantitative analysis of cell and tissue movement in a series of digitized video images. The result is a vector velocity field showing average direction and velocity of movement for every pixel in the frame. We apply this method to the analysis of cell movement during different stages of the Dictyostelium developmental cycle. We analysed time-lapse video recordings of cell movement in single cells, mounds and slugs. The program can correctly assess the speed and direction of movement of either unlabelled or labelled cells in a time series of video images depending on the illumination conditions.

Our analysis of cell movement during multicellar devel- opment shows that the entire morphogenesis of Dic- tyostelium is characterized by rotational cell movement. The analysis of cell and tissue movement by the velocity field method should be applicable to the analysis of mor- phogenetic processes in other systems such as gastrulation and neurulation in vertebrate embryos.
Retention and retrieval: both mechanisms cooperate to maintain calreticulin in the endoplasmic reticulum
Authors: Sönnichsen Birte , Füllekrug Joachim , Van Phuc Nguyen , Diekmann Wilfried , Robinson David G. , Mieskes Gottfried
Published: April 2021

Many soluble resident proteins of the endoplasmic reticulum share a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Current opinion favours a model in which these proteins can escape from the endoplasmic reticulum (ER) by bulk flow and are recognized and sorted in the Golgi apparatus by binding to a specific KDEL- receptor, which returns them to the ER. Through bio- chemical, morphological and mutational analysis we have studied the mechanisms that determine the localization of calreticulin, a soluble 60 kDa KDEL-protein of the ER. Immunogold labelling established the ER localization of calreticulin in transfected and nontransfected COS cells. Although the ER cisternae in transfected cells were enormously dilated and heavily labelled by gold particles we found no significant label in any other compartment. In vivo pulse chase experiments with [35S]methionine followed by biochemical fractionation of calreticulin overexpressing COS cells (50- to 100-fold) revealed that only a minor part of labelled calreticulin leaves the ER. Retrieval from the Golgi was confirmed by a partial redistribution of the endogenous KDEL-receptor as shown by double immuno- fluorescence. These data suggest a KDEL-independent retention of calreticulin in the ER. Further supporting evidence has come from morphological in vivo studies using calreticulin-transfected and vesicular stomatitis virus (ts045)-infected COS cells. Stimulation of vesicular transport from the ER by releasing the temperature- dependent transport block for the viral G-protein resulted in a small but significant appearance of calreticulin in a post-ER compartment. In contrast a calreticulin mutant, which lacked the Ca2+-binding domain but included the KDEL sequence, could escape from the ER to a much higher extent. Secretion of the nonmutated calreticulin was very low (1-2% of total calreticulin in 3 hours) compared to the mutated form (18% in 3 hours). Deletion of the KDEL sequence led to an increase in secretion to 29% over a 3 hour period, which is much less than expected for a secretory protein. Taken together these results strongly support the hypothesis of two independently operating retention/retrieval mechanisms for calreticulin: one providing for direct retention in the ER with a very high capacity and having Ca2+-dependent properties; the other a KDEL-based retrieval system for escaped calreticulin present in the Golgi apparatus.
Ultrastructure of the proteoliaisin-ovoperoxidase complex and its spatial organization within the Strongylocentrotus purpuratus fertilization envelope
Authors: Mozingo Nancy M. , Somers Cynthia E. , Chandler Douglas E.
Published: April 2021

Ovoperoxidase is a cortical granule-derived enzyme that hardens the sea urchin fertilization envelope by catalyzing the formation of dityrosine residues. Ovoperoxidase works in concert with a second protein, proteoliaisin, which anchors ovoperoxidase to the nascent fertilization envelope in a divalent cation-dependent manner. In this study, we examined the Ca2+-dependent interaction of proteoliaisin with ovoperoxidase in rotary-shadowed Pt replicas. Ovoperoxidase, a uniformly sized globular molecule, binds to a distal portion of rod-shaped proteoliaisin when low concentrations of Ca2+ are present. Higher Ca2+ concentrations lead to the formation of extended proteoliaisin strands that are decorated along their lengths with ovoper-oxidase. Using immunogold labeling, we also examined the assimilation of these two proteins into the fertilization envelope in quick-frozen, deeply etched samples. Both proteins are abundant in the fertilization envelope as early as one minute after fertilization. Coincident with paracrystalline coating of the envelope, the labeling density is markedly reduced, suggesting that antigenic sites may be masked by the paracrystalline coat. This suggests that the ovoperoxidase-proteoliaisin complex resides within the central portion of the fertilization envelope, rather than in the paracrystalline coat.
The fission yeast cdc19+ gene encodes a member of the MCM family of replication proteins
Authors: Forsburg Susan L. , Nurse Paul
Published: April 2021

We have cloned and characterized the fission yeast cdc19+ gene. We demonstrate that it encodes a structural homologue of the budding yeast MCM2 protein. In fission yeast, the cdc19+ gene is constitutively expressed, and essential for viability. Deletion delays progression through S phase, and cells arrest in the first cycle with an apparent 2C DNA content, with their checkpoint control intact. The temperature-sensitive cdc19-P1 mutation is synthetically lethal with cdc21-M68. In addition, we show by classical and molecular genetics that cdc19+ is allelic to the nda1+ locus. We conclude that cdc19p plays a potentially conserved role in S phase.
Dinoflagellates have a eukaryotic nuclear matrix with lamin-like proteins and topoisomerase II
Authors: Mínguez Ana , Franca Susana , Díaz de la Espina Susana Moreno
Published: April 2021

Unicellular Dinoflagellates represent the only eukaryotic Phylum lacking histones and nucleosomes. To investigate whether Dinoflagellates do have a nuclear matrix that would modulate the supramolecular organization of their non-nucleosomal DNA and chromosomes, cells of the free- living unarmored Dinoflagellate Amphidinium carterae were encapsulated in agarose microbeads and submitted to sequential extraction with non-ionic detergents, nucleases and 2 M NaCl. Our results demonstrate that this species has a residual nuclear matrix similar to that of vertebrates and higher plants. The cytoskeleton-nuclear matrix complex of A. carterae shows a relatively intricate polypeptide pattern. Immunoblots with different antibodies reveal several intermediate filament types of proteins, one of which is immunologically related to vertebrate lamins, confirming that these proteins are ancestral members of the IF family, which is highly conserved in eukaryotes. A topoi- somerase II homologue has also been identified in the nuclear matrix, suggesting that these structures could play a role in organizing the Dinoflagellate DNA in loop domains. Taken together our results demonstrate that the nuclear matrix is an early acquisition of the eukaryotic nucleus, independent of histones and nucleosomes in such a way that the mechanisms controlling the two levels of organization in eukaryotic chromatin would be molecularly and evolutionarily independent.
The effects of the neuN and neuT genes on differentiation and transformation of mammary epithelial cells
Authors: Lucassen E. , Andres A.-C. , Reichmann E. , Entwistle A. , Noble M.
Published: April 2021

Overexpression of the proto-oncogene product, p185neuN, in a non-tumorigenic mammary epithelial line (31E) facilitates aspects of lactogenic differentiation. Formation of branching cords and induction of β-casein synthesis by 31E cells normally require co-culture of these cells with fibroblasts, or the presence of collagen or fibronectin. In contrast, 31E cells expressing p185neuN spontaneously form branching cords when grown on tissue culture plastic and can synthesize β-casein in the absence of exogenous substrates or feeder layers. Under these conditions, the cells deposit laminin and fibronectin, indicating a possible role for p185neuN in the deposition of extracellular matrix proteins. Overexpression of the corresponding oncogene product, p185neuT, has markedly different effects. Expression of p185neuT does not facilitate the formation of branching cords or the synthesis of β-casein when grown on tissue culture plastic, although these cells do deposit laminin and fibronectin. Confocal microscopy indicates a significant difference in the distribu-tion of laminin and fibronectin in 31E cells expressing p185neuT compared to those expressing p185neuN. The effects of p185neuN and p185neuT expression on cell transformation depend on cell type. Expression of both p185neuN and p185neuT increases anchorage-independent growth of 31E cells, but only p185neuT induces anchorage-independent growth of NIH 3T3 fibroblasts. This lineage specificity in the action of p185neuN may be related to obser-vations that overexpression of p185c-erbB-2 (the human homologue of p185neuN) is only associated with the devel-opment of human epithelial cancers. The effects of p185neuN on laminin deposition by 31E cells may be relevant to the transforming ability of p185neuN, since laminin can induce anchorage-independent growth of mouse mammary cells. These results suggest that p185neuN and p185neuT could exert their effects on differentiation and transformation of mammary epithelial cells in part by promoting the deposition of extracellular matrix proteins.
Function of the syndecans - a family of cell surface proteoglycans
Authors: Elenius Klaus , Jalkanen Markku
Published: April 2021

Cell surface proteoglycans are membrane-anchored glycoproteins that contain covalently linked glycosaminoglycan sidechains. They interact via their extracellular part with various effector molecules such as growth factors, extracellular matrix components, other cell surface molecules and proteins involved in the regulation of blood coagulation. This together with numerous examples of strictly regulated expression patterns during embryogenesis and malignant transformation has lead to the attribution of several specific functions to cell surface proteoglycans. Molecular biology techniques that have made it possible to identify and characterize individual proteoglycans, may also turn out to be crucial in determining the relevance of the several suggested functions in vivo. This review will mainly focus on the functional aspects of one well-characterized group of cell surface proteoglycans, the members of the syndecan gene family.
The pericentriolar lattice of PtK2 cells exhibits temperature and calcium modulated behavior
Authors: Baron Andre T. , Suman Vera J. , Nemeth Edward , Salisbury Jeffrey L.
Published: April 2021

In this study, we demonstrate that manipulations of temperature and free calcium alter the morphology of the centrin-containing pericentriolar lattice of PtK2 cells. Immunofluorescence microscopy reveals that low-temperature incubation (4°C) causes anti-centrin-labeled pericentrosomal spots to coalesce in the peripheral cytoplasm, and fuses small spots into larger spots near the cell center. At electron microscopic resolution, well-formed pericentriolar satellites appear around the centrioles in response to incubation at 4°C. Elevated free calcium enhances these low-temperature-dependent effects. The data suggest that peri-centrosomal spots correspond to one or more pericentriolar satellites, and that pericentriolar satellites and centrosomal matrix are interconvertable forms of the same material. Transient elevation of intracellular free calcium at 37°C from a basal level of 3.7×10−8 M to a peak level of 2.0×10−7 M within 30 seconds with ionomycin results in a 35% increase in pericentrosomal spot number throughout the cytoplasm. The number of pericentrosomal spots is 50% larger 2 minutes after ionomycin addition; these spots are also nearer to the cell center as compared to 30 seconds after ionomycin addition. As intracellular free calcium returns to a basal level over 5 minutes, the number of spots and their cellular distribution resume a pretreatment value and pattern. We interpret these observations to indicate movement of pericentrosomal spots toward the cell center in response to the flux in intracellular free calcium. Alternatively, it is possible that no movement has occurred, but that the rise in free calcium has unmasked an epitope responsive to our anti-centrin antiserum. Regardless of the interpretation, we conclude that the pericentriolar lattice exhibits calcium-modulated behavior.
Evidence that kinetochore microtubules in crane-fly spermatocytes disassemble during anaphase primarily at the poleward end
Authors: Wilson Paula J. , Forer Arthur , Leggiadro Cindy
Published: April 2021

Anaphase chromosome motion involves the disassembly of kinetochore microtubules. We wished to determine the site of kinetochore microtubule disassembly during anaphase in crane-fly spermatocytes.

In crane-fly spermatocyte spindles, monoclonal antibody 6-11B-1 to acetylated α-tubulin labels kinetochore microtubules almost exclusively, with an area immediately adjacent to the kinetochore being weakly or not labelled. This ‘gap’ in acetylation at the kinetochore serves as a natural marker of kinetochore microtubules in the kinetochore fibre. We measured the length of the gap on kinetochore fibres in metaphase and anaphase in order to deduce the fate of the gap during anaphase; we used this information to determine where kinetochore microtubules disassemble in anaphase. Gap lengths were measured from confocal microscope images of fixed spermatocytes dual labelled with 6-11B-1 to acetylated α-tubulin and YL1/2 to tyrosinated α-tubulin, the latter being used to determine the positions of kinetochores.

In metaphase the average gap length was 1.7 µm. In anaphase, the gap appeared to decrease in length abruptly by about 0.4 µm, after which it decreased in length by about 0.2 µm for every 1 µm that the chromosome moved poleward. PacMan models of chromosome movement predict that this ‘gap’ in staining should disappear in anaphase at a rate equal to that of chromosome movement. Thus, our results do not support theories of chromosome motion that require disassembly solely at the kinetochore; rather, in crane-fly spermatocytes kinetochore microtubule disassembly in anaphase seems to take place primarily at the poles.
The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts
Authors: Hill Peter A. , Murphy Gillian , Docherty Andrew J. P. , Hembry Rosalind M. , Millican T. Andrew , Reynolds John J. , Meikle Murray C.
Published: April 2021

We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki<20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants cocultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices.

Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10—8 M) selective for the inhibition of human gelatinases A and B. CT435 (10−5 M) and CT543 (10−5 M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1α (IL-1α; 10−10 M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1α-stimulated cultures, with maximal action at 10−5 M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1α-stimulated secretion of the lysosomal enzyme β-glu-curonidase.

Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1α stimulation.

These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are released into the sub-osteoclastic resorption zone where they participate in bone collagen degradation.
Replicon clusters may form structurally stable complexes of chromatin and chromosomes
Authors: Sparvoli Elio , Levi Marisa , Rossi Emanuele
Published: April 2021

Nuclear DNA replication was monitored ‘in situ’ in pea nuclei with the bromodeoxyuridine antibody technique. The labelling appeared to be restricted to a number of finely distinct spots. The labelling was followed through three subsequent cell cycles in meristematic and differentiating pea root cells. The results show that the spots as seen just after the labelling persist distinctly over the mitotic chromosomes as well as in the nuclei of the following cell cycles up to 44 hours after the pulse. Moreover, they are also present in the nuclei of differentiating cells. The spots over the mitotic chromosomes in specific cases give rise to a dynamic banding. Nuclei of the second and third cycle show absence of labelling in specific zones, owing to the segregation of the labelled strands of chromosomal DNA. The maintenance of the spotted appearance of the replication clusters through all stages of the three subsequent cell cycles may be an indication in favour of the hypothesis that such clusters represent structurally stable replicon complexes held together by the nuclear matrix and the chromosome scaffold.
Sequence analysis of MAP2 function in living cells
Authors: Ferralli Jacqueline , Doll Thierry , Matus Andrew
Published: April 2021

Microtubule-associated protein 2 (MAP2) is an abundant neuron-specific protein that binds to microtubules through a domain near its carboxyl terminus that contains either three or four similar repeats of a 31 amino acid motif. When expressed in non-neuronal cells by transfection MAP2 stabilises microtubules and induces their rearrangement into long bundles that are capable of supporting process outgrowth. To investigate which elements in the MAP2 sequence are involved in these functions we have constructed a series of deletion mutants of the short embryonic form of MAP2, MAP2c, and transfected them into non-neuronal cells. This showed that the strength of binding to microtubules increased with the number of repeats present in the construct. However, the repeat domain itself was insufficient for microtubule binding, which required in addition contiguous sequences either amino-terminal or carboxyl-terminal to the repeats themselves. Particularly on the amino-terminal side of the repeats, where there is a proline-rich domain, step-wise increases in the length of neighbouring sequence produced a gradual increase in microtubule binding. The apparent strength of binding to microtubules produced by mutant MAP2 forms was further correlated with the degree of bundling they induced as well as with the ability of the resulting microtubules to support process outgrowth. These results indicate that the interaction of MAP2 with microtubules is mediated by the combined action of several weak binding sites, including each of the repeat motifs and elements in the sequences on either side of them, whose additive effect produces the strong binding of the native MAP2 molecule. The results further indicate that both the bundling and stiffening of microtubules by MAP2 are correlated with the strength of its binding to them and suggest that these properties are a direct result of microtubule stabilisation.
Dispersive locomotion of human neutrophils in response to a steroid-induced factor from monocytes
Authors: Chettibi S. , Lawrence A. J. , Young J. D. , Lawrence P. D. , Stevenson R. D.
Published: April 2021

A monocyte-derived factor that stimulates the locomotion of human neutrophils on an albumin-coated glass surface has been prepared from the culture supernatant of dexamethasone-treated human monocytes and called STMS (steroid-treated monocyte supernatant). A modified cell tracking program has been developed and the parameters of locomotion determined by the analysis of Gail and Boone for cells moving in a persistent random walk. Cells moving in uniform concentrations of STMS, interleukin- 8 (IL-8) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) chosen to give a sub-maximal speed of locomotion show persistent, random and constrained random diffusion, respectively, with augmented diffusion coefficients of 0.8±0.1, 0.14±0.02 and 0.12±0.03 µm2 per second for STMS, IL-8 and fMLP, respectively. The augmented diffusion coefficient and the underlying persistence are therefore sensitive quantitative assay parameters for STMS activity and the qualitative characteristics of locomotion allow STMS activity to be distinguished from that of all other factors tested. The contribution of lowered adhesion to locomotion was examined in a novel tilt-assay, which demonstrated that cells in the presence of STMS, but not other factors, moved down slope with significantly increased speed while maintaining contact with the substratum. The results were interpreted in terms of the bipolar form of STMS-treated cells, contrasting with multipolar forms in response to other agents. This together with low adhesiveness plus an inherent tendency of a single locomotor focus to continue motion in its original direction has been used to explain the difference between response to STMS and other factors. STMS has been proposed either to prevent directed locomotion of neutrophils to an inflammatory site or to promote dispersive locomotion away from such a site and perhaps to inhibit neutrophil transmigration between endothelial cells.
Transport of a lysosomal membrane glycoprotein from the Golgi to endosomes and lysosomes via the cell surface in African trypanosomes
Authors: Brickman Marla Jo , Balber Andrew E.
Published: April 2021

gp57/42 is a membrane glycoprotein localized in the trans - Golgi, flagellar pocket region of the cell surface, endosomes and lysosomes of bloodstream forms of Trypanosoma brucei rhodesiense. Pulse-chase immunoprecipitation experiments revealed that gp57/42 acquires a unique N-linked oligosaccharide recognized by the CB1 monoclonal antibody 20-30 minutes after protein synthesis, probably in the trans - Golgi. We refer to gp57/42 molecules that carry the CB1 eptiope as CB1-gp. Pulse labeled CB1-gp contained only one core protein, p57, when chase times were 30 minutes or less. As time of chase increased from 30 to 60 minutes, a new polypeptide, p42, appeared in N-glycanase-treated CB1 immunoprecipitates. Since p57 and p42 share 10 of 13 methionyl peptides, we conclude that p42 is a fragment of p57. Cleavage of p57 to p42 was not inhibited when cells were chased in two thiol protease inhibitors or in 3,4-diiso-coumarin, but was inhibited by leupeptin. Cell surface biotinylation was used to determine if newly synthesized CB1-gp was transported from the Golgi to the surface. When cells were pulse labeled and chased for 30 minutes, as much as 40% of the radiolabeled CB1-gp could be biotinylated on the cell surface. The amount of CB1-gp that could be biotinylated decreased when chases were extended from 30 to 60 minutes, suggesting that pulse labeled CB1-gp left the surface. In contrast, pulse labeled variant surface glycoprotein molecules continued to accumulate on the surface where they could be biotinylated between 30 and 60 minutes of chase. Biotinylated CB1-gp derived from cells chased for 30 minutes contained p57 but no p42. However, when labeled cells were biotinylated after a 30 minute chase and then incubated another 30 minutes at 37°C, the biotinylated CB1-gp contained both p57 and p42. The p57 in biotinylated CB1-gp was not cleaved to p42 if the additional incubation was done at 4 or 12°C. This suggests that transport to a compartment where processing occurs and/or the processing enzymes are inhibited by low temperature. When surface biotinylation was done after a 60 minute chase, p42 was detected in biotinylated CB1-gp, suggesting that CB1-gp molecules had passed through the processing compartment and then appeared on the cell surface. Thus, a major portion of the newly synthesized CB1-gp is routed from the Golgi to endocytic compartments via the cell surface. In trypanosomes this process involves a unique surface domain, the flagellar pocket. Otherwise, this pathway of lysosomal membrane glycoprotein transport has been conserved in evolution from the trypanosomes to the vertebrates.
Functional consequences in yeast of single-residue alterations in a consensus calmodulin
Authors: Harris Eva , Watterson D. Martin , Thorner Jeremy
Published: April 2021

A synthetic gene encoding a ‘consensus’ calmodulin (synCaM) was able to substitute for the Saccharomyces cerevisiae calmodulin gene (CMD1), even though synCaM is only 60% identical in primary amino acid sequence to yeast Cmd1. Twelve different synCaM mutants were also expressed in yeast. Seven of the 12 mutant synCaMs supported germination and growth of Cmd1-deficient spores. Five of the 12 mutant synCaMs were incapable of supporting germination of Cmd1-deficient spores and, of these, four were also incapable of supporting vegetative growth of Cmd1-deficient haploid cells. The five nonfunctional synCaM mutants were expressed at levels equivalent to, or higher than, the seven synCaM mutants that were able to substitute for Cmd1; thus, the inability to function was not simply due to inadequate expression or rapid degradation. All nonfunctional synCaM mutants shared a single charge reversal mutation in the central helix (E84K), which was found to be sufficient to confer the lethal phenotype. The ability of another mutant synCaM (S101F) to support growth of Cmd1-deficient cells was dependent on cell ploidy. Another mutant (K115Y) supported spore germination and vegetative growth, but not meiosis and sporulation. The terminal phenotype of cells lacking a functional calmodulin included a dramatic accumulation of polymerized microtubules.
Inductive effect of copper deficiency on the reversion of dedifferentiated rat hepatoma cells and on gene amplification
Authors: Renault Eric , Deschatrette Jean
Published: April 2021

Cells of a dedifferentiated rat hepatoma clone were submitted in vitro to copper deficiency. This treatment caused inhibition of cell growth. In addition, in treated cultures, the frequency of differentiated revertants selected in glucose-free medium was drastically increased when compared with the spontaneous frequency. The maximum effect was observed when cell proliferation spontaneously resumed after 20 days of copper deficiency. Furthermore, a copper depletion/replenishment protocol applied before the selection of revertants reduced the period of time of copper deficiency that was necessary to provoke the reversion process.

It has been previously demonstrated that cell growth arrest and reinitiation may induce gene amplification events. Amplification of the dihydrofolate reductase gene as an indicator of such events was tested during the copper deficiency treatment. The frequency of cells resistant to increasing methotrexate concentrations due to gene amplification was enhanced by the treatment, just as was the frequency of differentiated revertants. These results suggest that in rat hepatoma cells the phenotypic transition to the stable differentiated state involves gene amplification and/or genome rearrangement.
ATP depletion: a novel method to study junctional properties in epithelial tissues: cytoskeleton: I. Rearrangement of the actin
Authors: Bacallao Robert , Garfinkel Alan , Monke Steven , Zampighi Guido , Mandel Lazaro J.
Published: April 2021

The effect of cellular injury caused by depletion of intracellular ATP stores was studied in the Madin-Darby canine kidney (MDCK) and JTC cell lines. In prior studies, it was shown that ATP depletion uncouples the gate and fence functions of the tight junction. This paper extends these observations by studying the changes in the actin cytoskeleton and tight junction using electron microscopy and confocal fluorescence microscopy in combination with computer-aided three-dimensional reconstruction. Marked regional differences in the sensitivity to the effects of ATP depletion were observed in the actin cytoskeleton. Actin depolymerization appears to first affect the cortical actin network running along the apical basal axis of the cell. The next actin network that is disrupted is the stress fibers found at the basal surface of the cell. Finally, the actin ring at the level of the zonulae occludens and adherens is compromised. The breakup of the actin ring correlates with ultrastructural changes in tight junction strands and the loss of the tight junction’s role as a molecular fence. During the process of actin network dissolution, polymerized actin aggregates form in the cytoplasm. The changes in the junctional complexes and the potential to reverse the ATP depletion suggest that this may be a useful method to study junctional complex formation and its relationship to the actin cytoskeletal network.