bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubule assembly and kinetochore directional instability in vertebrate monopolar spindles: Implications for the mechanism of chromosome congression
Authors: Cassimeris Lynne , Rieder Conly L. , Salmon E. D.
Published: April 2021

We have proposed previously a kinetochore motor-polar ejection model for chromosome congression to the metaphase plate where forces generated at the kinetochore are antagonized by away-from-the pole forces generated within each half-spindle on the chromosome arms. This model was based in large part on observations of the behavior of chromosomes on monopolar spindles. In these cells chromosomes typically become attached to the pole by only one kinetochore fiber. These mono-oriented chromo-somes move to positions away from the pole even though they are pulled poleward at their kinetochores. Their arms are also ejected away from the pole when severed from the centromere. Here we have characterized further the prop-erties of monopolar spindles in newt lung epithelial cells to determine the similarities between monopolar and bipolar spindles. We found no significant differences between monopolar and bipolar spindles over the parameters examined, which included: microtubule dynamics as measured by fluorescence redistribution after photo-bleaching; the ability of polar microtubule arrays to push chromosome arms away from the pole; the dependence of chromosome position relative to the pole on microtubule assembly; the number of kinetochore microtubules per kinetochore; and the directional instability of kinetochore motion during chromosome oscillations poleward and away-from-the-pole. As in bipolar spindles, kinetochore directional instability is characterized by abrupt switching between constant velocity phases of poleward and away-from-the-pole motion. From these data we conclude that the mechanism(s) responsible for chromosome positioning in monopolar spindles are fundamentally the same as those in bipolar spindles; only the geometry of the two spindle forms and the interplay between sister kinetochore direc-tional instabilities are different. We also found no correla-tion in the kinetochore-to-pole distance with kinetochore microtubule number in monopolar spindles, but a strong qualitative correlation with microtubule density. This finding indicates that oscillations of mono-oriented chro-mosomes in both monopolar and bipolar spindles occur because chromosomes persist in poleward motion until they reach a density of polar microtubules sufficiently high to promote switching to away-from-the-pole motion. As the kinetochore and chromosome arms move away-from-the-pole, microtubule density decreases and the kinetochore switches to poleward motion, pulling the chromosome arms back into regions of higher microtubule density. The mechanism regulating kinetochore switching between poleward and away-from-the-pole motion is poorly under-stood, but may depend on tension at the kinetochore generated by pushing forces on the chromosome arms produced by the polar microtubule arrays.
The cell adhesion molecule, connectin, and the development of the Drosophila neuromuscular system
Authors: Meadows Lisa A. , Gell David , Broadie Kendal , Gould Alex P. , White Robert A. H.
Published: April 2021

The connectin gene of Drosophila has been identified as a candidate direct target of homeotic gene control and has also been implicated in the formation of specific neuro-muscular connections. The gene product, connectin, is a member of the leucine-rich repeat protein family and we show that it is attached to the cell surface via a glycosyl-phosphatidylinositol linkage and that it can mediate homotypic cell-cell adhesion in vitro. The expression of connectin protein during Drosophila embryogenesis provides support for a role in adhesion in vivo. In the central nervous system, it is initially expressed on longitu-dinal glia and on a few identified neurons. These cells extend processes and connect up to form a continuous scaffold of connectin-expressing cells, presaging the devel-opment of axonal pathways. Later, connectin is expressed on specific axons as they track along the connectin scaffold. Glial expression then declines and connectin appears on axons that fasciculate with pre-existing connectin-positive bundles. Thus scaffold formation, axon pathfinding and fasciculation involve specific contacts between connectin-positive cells. The timing and pattern of connectin expression suggest that it may play an important role in mediating specific interactions through homotypic cell adhesion.
Cloning of a cdna encoding human centrin, an ef-hand protein of centrosomes and mitotic spindle poles
Authors: Errabolu Ramesh , Sanders Mark A. , Salisbury Jeffrey L.
Published: April 2021

A human cDNA expression library was screened using anti-centrin antibodies to obtain a cDNA clone encoding human centrin. The cDNA clone contains an open reading frame of 516 base pairs and predicts a product of 172 amino acids with a calculated molecular mass of 19,528 and a pI of 4.61. Sequence analysis demonstrates that human centrin and centrins from higher plants, protozoa, algae, Xenopus and the yeast CDC31 gene product are closely related members of a subfamily of the EF-hand superfamily of calcium-binding proteins. The human centrin sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation and western blot studies from HeLa cells confirm that human centrin is a protein of ∼20,000 Mr as predicted from the cDNA clone. Indirect immunofluorescence analysis of HeLa cells demonstrates that centrin is localized at the cen-trosome of interphase cells and that it redistributes to the region of the spindle poles during mitosis. When taken together with earlier genetic studies, these results demon-strate that centrin is a ubiquitous component of centro-somes and mitotic spindle poles of diverse organisms and suggest that centrin plays a role in centrosome separation at the time of mitosis.
Distribution of hyaluronan in the epiphysial growth plate: turnover by CD44-expressing osteoprogenitor cells
Authors: Pavasant Prasit , Shizari Tony M. , Underhill Charles B.
Published: April 2021

In the present study, we have examined the distribution of both hyaluronan and its receptor, CD44, during the process of endochondral ossification in the mouse tibia. Histochemical staining revealed that a large amount of hyaluronan was present in the lacunae located in the zone of hypertrophy, but it was greatly reduced or absent from the zone of erosion. In addition, hyaluronan was present in the cytoplasm of osteoprogenitor cells located in the zone of erosion. These cells also expressed CD44 on their surfaces, as revealed by double-label immunohistochem-istry. These results suggested that the osteoprogenitor cells may use CD44 to bind and internalize hyaluronan, and subsequently degrade it with lysosomal enzymes. To test this possibility, we examined the human cell line, MG-63, which closely resembles osteoprogenitor cells. These cells produced several different forms of CD44, as determined by western blotting (85, 116 and 150 kDa). In addition, the binding of isotopically labeled hyaluronan to detergent extracts of these cells was blocked by a monoclonal antibody to CD44. Similarly, the degradation of hyaluronan by these cultured cells was also inhibited by a mono-clonal antibody to CD44. To determine if these cells could remove hyaluronan from the growth plate, the cells were cultured directly on top of thin sections of the epiphysial region of long bone. After 16 hours, the sections were stained for hyaluronan. The MG-63 cells removed signifi-cant amounts of hyaluronan present in the zone of hyper-trophy, and this effect was blocked by an excess of soluble hyaluronan and by a monoclonal antibody to CD44. Taken together, these results suggest that, under physiological conditions, osteoprogenitor cells in the zone of erosion can use a CD44-dependent mechanism to remove hyaluronan.
Programmed cell death in Dictyostelium
Authors: Cornillon Sophie , Foa Colette , Davoust Jean , Buonavista Nathalie , Gross Julian D. , Golstein Pierre
Published: April 2021

Programmed cell death (PCD) of Dictyostelium discoideum cells was triggered precisely and studied quantitatively in an in vitro system involving differentiation without morphogenesis. In temporal succession after the triggering of differentiation, PCD included first an irreversible step leading to the inability to regrow at 8 hours. At 12 hours, massive vacuolisation was best evidenced by confocal microscopy, and prominent cytoplasmic condensation and focal chromatin condensation could be observed by electron microscopy. Membrane permeabilization occurred only very late (at 40-60 hours) as judged by propidium iodide staining. No early DNA fragmentation could be detected by standard or pulsed field gel electrophoresis. These traits exhibit some similarity to those of previously described non-apoptotic and apoptotic PCD, suggesting the hypothesis of a single core molecular mechanism of PCD emerging in evolution before the pos-tulated multiple emergences of multicellularity. A single core mechanism would underly phenotypic variations of PCD resulting in various cells from differences in enzymatic equipment and mechanical constraints. A pre-diction is that some of the molecules involved in the core PCD mechanism of even phylogenetically very distant organisms, e.g. Dictyostelium and vertebrates, should be related.
Synaptonemal complex proteins: occurrence, epitope mapping and chromosome disjunction
Authors: Dobson Melanie J. , Pearlman Ronald E. , Karaiskakis Angelo , Spyropoulos Barbara , Moens Peter B.
Published: April 2021

We have used polyclonal antibodies against fusion proteins produced from cDNA fragments of a meiotic chromosome core protein, Cor1, and a protein present only in the synapsed portions of the cores, Syn1, to detect the occurrence and the locations of these proteins in rodent meiotic prophase chromosomes. The 234 amino acid Cor1 protein is present in early unpaired cores, in the lateral domains of the synaptonemal complex and in the chromosome cores when they separate at diplotene. A novel observation showed the presence of Cor1 axial to the metaphase I chromosomes and substantial amounts of Cor1 in association with pairs of sister centromeres. The centromere-associated Cor1 protein becomes dissociated from the centromeres at anaphase II and it is not found in mitotic metaphase centromeres. The extended presence of Cor1 suggests that it may have a role in chromosome disjunction by fastening chiasmata at metaphase I and by joining sister kinetochores, which ensures co-segregation at anaphase I. Two-colour immunofluorescence of Cor1 and Syn1 demonstrates that synapsis between homologous cores is initiated at few sites but advances rapidly relative to the establishment of new initiation sites. If the rapid advance of synapsis deters additional initiation sites between pairs of homologues, it may provide a mechanism for positive recombi- nation interference. Immunogold epitope mapping of anti- bodies to four Syn1 fusion proteins places the amino terminus of Syn1 towards the centre of the synaptonemal complex while the carboxyl terminus extends well into the lateral domain of the synaptonemal complex. The Syn1 fusion proteins have a non-specific DNA binding capacity. Immunogold labelling of Cor1 antigens indicates that the lateral domain of the synaptonemal complex is about twice as wide as the apparent width of lateral elements when stained with electron-dense metal ions. Electron microscopy of shadow-cast surface-spread SCs confirms the greater width of the lateral domain. The implication of these dimensions is that the proteins that comprise the synaptic domain overlap with the protein constituents of the lateral domains of the synaptonemal complex more than was apparent from earlier observations. This arrangement suggests that direct interactions might be expected between some of the synaptonemal complex proteins.
The role of duplication of tumour-derived chromosome 15 carrying the rearranged pvt-1 gene in the transformed phenotype of YACUT T-cell lymphoma × G4 T-cell line somatic cell hybrids in dictating the terminal differentiation program of the parental G4 cell
Authors: Kubota Koichi , Nakazato Kazumi
Published: April 2021

Fusion of the YACUT T-cell lymphoma with the Mls-1a-antigen-specific non-tumorigenic T-cell line G4 was previously reported to produce growth-arrested hybrids that could be induced to proliferate in the presence of Mls-1a antigen. The proliferation-suppressed hybrid lines exhibited phenotypic changes as follows: the usually high levels in YACUT of J11d antigen, IL-2 receptor, and c-myb expression, which are markers of immature T cells, were all down-regulated; the G4 T-cell function, i.e., contact helper activity for B-cell proliferation in T/B cell collabo-ration, was retained. Furthermore, fusion of the YACUT lymphoma with a killer T-cell line produced growth-arrested and tetraploid somatic cell hybrids having killer activity. Thus, in addition to the transformed phenotype (autonomous proliferation in vitro), the antigen-specific non-tumorigenic T-cell line genomes introduced into the YACUT lymphoma suppressed the immature phenotypes of YACUT and imposed their own programming of terminally differentiated traits on the hybrids.

Prolonged growth of the proliferation-suppressed hybrid lines by repeated antigenic stimulation was previously reported to result in the appearance of transformed hybrids, which was accompanied by both a reversion of c-myc expression to the levels of YACUT and an increase in the number of chromosome 15. The present study revealed that the amplification of chromosome 15 resulted from the duplication of the tumour-derived chromosome 15 carrying the rearranged pvt-1 gene. However, the differentiated phenotypes of the hybrids remained mostly unchanged upon cell transformation. These results indicate that the duplication of the tumour-derived chromosome 15 plays an important role in the appearance of the trans-formed phenotype without abrogating the imposition of the terminally differentiated traits on the YACUT × G4 hybrids.
Differential effects of compartment deacidification on the targeting of membrane and soluble proteins to the vacuole in yeast
Authors: Morano Kevin A. , Klionsky Daniel J.
Published: April 2021

Lysosomal/vacuolar protein targeting is dependent on compartment acidification. In yeast, sorting of soluble vacuolar proteins such as carboxypeptidase Y is sensitive to acute changes in vacuolar pH. In contrast, the vacuolar membrane protein alkaline phosphatase is missorted only under conditions of chronic deacidification. We have undertaken a temporal analysis to define further the relationship between compartment acidification and sorting of soluble and membrane vacuolar proteins. Depletion of either the Vma3p or Vma4p subunits of the yeast vacuolar ATPase over time resulted in loss of vacuolar ATPase activity and vacuolar acidification. A kinetic delay in processing of carboxypeptidase Y occurred concomitant with these physiological changes while transport of alkaline phosphatase remained unaffected. Carboxypeptidase S, another vacuolar hydrolase that transits through the secretory pathway as an integral membrane protein, displayed a pH sensitivity similar to that of soluble vacuolar proteins. These results indicate that compartment acidification is tightly coupled to efficient targeting of proteins to the vacuole and that there may be multiple distinct mechanisms for targeting of vacuolar membrane proteins.
Recruitment of antigenic gamma-tubulin during mitosis in animal cells: presence of gamma-tubulin in the mitotic spindle
Authors: Lajoie-Mazenc Isabelle , Tollon Yvette , Detraves Claire , Julian Monique , Moisand André , Gueth-Hallonet Catherine , Debec Alain , Salles-Passador Isabelle , Puget Alain , Mazarguil Honoré , Raynaud-Messina Brigitte , Wright Michel
Published: April 2021

It has been claimed repeatedly that γ-tubulin is exclusively localized at the spindle poles in mitotic animal cells, where it plays a role in microtubule nucleation. In addition to this localization, we have observed a γ-tubulin-specific staining of the mitotic spindle in several animal cells (human, kangaroo rat, mouse, Chinese hamster, Xenopus and Drosophila) using five polyclonal antibodies raised against unique γ-tubulin sequences and four different fixation protocols. In HeLa and PtK2 cells, γ-tubulin was detected in the mitotic spindle from late prometaphase to telophase. In contrast, in other cell types, it was detected in metaphase only. In all cases we failed to detect γ-tubulin in the short aster microtubules at the spindle poles. Electron microscopic observation revealed that at least part of the γ-tubulin localized on the surface of spindle microtubules with a preferential distribution along kinetochore microtubules. In HeLa cells, the amount of antigenic γ-tubulin was fairly constant in the spindle poles during mitosis from prometaphase to telophase. In contrast, γ-tubulin appeared in the mitotic spindles in prometaphase. The amount of γ-tubulin decreased in telophase, where it relocalized in the interzone. In metaphase cells about 15-25% of the total fluorescence was localized at the spindle poles, while 75-85% of the fluorescence was distributed over the rest of the spindle. These results suggest that the localization and timing of γ-tubulin during the cell cycle is highly regulated and that its physiological role could be more complex and diverse than initially assumed.
TRiC-P5, a novel TCP1-related protein, is localized in the cytoplasm and in the nuclear matrix
Authors: Joly Erik C. , Tremblay Esther , Tanguay Robert M. , Wu Yang , Bibor-Hardy Viviane
Published: April 2021

We have recently reported the cloning of a novel protein, TRiC-P5, with significant homology with protein 1 of the t-complex (TCP1). In the present study, the cellular localization of TRiC-P5 in Raji cells has been determined using an antiserum raised against a 18.5 kDa fusion protein. Results from cell fractionation and immunoblot studies indicate that TRiC-P5 is mainly localized in the cytoplasm. In addition, a significant part of TRiC-P5 is also found in the nucleus where it is attached to the nuclear matrix, a complex filament network involved in essential cellular functions such as DNA replication, and RNA transcription and maturation. Immunofluorescence experiments using the anti-TRiC-P5 antibodies confirm these results. We also provide evidence that, in the cytoplasm, TRiC-P5 is part of a large protein complex, most probably the TCP1-ring complex (TRiC), a hetero-oligomeric ring complex that plays a role of molecular chaperone in the folding of actin and tubulin.
Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis
Authors: Morse-Gaudio Michele , Risley Michael S.
Published: April 2021

The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing sper-matogenic cells of Xenopus laevis to gain an insight into the roles of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo IIα and β, and an antipeptide antibody to human topo IIα, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo IIα. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 μM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.
cAMP does not regulate [Ca2+]i in human tracheal epithelial cells in primary culture
Authors: Davis Pamela B. , Perez Catherine L. Silski and Aura
Published: April 2021

Human tracheal epithelial cells in primary culture respond to different receptor agonists with different peak intracel- lular calcium concentrations. From resting concentration 138±13 nM, bradykinin (0.1 μM) produces an increase to a maximum of 835±195 nM, histamine (10 μM) to 352±51 nM, and ATP (5-500 μM) to more than 1500 nM. Nine of 14 cultures also responded to isoproterenol (10 μM), though with a smaller increase, to 210±29 nM. A response was observed with isoproterenol, and epinephrine, but not norepinephrine, phenylephrine or methoxamine, was inhibited by propranolol but not phentolamine, and so this appeared to be a β-adrenergic response. However, no response could be detected to adenosine, prostaglandin E2 or forskolin, agents that activate adenylate cyclase, or to permeant analogs of cAMP (CPT-cAMP or db-cAMP). The intracellular calcium response to isoproterenol did not follow either the time-course or the desensitization pattern of the cAMP response. Thus, this response to isoproterenol is not mediated by cAMP. No relation was demonstrated between cAMP production by other agonists and the response of intracellular calcium. Pretreatment with agents that increase cAMP did not affect the calcium responses to ATP or bradykinin. Thus, cAMP does not regulate intra- cellular calcium concentration in human tracheal epithelial cells. The variation in peak intracellular calcium responses to various agonists may be explained by the presence of multiple second messengers (other than cAMP), multiple intracellular pools of calcium, or cell heterogeneity. The agonists tested had the same relative potency in cells from patients with cystic fibrosis as in non-cystic fibrosis cells.
The network organization and the phosphorylation of cytokeratins are concomitantly modified by forskolin in the enterocyte-like differentiated Caco-2 cell line
Authors: Baricault Laurent , Néchaud Béatrice de , Sapin Catherine , Codogno Patrice , Denoulet Philippe , Trugnan Germain
Published: April 2021

Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional charac-teristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organiz-ation of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn’t interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantita-tively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcelluar distribution of cytoker-atin by changing their phosphorylation status and therefore to study their potential cellular functions.
Ectoenzyme regulation by phenotypically distinct fibroblast sub-populations isolated from the human mammary gland
Authors: Atherton Amanda J. , O’Hare Michael J. , Buluwela Lakjaya , Titley Jenny , Monaghan Paul , Paterson Hugh F. , Warburton Michael J. , Gusterson Barry A.
Published: April 2021

Inter- and intralobular mammary fibroblasts have been separated from normal human breast tissue and cultured to study the differential expression of ectoenzymes present within the stroma of the normal gland and associated with breast cancers. Specific ectoenzymes were identified by indirect immunofluorescence and quantified by flow cytometry and semi-quantitative PCR. A consistent difference was noted between the two fibroblast sub-populations at early passage in respect of dipeptidyl peptidase IV (DPP IV) and aminopeptidase N (APN) expression. Early passage intralobular fibroblasts were positive for APN but negative for DPP IV, as seen in the intact tissue. However, with continued sub-culture they gradually began to express DPP IV, until at later passages they became indistinguishable from the interlobular fibroblasts, which were APN and DPP IV-positive at all stages in culture, as they are in intact tissue. Neutral endopeptidase (NEP/CALLA/CD10) is not expressed by normal adult breast fibroblasts but is found in the stroma associated with over 60% of breast cancers.

It was up-regulated in vitro on both inter- and intralobular fibroblasts, with final levels that were significantly (<14 times) higher on the former in all pairs of preparations from individual donors analysed. This difference persisted with continued passage, and levels of the ectoenzyme and its messenger RNA were further up-regulated by hydro-cortisone in both populations. These results demonstrate that phenotypically distinct cultures of human mammary fibroblast sub-populations can be used to study the regulation of these stromal ectoenzymes. Such cultures also provide a means of investigating the mechanisms and functional significance of changes occurring as the result of neoplasia, as well as enabling the individual roles of inter- and intralobular fibroblasts in epithelial/mesenchymal interactions to be evaluated.
Dissociation of minor satellite from the centromere in mouse
Authors: Vig Baldev K. , Latour Derek , Frankovich Jennifer
Published: April 2021

The minor satellite DNA of mouse is believed to constitute the centromere. We report that centromeres of some chromosomes in the Cl1D cells of mouse are not associated with this DNA even though the latter is present on these chromosomes. The satellite DNA was detected distally from the centromere and could not be mistaken as a component of the centromere. We also report that the site of the primary constriction may not always coincide with the site of the anti-kinetochore antibody reaction. Whereas the regions containing the major satellite decondense upon treatment with bisbenzimidazole (Hoechst 33258), the sites carrying minor satellite resist decondensing.
Overexpression of a truncated cyclin B gene arrests Dictyostelium cell division during mitosis
Authors: Luo Qian , Michaelis Christine , Weeks Gerald
Published: April 2021

A cyclin gene has been isolated from Dictyostelium discoideum and the available evidence indicates that the gene encodes a B type cyclin. The cyclin box region of the protein encoded by the gene, clbl, has the highest degree of sequence identity with the B-type cyclins of other species. Levels of cyclin B mRNA and protein oscillate during the cell cycle with maximum accumulation of mRNA occurring prior to cell division and maximum levels of protein occurring during cell division. Overexpression of a N-ter- minally truncated cyclin B protein lacking the destruction box inhibits cell growth by arresting cell division during mitosis. The gene is present as a single copy in the Dictyostelium genome and there is no evidence for any other highly related cyclin B genes.
Identification of a large complex containing the integrin α6β 1 laminin receptor in neural retinal cells
Authors: Curtis Ivan de , Gatti Giuliana
Published: April 2021

Integrin α6β 1 is a laminin receptor involved in adhesion and neurite extension of retinal neurons on laminin. The present study was carried out to identify potential interactions between the α 6β 1receptor and cellular proteins that may be involved in integrin signaling and function. For this purpose we have used a biochemical approach based on the solubilization of retinal neurons cultured on laminin with nonionic detergents, followed by centrifugation on sucrose velocity gradients. Analysis of the distribution of the α6β 1integrin subunits in the gradients showed that they migrate as a large complex after extraction of cells with octylglucoside, but not after Triton X-100 extraction. Cytoskeletal proteins known to localize in adhesion plaques did not comigrate with α 6β 1in octylglucoside gradients, while a set of polypeptides whose tyrosine phosphorylation was enhanced by culture on laminin colocalized with α6β1on the gradients after octylglucoside solubilization. Culture of retinal neurons on bovine serum albumin, a nonadhesive substratum, partially affected the gradient distribution of the receptor after octylglucoside extraction. Furthermore, analysis of the gradient distribution of two alternatively spliced isoforms of the α 6 subunit, α 6-cytoA and α6-cytoB, showed that the effect of non-adhesion on the sedimentation properties of the two integrin α6 isoforms was more dramatic for α 6-cytoB than α6-cytoA. These differences in the sedimentation behaviour indicate distinct biochemical properties of the two α6 isoforms that, together with previous observations on their differential distribution in the developing retina, may reflect functional specificities.
Heparan sulfate proteoglycans as transducers of FGF-2 signalling
Authors: Quarto Natalina , Amalric François
Published: April 2021

The fibroblast growth factor-2 (FGF-2) low-affinity binding sites, heparan sulfate proteoglycans (HSPGs), function as modulators of FGF-2 activity. It is noteworthy that HSPG binding protects FGF-2 from denaturation and proteolytic degradation, provides a matrix-bound or cellsurface reservoir of this factor for the cells and is required for the activation of FGF high-affinity receptors. In our study we investigated the biological meaning of FGF-2 internalization mediated through its low-affinity binding sites, HSPGs.

Using as model system L6 myoblasts lacking endogenous FGF receptors (FGFRs), we demonstrated that these cells internalize FGF-2 efficiently through an HSPG-mediated pathway. FGF-2 internalization occurring through HSPGs was paralleled by an increase in the activity of urokinase plasminogen activator (u-PA). The u-PA-inducing activity of FGF-2 was strictly correlated to its internalization, as chlorate treatment, which causes a strong inhibition of FGF-2 internalization, abrogated the u-PA-inducing activity of FGF-2. In addition, expression of functional FGF high-affinity receptors (FGFR-1) did not enhance u-PA in L6 myoblasts upon FGF-2 stimulation. According to our results we propose that FGF-2 internalization mediated through HSPGs may transduce FGF-2 signalling such as u-PA-activity stimulation. Thus, HSPGs may act as direct transducers of FGF signalling and indeed, different FGF-signalling pathways must exist.
Immunocytochemical and in situ hybridization studies of the heparan sulfate proteoglycan, glypican, in nervous tissue
Authors: Karthikeyan Laina , Flad Manuela , Engel Margit , Meyer-Puttlitz Birgit , Margolis Richard U. , K. Margolis Renée
Published: April 2021

Using immunocytochemistry and in situ hybridization histochemistry, we have investigated in embryonic and postnatal rat nervous tissue the localization and cellular sites of synthesis of glypican, a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan. Glypican immunoreactivity is present in the marginal layer (prospective white matter) and in the dorsal root entry zone of E13-16 spinal cord, as well as in the optic nerve and retina at this stage, but does not appear at significant levels in brain until approximately E19. The proteoglycan shows a wide distribution in grey matter and axonal projections of postnatal brain, including the hippocampal formation, the parallel fibers of cerebellar granule cells, and in the medulla and brainstem. Northern analysis demonstrated high levels of glypican mRNA in brain and skeletal muscle, and in rat PC12 pheochromocytoma cells. In situ hybridization histochemistry showed that glypican mRNA was especially prominent in cerebellar granule cells, large motor neurons in the brainstem, and CA3 pyramidal cells of the hippocampus. Our immunocytochemical and in situ hybridization results indicate that glypican is predominantly a neuronal membrane proteoglycan in the late embryonic and postnatal rat central nervous system.
Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers
Authors: Wang Jingsong , Satoh Minoru , Pierani Alessandra , Schmitt Jacky , Chou Chih-Hao , Stunnenberg Hendrik G. , Roeder Robert G. , Reeves Westley H.
Published: April 2021

The Ku autoantigen is a heterodimer of 70 kDa (p70) and ~80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respec tively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.
Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication
Authors: Hutchison C.J. , Bridger J.M. , Cox L.S. , Kill I.R.
Published: April 2021

The major residual structure that remains associated with the nuclear envelope following extraction of isolated nuclei or oocyte germinal vesicles with non-ionic detergents, nucleases and high salt is the lamina (Fawcett, 1966; Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The nuclear lamina is composed of intermediate filament proteins, termed lamins (Gerace and Blobel, 1980; Shelton et al., 1980), which polymerise to form a basket-weave lattice of fibrils, which covers the entire inner surface of the nuclear envelope and interlinks nuclear pores (Aebi et al., 1986; Stewart and Whytock, 1988; Goldberg and Allen, 1992). At mitosis, the nuclear envelope and the lamina both break down to allow chromosome segregation. As a consequence, each structure has to be rebuilt during anaphase and telophase, allowing cells an opportunity to reposition chromosomes (Heslop-Harrison and Bennett, 1990) and to reorganise looped chromatin domains (Franke, 1974; Franke et al., 1981; Hochstrasser et al., 1986), which may in turn control the use of subsets of genes. Because of the position that it occupies, its dynamics during mitosis and the fact that it is an essential component of proliferating cells, the lamina has been assigned a number of putative roles both in nuclear metabolism and in nuclear envelope assembly (Burke and Gerace, 1986; Nigg, 1989). However, to date there is little clear cut evidence that satisfactorily explains the function of the lamina in relation to its structure. In this Commentary we will describe some of the recent work that addresses this problem and attempt to provide a unified model for the role of lamins in nuclear envelope assembly and for the lamina in the initiation of DNA replication.
The crypt cycle in mouse small intestinal epithelium
Authors: Li Y. Q. , Roberts S. A. , Paulus U. , Loeffler M. , Potten C. S.
Published: April 2021

We have used a mutation-induced marker system in the intestine of mice heterozygous at the Dlb-1 locus, which determines the expression of binding sites for the lectin Dolichos biflorus agglutinin, and the frequency of clustering of mutated crypts with time as a means of investigating the frequency of the crypt fission process and the crypt cycle. Whole-mount preparations from heterozygous Dlb-1b/Dlb-1a mice were stained with a peroxidase conjugate of Dolichos biflorus agglutinin. Mutations at the Dlb-1b locus in crypt stem cells result in loss of DBA-Px binding in these cells and subsequently their progeny, which eventually results in a rare isolated single, unstained crypt. The sub-sequent development of pairs, triplets and clusters of negative staining crypts has been assumed to be the result of crypt fission. The frequency of these fission events has been measured in control untreated mice. These negative crypts are the result of spontaneous mutations. We have also looked at mutated crypts after treatment with N-nitroso-N-ethylurea or N-methyl-N′-nitro-N-nitrosoguanidine of young adult mice, which elevates the number of mutations.

Our results suggest that the crypt cycle in control animals is very long, 187±44 weeks (3.6 years, i.e. essentially the life of a laboratory mouse). This implies that about a third of the crypts may divide once in the life of a mouse. After sufficient time for conversion of mixed crypts to monophenotypic crypts after mutagen treatment several clusters of negative crypts were seen. This suggests that agents like N-methyl-N′-nitro-N-nitrosoguanidine cause sufficient damage to actually destroy some crypts, which is confirmed by crypt survival studies, and this induces local crypt repopulation and fission.
Nuclear distribution of proliferating cell nuclear antigen (PCNA) in fertilized eggs of the starfish Asterina pectinifera
Authors: Nomura Akira
Published: April 2021

Previous studies (Nomura et al. (1991)Dev. Biol. 143, 289-296 (1993) Dev. Biol. 159, 288-297) determined the time of DNA replication period (S phase) in starfish eggs fertilized either during or after oocyte maturation. Here proliferating cell nuclear antigen (PCNA) localized within nuclei of starfish eggs was detected with an anti-PCNA human antiserum. Using a confocal laser scanning microscope, a three-dimensional structure of the PCNA region was analyzed.

In eggs fertilized during maturation, PCNA started to localize within the nuclei at the same time as the initiation of the first S phase. During the S phase, the distribution of localized PCNA in a three-dimensional view coincided with the chromatin distribution. After the S phase, PCNA remained localized within the nuclei, but its distribution no longer coincided with the chromatin distribution.

In eggs fertilized after maturation, however, PCNA started to localize within the female pronuclei about 10 minutes ahead of the first S phase. Localized PCNA occupied only a limited region of the nuclei without diffusing over the whole nuclear area. Chromatin distributed around the peripheral region of the nuclei mostly outside the PCNA region. When the first S phase was initiated, the chromatin distribution became coincident with the PCNA region. Later behavior of PCNA was the same as that of the eggs fertilized during maturation. The precocious localization of PCNA in those eggs fertilized after maturation simply demonstrates that the ‘postactivation process’ for preparing DNA replication is triggered by fertilization and PCNA localization and S phase are sequentially initiated with a time-lapse. On the other hand, the simultaneous occurrence of them seen in those eggs fertilized during maturation indicates that the postactivation process must be going on in parallel with the maturation process.
Changes in yolk platelet pH during Xenopus laevis development correlate with yolk utilization: A quantitative confocal microscopy study
Authors: Fagotto François , Maxfield Frederick R.
Published: April 2021

The variations of the pH in Xenopus yolk platelets have been estimated by fluorescence confocal microscopy and computer image processing. For pH measurements in vitellogenic oocytes, the pH-sensitive fluorescent dye, DM-NERF, was coupled to vitellogenin, and the DM-NERF-vitellogenin was taken up by oocytes via receptor-mediated endocytosis. Dual emission ratio measurements of internalized DM-NERF-vitellogenin indicated that the mature yolk platelets are mildly acidic (pH 5.6). Their precursors, the primordial yolk platelelets, have a similar pH. This pH is probably sufficiently low for the partial cleavage of vitellogenin to yolk proteins, but not for yolk degradation. The yolk platelet pH at various developmental stages was estimated by measuring the accumulation of Acridine Orange, both in isolated yolk platelets and in disaggregated embryonic cells. During oogenesis, the yolk platelets accumulated a constant amount of Acridine Orange, corre-sponding to a pH of around 5.7. During embryogenesis, however, yolk platelets became progressively much more acidic (pH <5). Acidification correlated with yolk degradation in the various tissues examined, and yolk utilization was blocked when acidification was inhibited with bafilomycin, an inhibitor of vacuolar H+-ATPase. Bafilomycin also inhibited differentiation of cells isolated from stage 13–15 embryos. These data show that the yolk platelet pH is developmentally regulated and is involved in triggering yolk degradation. Also, yolk acidification and degradation appeared to be associated with cell differentiation and with the formation of the endosomal/lysosomal compartment, typical of adult cells, but absent in early embryos.
Quantitative measurement of α6β1 and α6β4 integrin internalization under cross-linking conditions: a possible role for α6 cytoplasmic domains
Authors: Gaietta Guido , Redelmeier Thomas E. , Jackson Michael R. , Tamura Richard N. , Quaranta Vito
Published: April 2021

In epithelial cells integrins are segregated on discrete domains of the plasma membrane. Redistribution may also occur during migration or differentiation. However, little is known about the mechanisms that control such redistribution. Receptor internalization may be a part of one such mechanism. We developed a quantitative assay and measured internalization of two epithelial integrin heterodimers, α6β1 and α6β4, induced by cross-linking with specific antibodies. α6β1 is a receptor for EHS laminin, while α6β4 is a receptor for a component of the basement membrane. α6β4 plays an important role in the establishment of hemidesmosomes, and becomes redistributed on the epithelial cell surface when cells are in a migratory phase. We report that α6β4 is efficiently internalized in human keratinocytes. More than 25% of cell surface α6β4 was internalized at 30 minutes, after cross-linking with A9, an anti-β4 monoclonal antibody. α6β1 is also internalized, in melanoma and teratocarcinoma cells, with maximum values of 20% of total receptors expressed at the cell surface. No significant difference was observed between the α6 isoforms A and B in these assays. To determine whether α6 cytoplasmic domains could influence integrin endocytosis, we prepared chimeric constructs with the extracellular domain of a reporter protein (CD8), and the cytoplasmic domains of either α6A or α6B. Both α6 cytoplasmic domains but not a control cytoplasmic domain promoted internalization of the chimeric proteins, after cross-linking with antibody. Internalization of α6 integrins may have a role in redistributing these receptors at the cell surface. Furthermore, the cytoplasmic domains of α6 may be involved in regulating integrin internalization.
Identification of a novel protein from adult chicken brain that inhibits neurite outgrowth
Authors: Clarke Graham A. , Moss Diana J.
Published: April 2021

Glycoproteins that inhibit neurite outgrowth may guide growth cones during development by acting as a barrier and closing off inappropriate routes. Their continued expression in the adult central nervous system may be a key factor in preventing regeneration of central nervous system neurons. A glycoprotein of 55 kDa has been isolated from the detergent-insoluble membrane skeleton from adult chicken brain. Initial experiments showed that dorsal root ganglion neurons would not adhere to or extend neurites on a substratum coated with GP55. Furthermore, GP55 will act as a barrier to the advance of established growth cones in the presence of poly-L-lysine, laminin or G4. Central nervous system neurons from forebrain as well as dorsal root ganglion neurons from the peripheral nervous system are inhibited by GP55. GP55 is also effective in blocking the initial adhesion of neurons to a substratum of poly-L-lysine and, particularly, laminin. In contrast to the inhibition of neurite outgrowth, neuronal adhesion is concentration independent over the range tested. A preliminary investigation of the mechanism by which GP55 inhibits outgrowth suggests that a pertussis toxin-sensitive G protein is required. Preliminary evidence suggests that GP55 is anchored in the membrane by a glycosyl phosphatidylinositol moiety. GP55 is distinct from previously identified inhibitory proteins, based on the source and molecular mass, and is thus a new member of this rapidly expanding family.