bluuebunny/crossref_metadata_2025 huggingface dataset:
Redistribution of cytoplasmic components during germinal vesicle breakdown in starfish oocytes
Authors: Terasaki Mark
Published: April 2021

The starfish oocyte is relatively clear optically, and its nucleus, termed the germinal vesicle, is large. These characteristics allowed studies by confocal microscopy of germinal vesicle breakdown during maturation in living oocytes. Three fluorescent probes for cytoplasmic components were used: fluorescein 70 kDa dextran, which does not cross the nuclear pore of immature oocytes and probably behaves in the same way as soluble cytosolic proteins, YOYO-1, which was used to localize ribosomes, and DiI which labels the nuclear envelope and endoplasmic reticulum. The first change observable by transmitted light microscopy during maturation is a wrinkling of the germinal vesicle envelope. Several minutes before the wrinkling, the 70 kDa dextran began to enter the germinal vesicle; the ribosomes did not enter during this period. The dextran is likely to be passing through nuclear pores whose size limit has increased but which still exclude ribosomes.

At the time of the wrinkling of the germinal vesicle envelope, both 70 kDa dextran and ribosomes entered as a massive wave. The characteristics of this entry indicate that the permeability barrier of the nuclear envelope bilayer has been disrupted. The disruption of the permeability barrier occured in a local region rather than around the entire periphery. Also, the disruption was observed more often on the animal pole side of the germinal vesicle (26/34 oocytes). The endoplasmic reticulum entered the nuclear region more slowly. Cytochalasin B inhibited this movement and also inhibited characteristic endoplasmic reticulum movements seen at high magnification. The effects of cytochalasin indicate that mixing of endoplasmic reticulum with nuclear space is an active process involving actin filaments.
Cleavage furrow: timing of emergence of contractile ring actin filaments and establishment of the contractile ring by filament bundling in sea urchin eggs
Authors: Mabuchi I.
Published: April 2021

Cleavage furrow formation at the first cell division of sea urchin and sand dollar eggs was investigated in detail by fluorescence staining of actin filaments with rhodamine-phalloidin of either whole eggs or isolated egg cortices. Cortical actin filaments were clustered at anaphase and then the clusters became fibrillar at the end of anaphase. The timing when the contractile ring actin filaments appear was precisely determined in the course of mitosis: accumulation of the contractile ring actin filaments at the equatorial cell cortex is first noticed at the beginning of telophase (shortly before furrow formation), when the chromosomal vesicles are fusing with each other. The accumulated actin filaments were not well organized at the early stage but were organized into parallel bundles as the furrowing progressed. The bundles were finally fused into a tightly packed filament belt. Wheat germ agglutinin (WGA)-binding sites were distributed on the surface of the egg in a manner similar to the actin filaments after anaphase. The WGA-binding sites became accumulated in the contractile ring together with the contractile ring actin filaments, indicating an intimate relationship between these sites and actin filament-anchoring sites on the plasma membrane. Myosin also appeared in the contractile ring together with the actin filaments. The ‘cleavage stimulus’, a signal hypothesized by Rappaport (reviewed by R. Rappaport (1986) Int. Rev. Cytol. 105, 245–281) was suggested to induce aggregation or bundling of the actin filaments in the cortical layer.
The nullo protein is a component of the actin-myosin network that mediates cellularization in Drosophila melanogaster embryos
Authors: Postner Marya A. , Wieschaus Eric F.
Published: April 2021

After the 13th nuclear division cycle of Drosophila embryogenesis, cortical microfilaments are reorganized into a hexagonal network that drives the subsequent cellularization of the syncytial embryo. Zygotic transcription of the nullo and serendipity-α genes is required for normal structuring of the microfilament network. When either gene is deleted, the network assumes an irregular configuration leading to the formation of multinuceate cells. To investigate the role of these genes during cellularization, we have made monoclonal antibodies to both proteins. The nullo protein is present from cycle 13 through the end of cellularization. During cycle 13, it localizes between interphase actin caps and within metaphase furrows. In cellularizing embryos, nullo co-localizes with the actin-myosin network and invaginates along with the leading edge of the plasma membrane. The serendipity-α (sry-α) protein co-localizes with nullo protein to the hexagonal network but, unlike the nullo protein, it localizes to the sides rather than the vertices of each hexagon. Mutant embryos demonstrate that neither protein translationally regulates the other, but the localization of the sry-α protein to the hexagonal network is dependent upon nullo.
Immunocytochemical detection of extracellular annexin II in cultured human skin keratinocytes and isolation of annexin II isoforms enriched in the extracellular pool
Authors: Ping Ma Alice Shau , Bell Daniel J. , Mittal Anshu A. , Harrison Harold H.
Published: April 2021

Monoclonal antibodies were raised against trypsinized human skin epidermal cells and selected for their staining of the epidermal cells in a cell periphery pattern. One antibody, CP-1, immunoprecipitated a 36 kDa protein that was identified as annexin II heavy chain by microsequencing of a CNBr-generated peptide fragment from the antigen and by cross-identification with another anti-annexin II antibody. In addition to staining a broad cell periphery band in keratinocytes, CP-1 also detected annexin II outside and in between the top layer cells before cell permeabilization. Double-labeling of annexin II and F-actin revealed a distinct topographical relationship between the two, with intercellular annexin II flanked by the submembranously located actin of the juxtapositioned cells. Annexin II was isolated from cultured keratinocytes via immunoaffinity column chromatography in one step, using the same monoclonal antibody CP-1 and was found to be resolved into multiple isoforms when analyzed by two-dimensional gel electrophoresis. The predominant components of annexin II were basic, with pI of 6.5-8.5, and some of them formed disulfide-linked monomeric multimers under non-reducing conditions. Acidic annexin II isoforms with pI 5.4-5.8 were barely detectable among the total annexin II isolated but were selectively enriched in an extracellular pool created by 0.05% ethylenedi-aminetetraacetic acid (EDTA) dispersion of the cultured cells into single cell suspensions. Furthermore, they can be separated from the rest of annexin II by using a different elution condition. A 46 kDa protein, the identity of which is unclear, co-eluted with the acidic isoforms in the EDTA washes. These acidic isoforms, which co-eluted with the 46 kDa protein, are suspected of corresponding to the extra-cellular annexin II detected immunocytochemically.
Microtubules are required in amoeba chemotaxis for preferential stabilization of appropriate pseudopods
Authors: Ueda Masahiro , Ogihara Satoshi
Published: April 2021

Amoebae of Physarum polycephalum exhibit chemotactic responses to glucose and to cAMP. The chemotaxing amoebae exhibit alternating locomotive movements: relatively linear locomotion and movements that change the direction of the locomotion. Such locomotive activity is tightly coupled with the changes in the number and the positions of the pseudopods; cells have one pseudopod at the leading edge during their linear locomotion, while they have multiple pseudopods when they are changing the direction of locomotion. Treatment of cells with microtubule-disrupting reagents inhibited the chemotaxis of the cells. To characterize the role of the microtubule system in chemotaxis, we quantitatively analyzed the relationship between the positions of multiple pseudopods of the amoebae and the relative stability of the pseudopods during reorientation. No significant differences were observed in the pseudopod dynamics between the untreated and the treated amoebae. In both cases, one pseudopod at the leading edge continued to expand during linear locomotion. It then split into two to three pseudopods in the reorientation phase, and the positions of the multiple pseudopods were random. Among multiple pseudopods, however, the pseudopods closer to the microneedle tip were selectively stabilized more often than those distant from the tip in the presence of the microtubule system. By contrast, such preferential stabilization of the appropriate pseudopods was completely abolished by microtubule inhibitors. The microtubule-dependent selection of appropriately located pseudopods enables amoebae to turn correctly at the reorientation step.
Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules
Authors: Steffen Walter , Fajer Elizabeth A. , Linck Richard W.
Published: April 2021

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific-or crossspecific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated α-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidis-sima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48–50 kDa in isolated spindles and centrosomes from CHO cells.
Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle
Authors: Hozak Pavel , Jackson Dean A. , Cook Peter R.
Published: April 2021

Sites of replication in synchronized HeLa cells were visualized by light and electron microscopy; cells were perme-abilized and incubated with biotin-16-dUTP, and incorporation sites were immunolabelled. Electron microscopy of thick resinless sections from which ∼90% chromatin had been removed showed that most DNA synthesis occurs in specific dense structures (replication factories) attached to a diffuse nucleoskeleton. These factories appear at the end of G1-phase and quickly become active; as S-phase progresses, they increase in size and decrease in number like sites of incorporation seen by light microscopy. Electron microscopy of conventional thin sections proved that these factories are a subset of nuclear bodies; they changed in the same characteristic way and contained DNA polymerase α and proliferating cell nuclear antigen. As replication factories can be observed and labelled in non-permeabi-lized cells, they cannot be aggregation artifacts. Some replication occurs outside factories at discrete sites on the diffuse skeleton; it becomes significant by mid S-phase and later becomes concentrated beneath the lamina.
Regulation of chemical stress-induced hsp70 gene expression in murine L929 cells
Authors: Liu Richard Y. , Corry Peter M. , Lee Yong J.
Published: April 2021

We have investigated the regulation mechanism of chemical stress-induced hsp70 gene expression in murine L929 cells. Our data show that chemical treatments including sodium arsenite, cadmium chloride and sodium salicylate, induced significant synthesis of hsp70 and its mRNA. The induced hsp70 gene expression appears to be regulated at the transcriptional level. A factor (CHBF), which constitutively binds to the heat shock element (HSE) at 37°C, functions like a negative regulator and the heat-induced heat shock factor (HSF) acts as an activator. The chemical treatments that induce significant hsp70 synthesis activate HSF binding to HSE but also dissociate the HSE-CHBF complex. Some chemical treatments, e.g. IPTG, which fail to activate hsp70 gene transcription, still activate HSF binding to HSE. However, in this case, the HSE-CHBF complex remained like that of untreated control cells.
Apical plasma membrane proteins are not obligatorily stored in secretory granules in exocrine cells
Authors: Colomer Veronica , Rindler Michael J. , Lowe Anson W.
Published: April 2021

Exocrine cells are epithelial cells in which secretory granules undergo fusion with the apical plasma membrane upon secretagogue stimulation. Several apical plasma membrane proteins have been found in secretory granules in cells from pancreas and salivary glands raising the possibility that incorporation into secretory granules followed by exocytosis of the granules accounts for their insertion into the apical plasma membrane. To test this hypothesis, we have expressed the influenza hemagglutinin (HA) in pancreatic AR42J cells, which make zymogen-like granules upon incubation with dexamethasone. The influenza virus HA is known to be specifically targeted to the apical plasma membrane of epithelial cells that lack a regulated pathway and is also known to be excluded from secretory granules in virally-infected pituitary AtT20 cells. Localization of the protein by immunofluorescence microscopy revealed that it accumulated at the plasma membrane of the transfected AR42J cells. HA was not observed in the amylase-rich secretory granules. By immunolabeling of ultrathin cryosections of the transfected cells, HA was also found exclusively on the cell surface, with label over secretory granules not exceeding that seen in control, untransfected cells. In addition, in cell fractionation experiments performed on radiolabeled AR42J cell transformants, HA was not detectable in the secretory granule fractions. These results indicate that HA is not efficiently stored in mature secretory granules and is likely to reach the cell surface via constitutive transport pathways.
Movement of axoplasmic organelles on actin filaments assembled on acrosomal processes: evidence for a barbed-end-directed organelle motor
Authors: Langford George M. , Kuznetsov Sergei A. , Johnson Dale , Cohen Darien L. , Weiss Dieter G.
Published: April 2021

The directionality of the actin-dependent motors on squid axoplasmic organelles was determined using actin filaments assembled on the barbed ends of acrosomal processes. Acrosomal processes were isolated from Limulus polyphemus sperm and incubated in monomeric actin under conditions that promoted barbed end assembly only. Newly assembled actin was stabilized and stained with rhodamine-phalloidin and the presence of filaments at the barbed ends of the acrosomal processes was verified by flu-orescence microscopy and negative contrast electron microscopy. Axoplasmic organelles that dissociated from extruded axoplasm were observed by video microscopy to move along the newly assembled actin filaments at an average velocity of 1.1±0.3 μm/second. All organelles moved in the direction away from the acrosomal fragment and towards the tip of the actin filaments. Therefore, the actin-dependent organelle motor on axoplasmic organelles is a barbed-end-directed motor like other myosins analyzed. These findings support the conclusions that axo-plasmic organelles are driven by a myosin-like motor along actin filaments and that these filaments as well as micro-tubules function in fast axonal transport.
The endless story of the glial fibrillary acidic protein
Authors: Chen Wan-Jui , Liem Ronald K. H.
Published: April 2021

All intermediate filament proteins consist of an α-helical rod domain flanked by non-helical N-terminal head and C-terminal tail domains. The roles of the non-helical domains of various intermediate filament proteins in the assembly and co-assembly of higher-order filamentous structures have been studied by many groups but with quite contradictory results. Type III intermediate filaments are unique in that they can form homopolymers both in vitro and in vivo. The expression and assembly characteristics of carboxy- and amino-terminal deletion mutants of glial fibrillary acidic protein (GFAP), an astrocyte-specific type III intermediate filament protein, were examined by transient transfections of either vimentin-positive or vimentin-negative variants of human adrenocarcinoma-derived SW13 cell lines. Whereas complete deletion of the C-terminal tail domain of GFAP results in the formation of polymorphic aggregates, both intranuclear and cytoplasmic in self-assembly experiments, efficient co-assembly of these tail-less GFAP mutants with vimentin can be achieved as long as the KLLEGEE sequence at the C-terminal end of the rod domain is preserved. Up to one-fifth of the C-terminal end of the tail domain can be deleted without affecting the capability of GFAP to self-assemble. The highly conserved RDG-containing motif in the tail domain may be important for self-assembly but is not sufficient. The entire head domain seems to be required for self-assembly. All N-terminal deletion mutants of GFAP share the same phenotype of diffuse cytoplasmic staining when expressed in vimentin-negative SWI3 cells. Although co-assembly with vimentin can still be achieved with completely head-less GFAP, preservation of some of the head domain greatly enhanced the efficiency. Our results form the basis for further, more detailed mapping of the essential regions in filament assembly of GFAP and other type III IFs.
Cell growth-promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2)
Authors: Hayakawa Taro , Yamashita Kyoko , Ohuchi Eiko , Shinagawa Akira
Published: April 2021

Human tissue inhibitor of metalloproteinases-2 (TIMP-2) has a potent growth-promoting activity for wide range of human, bovine and mouse cells, having an optimal concentration (10 ng/ml, 0.46 nM) that is ten-times lower than that of TIMP-1 (Hayakawa et al. (1992)FEBS Lett. 298, 29). Neither TIMP-1 complexed with progelatinase B nor TIMP-2 complexed with progelatinase A, both of which have full inhibitory activity against active forms of matrix metalloproteinases (MMPs), showed any cell growthpromoting activity. On the contrary, both reductively alkylated TIMPs had no MMP inhibitory activity, but significantly stimulated cell proliferation. These facts clearly indicate that the cell-proliferating activity of TIMPs is independent of MMP inhibitory activity. We also demonstrated that [3H]thymidine was significantly incorporated into Raji cells, a Burkitt lymphoma cell line, in the presence of either 4 ng/ml of TIMP-1 or 0.1 ng/ml of TIMP-2. Under steady-state conditions at 4°C, high-(Kd=0.15 nM) and low-(35 nM) affinity binding sites for TIMP-2 were identified on Raji cells with 20,000 and 1.4×105 sites/cell, respectively. Both high- and low-affinity binding of 125I-TIMP-2 to Raji cells were competitively inhibited by unlabeled TIMP-2 but not by unlabeled TIMP-1, suggesting the presence of receptors for TIMP-2 independent from those for TIMP-1. TIMP-2 seems to be another new TIMP cell-growth factor in serum, besides TIMP-1.
Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells
Authors: Soole Kathleen L. , Jepson Mark A. , Hazlewood Geoffrey P. , Gilbert Harry J. , Hirst Barry H.
Published: April 2021

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E’ (EGE’) from Clostridium thermocellum and polarity of secretion of the chimeric EGE’-GPI protein was evaluated. The chimeric EGE’-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and sus-ceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE’ was localized almost exclu-sively to the apical cell surface, while in polarized intesti-nal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE’ was also detected at the basolateral membrane. Rates of delivery of EGE’-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE’ delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE’GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.
Leukemia inhibitory factor (LIF) inhibits angiogenesis in vitro
Authors: Pepper M. S. , Ferrara N. , Orci L. , Montesano R.
Published: April 2021

Using an in vitro model in which endothelial cells can be induced to invade a three-dimensional collagen gel to form capillary-like tubular structures, we demonstrate that leukemia inhibitory factor (LIF) inhibits angiogenesis in vitro. The inhibitory effect was observed on both bovine aortic endothelial (BAE) and bovine microvascular endothelial (BME) cells, and occurred irrespective of the angiogenic stimulus, which included basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), the synergistic effect of the two in combination, or the tumor promoter phorbol myristate acetate. LIF inhibited bFGF- and VEGF-induced proliferation in BAE and BME cells. In addition, LIF inhibited BAE but not BME cell migration in a conventional two-dimensional assay. Finally, LIF decreased the proteolytic activity of BAE and BME cells and increased their expression of plasminogen activator inhibitor-1. These results demonstrate that LIF inhibits angiogenesis in vitro, an effect that can be correlated with a LIF-mediated decrease in endothelial cell proliferation, migration and extracellular proteolysis.
Identification of the cells expressing cot proto-oncogene mRNA
Authors: Ohara Reiko , Hirota Seiichi , Onoue Hitoshi , Nomura Shintaro , Kitamura Yukihiko , Toyoshima Kumao
Published: April 2021

The cell types expressing cot proto-oncogene mRNA were identified by in situ hybridization (ISH) histochemistry. Among a variety of adult mouse tissues examined, four types of glandular cells expressing cot gene were identified: (1) granular duct cells in the submandibular and sublingual glands; (2) serous cells in the parotid gland; (3) peptic (chief) cells in gastric glands; and (4) goblet cells in colonic glands. Investigation of the developmentally regulated expression of cot mRNA using tissues of 14-day and 18-day embryos, newborn and weanling mice showed that cot gene is expressed only in morphologically differentiated and functionally activated cells of these four types. No other types of cells showing ISH signals were observed. Based on these results, cot gene expressions in cultured cells of colonic adenocarcinomas and gastric adenocarcinomas were examined. SW 480 and WiDr cells showed high expression of this gene and so should be useful for functional analysis of Cot kinase. The expression patterns of cot gene in tumor tissues of the parotid gland, and gastric and colonic glands were investigated. Two of the tissues over-expressed this gene markedly, suggesting that overproduction of Cot kinase may be one cause of their transformation.
Functional assessment of white and brown adipocyte development and energy metabolism in cell culture Dissociation of terminal differentiation and thermogenesis in brown adipocytes
Authors: Klaus Susanne , Ely Marina , Encke Dag , Heldmaier Gerhard
Published: April 2021

We investigated the effect of insulin, triiodothyronine (T3) and dexamethasone (a synthetic glucocorticoid) on differentiation, lipid metabolism and thermogenesis of preadipocytes isolated from white fat (WAT) and brown fat (BAT) from the Siberian dwarf hamster (Phodopus sungorus). Cell cultures from WAT and BAT were chronically treated with the above hormones alone or in any combination. After differentiation (day 8 or 9 of culture) we measured the following parameters: adipogenic index (number × size of adipocytes), protein content, lipolysis, cell respiration, and expression of the uncoupling protein UCP, which is unique to mitochondria of brown adipocytes. Insulin was the most important adipogenic factor for brown and white adipocytes and necessary for terminal differentiation, whereas dexamethasone alone completely inhibited differentiation. T3 had no effect on adipogenesis in WAT cultures, but further increased insulin stimulated adipogenesis in BAT cultures. Basal lipolysis was higher in WAT than in BAT cultures except when dexamethasone was present, which stimulated lipolysis in both culture types to the same extent. T3 had a pronounced dose dependent lipolytic effect on WAT cultures but very little effect on BAT cultures. Respiration rates were generally higher in differentiated adipocytes than in fibroblast like cells. T3 had no effect on thermogenesis in WAT cultures but increased thermogenesis in BAT cultures, and this was further elevated by insulin. UCP expression in BAT cultures could be detected by western blot in insulin treated, T3 treated and insulin + T3 treated cultures with highest expression in the latter. These results imply a possible dissociation of terminal differentiation and thermogenic function of brown adipocytes. In WAT cultures there was also a low level of UCP detectable in the insulin + T3 treated cultures. Immuno-fluorescence microscopy analysis revealed the presence of UCP in 10-15% of adipocytes from WAT cultures (in BAT cultures: 90%), indicating the presence of some brown preadipocytes in typical WAT deposits.
Expression of Drosophila lamin C is developmentally regulated: analogies with vertebrate A-type lamins
Authors: Riemer Dieter , Stuurman Nico , Berrios Miguel , Hunter Cecil , Fisher Paul A. , Weber Klaus
Published: April 2021

Vertebrate nuclear lamins form a multigene family with developmentally controlled expression. In contrast, invertebrates have long been thought to contain only a single lamin, which in Drosophila is the well-characterized lamin Dm0. Recently, however, a Drosophila cDNA clone (pG-IF) has been identified that codes for an intermediate filament protein which harbors a nuclear localization signal but lacks a carboxy-terminal CAAX motif. Based on these data the putative protein encoded by pG-IF was tentatively called Drosophila lamin C. To address whether the pG-IF encoded protein is expressed and whether it encodes a cytoplasmic intermediate filament protein or a nuclear lamin we raised antibodies against the recombinant pG-IF protein. The antibodies decorate the nuclear envelope in Drosophila Kc tissue culture cells as well as in salivary and accessory glands demonstrating that pG-IF encodes a nuclear lamin (lamin C). Antibody decoration, in situ hybridization, western and northern blotting studies show that lamin C is acquired late in embryogenesis. In contrast, lamin Dm0 is constitutively expressed. Lamin C is first detected in late stage 12 embryos in oenocytes, hindgut and posterior spiracles and subsequently also in other differentiated tissues. In third instar larvae lamins C and Dm0 are coexpressed in all tissues tested. Thus, Drosophila has two lamins: lamin Dm0, containing a CaaX motif, is expressed throughout, while lamin C, lacking a CaaX motif, is expressed only later in development. Expression of Drosophila lamin C is similar to that of vertebrate lamin A (plus C), which loses its CaaX motif during incorporation into the lamina.
Multinucleated cells can continuously generate mononucleated cells in the absence of mitosis: a study of cells of the avian osteoclast lineage
Authors: Solari F. , Domenget C. , Gire V. , Woods C. , Lazarides E. , Rousset B. , Jurdic P.
Published: April 2021

The multinucleated bone-resorbing osteoclast has a hematopoietic origin. We have demonstrated previously that osteoclasts are derived from the monocytic lineage by fusion of mononuclear macrophage precursors. Using an in vitro osteoclast differentiation model derived from pure populations of chick macrophage cultures, osteoclast-like multinucleated giant cells (MNGCs) can be formed by fusion following an active proliferation phase. However, after reaching a peak with 70% of the culture being MNGCs, a new round of expansion of the mononuclear cells is observed. The following experiments suggest that these mononuclear cells were derived directly from the MNGCs by a budding process, selectively from the central zone of the apical surface. After microinjection of the membrane-impermeable probe, Lucifer Yellow, into single MNGCs, initially only diffuse fluorescence, limited to the whole MNGC injected, was observed. However, after 24-48 hours fluorescent mononuclear cells were observed adjacent but distinct from the injected MNGC. To confirm that these mononuclear cells were indeed derived from a parent MNGC, single MNGCs were cloned into single wells. Within a week, the MNGC was surrounded by mononuclear cells, which eventually populated the entire well. These mononuclear cells could then give rise to a second generation of MNGCs following a three-week period of culture. To determine whether this process required mitosis, MNGCs were cultured for three days in the presence of the mitotic inhibitor, Ara-C, prior to microinjection with Lucifer Yellow. Fluorescent mononuclear cells were still seen to arise from a single injected MNGC under these conditions. Detailed observations by scanning electron microscopy and confocal microscopy indicated that these mononuclear cells arise by a budding process from the central zone of the apical cell surface. A continuum of nuclei was observed to exist in MNGCs, with a distinct and characteristic spatial localization of nuclei. Nuclei located at the basal surface were uniformly oval and regular in shape, being clustered in a central location in a single plane at the base of the MNGCs. Nuclei at the apical surface, in contrast, were clustered and irregular, and twisted in shape. Taken together, our data provide the first evidence that mononuclear cells can be generated from a multinucleated ‘differentiated’ cell type, by a budding process that is independent of mitosis.
Interaction of cdc2 and rum1 regulates Start and S-phase in fission yeast
Authors: Labib Karim , Moreno Sergio , Nurse Paul
Published: April 2021

The p34cdc2 kinase is essential for progression past Start in the G1 phase of the fission yeast cell cycle, and also acts in G2 to promote mitotic entry. Whilst very little is known about the G1 function of cdc2, the rum1 gene has recently been shown to encode an important regulator of Start in fission yeast, and a model for rum1 function suggests that it inhibits p34cdc2 activity. Here we present genetic data suggesting that rum1 maintains p34cdc2 in a pre-Start G1 form, inhibiting its activity until the cell achieves the critical mass required for Start, and find that in the absence of rum1 p34cdc2 has increased Start activity in vivo. It is also known that mutation of cdc2, or overexpression of rum1, can disrupt the dependency of S-phase upon mitosis, resulting in an extra round of S-phase in the absence of mitosis. We show that cdc2 and rum1 interact in this process, and describe dominant cdc2 mutants causing multiple rounds of S-phase in the absence of mitosis. We suggest that interaction of rum1 and cdc2 regulates Start, and this interaction is important for the regulation of S-phase within the cell cycle.
Molecular probing of the secretory pathway in peptide hormone-producing cells
Authors: Holthuis Joost C. M. , Jansen Eric J. R. , Riel Maarten C. H. M. van , Martens Gerard J. M.
Published: April 2021

The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of blackand whiteadapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC.

Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H+-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.
Cytokines modulate phagocytosis and intracellular digestion of collagen fibrils by fibroblasts in rabbit periosteal explants Inverse effects on procollagenase production and collagen phagocytosis
Authors: Zee Erwin van der , Everts Vincent , Hoeben Kees , Beertsen Wouter
Published: April 2021

Degradation of fibrillar collagen may occur in the extracellular space by enzymes, such as the metalloproteinase collagenase, or in the lysosomal apparatus of fibroblasts following phagocytosis. As the mechanisms involved in the regulation of the latter process are unknown, we investigated possible modulating effects of the cytokines epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interleukin-1α (IL-1α) and transforming growth factor-β (TGF-β) on both collagen phagocytosis and the release of collagenase in an in vitro model employing periosteal tissue explants. The data demonstrated that the level of intracellular collagen digestion could be influenced by cytokines: IL-1α inhibited and TGF-β enhanced phagocytosis of fibrillar collagen by periosteal fibroblasts, whereas the cytokines had an opposite effect on the release of procollagenase. In combination, IL-1α and TGF-β proved to have an antagonizing effect on either parameter. PDGF and EGF had no effect on phagocytosis or collagenase release. The level of phagocytosed collagen correlated positively with the actual breakdown of collagen as assessed by the release of hydroxyproline but negatively with the level of released procollagenase. Our findings demonstrated that cytokines are able to modulate both the phagocytosis of collagen fibrils by fibroblasts and their subsequent intracellular breakdown, as well as the release of procollagenase, an enzyme considered crucial for extracellular collagenolysis. Moreover, our data show a negative correlation between these two parameters. It is concluded that IL-1α, EGF and TGF-β may be important in modulating the contribution of the intracellular and extracellular route of collagen breakdown.
Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles
Authors: Adessi Céline , Chapel Agnès , Vinçon Mathilde , Rabilloud Thierry , Klein Gérard , Satre Michel , Garin Jérôme
Published: April 2021

Magnetic isolation of endocytic vesicles from Dictyostelium discoideum was accomplished after feeding the amoebae with iron oxide particles. Proteins associated with the endocytic vesicles were resolved by SDS-PAGE and digested ‘in-gel’ with endoproteinase Lys-C or Asp-N to generate peptides for amino acid sequencing. This strategy allowed the identification of the major protein constituents of the vesicles: namely, the A, B, D, E and 110 kDa subunits of a vacuolar type H+-ATPase, actin, a Rab 7-like GTPase, a p34 protein corresponding to a new cysteine proteinase and the 25 kDa product of a recently sequenced D. discoideum open reading frame.
The rab7 gtpase resides on a vesicular compartment connected to lysosomes
Authors: Méresse Stéphane , Gorvel Jean-Pierre , Chavrier Philippe
Published: April 2021

Rab GTPases belong to the Ras GTPase superfamily and are key regulators of membrane traffic. Among them, rab7 has been localized on late endosomes of NRK cells but its function remains unknown. In order to investigate its role, we generated stable HeLa cell lines that express either wild type or a GTPase-defective mutant of rab7 in an inducible manner. A morphological analysis of the intracellular localization of these proteins was performed by confocal laser microscopy. Here we show that, in HeLa cells, rab7 is present on a vesicular compartment that extends from the perinuclear area to the cell periphery and shows only a partial colocalization with the cation-independent mannose 6-phosphate receptor, a marker for late endosomes. The topology of this compartment is dependent on the microtubule network since nocodazole treatment results in its scattering throughout the cytoplasm. In addition, we observed that, in contrast to the wild-type protein, a rab7 mutant with a reduced GTPase activity is in part associated with lysosomal membranes. This observation was confirmed by subcellular fractionation in a Percoll gradient. Our data implicate rab7 as the first GTPase functioning on terminal endocytic structures in mammalian cells.
Drosophila ppy, a novel male specific protein serine/threonine phosphatase localised in somatic cells of the testis
Authors: Armstrong Christopher G. , Mann David J. , Berndt Norbert , Cohen Patricia T. W.
Published: April 2021

Drosophila protein phosphatase Y (PPY) displays 64% amino acid identity to protein serine/threonine phos-phatase 1 (PP1) and 39% to protein phosphatase 2A (PP2A). Here we show by expression of cDNA in bacteria, that PPY is a protein serine phosphatase and that its bio-chemical properties are distinct from PP1 in both substrate specificity and regulation by the thermostable inhibitory proteins inhibitor 1 and inhibitor 2. We also demonstrate that PPY is a novel testis specific protein phosphatase by analysis of both mRNA and protein distribution. More precise immunolocalisation within the testis, using affinity purified anti-PPY protein and anti-PPY peptide antibodies, shows that PPY is present in somatic cyst cells, which encase the germ cells. The predominant location of PPY is in the nuclei of both head and tail cyst cells throughout the length of the testis except for the apical tip. The distribution of PPY, coupled with its unique biochemical proper-ties, suggests that PPY may be required to prevent cyst cell division, increase transcription for provision of nutrients to the germ cells and/or provide a signal for spermatocyte differentiation.
Cdebp, a site-specific dna-binding protein of the ‘app-like’ family, is required during the early development of the mouse
Authors: Blangy Anne , Vidal Frédérique , Cuzin François , Yang Yin-Hua , Boulukos Kim , Rassoulzadegan Minoo
Published: April 2021

A murine protein, termed CDEBP, was previously shown to bind the double-stranded DNA motif GTCACATG, identical to the yeast centromeric element CDEI. The cDNA sequence showed three domains with extensive similarities to the amyloid β precursor protein (APP). The protein is homologous over its entire length to the human protein designated APPH. In situ immunofluorescence assays using antibodies raised against distinct parts of CDEBP detected discrete sites of accumulation inside the interphase nucleus, and the bulk of the protein was not associated with mitotic chromosomes. One of the complexes with double-stranded CDEI oligonucleotides detected by gel shift assay was not present when the protein had been selectively removed from nuclear extracts by immunoprecipitation. We reported previously that microinjection into one-cell mouse embryos of DNA fragments including the CDEI sequence results in an early arrest of development with abnormal nuclei containing variable amounts of DNA. The same characteristic figures were observed when embryos were treated with antisense oligonucleotides complementary to parts of the CDEBP coding region. Complexes between the CDEBP protein and CDEI sites in the mouse genome thus appear to play a critical role in the replication/segregation of the embryonic genome.
Scanning force microscopy of microtubules and polymorphic tubulin assemblies in air and in liquid
Authors: Vater W. , Fritzsche W. , Schaper A. , Böhm K. J. , Unger E. , Jovin T. M.
Published: April 2021

We have investigated microtubules (MTs) and polymor-phic assemblies, formed in vitro from isolated microtubule protein, by scanning force microscopy (SFM) in air and in liquid. Immobilization of MTs was achieved by placing a drop of the assembly solution on a polylysine-coated coverslip. After washing with taxol and air drying, the characteristic microtubular fibrous morphology appeared in the SFM. The MTs formed a network similar to that obtained by transmission electron microscopy (TEM). A height of ∼9.5 nm for dried MTs was computed from the surface topography. Glutaraldehyde fixation of the MTs yielded higher structures (∼14 nm), which swelled to ∼20 nm after rehydration, a value close to the MT diameter of ∼25 nm determined from TEM images of ultrathin sections. The protofilament pattern of the MTs and surface attached MT-associated proteins were not apparent from SFM, although the height along the long axis of the MTs appeared slightly modulated. In addition to MTs, various polymorphic tubulin assemblies including ribbons, hoops and double-walled MTs were visualized by SFM.
High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes
Authors: Moscatelli Alessandra , Casino Cecilia Del , Lozzi Luisa , Cai Giampiero , Scali Monica , Tiezzi Antonio , Cresti Mauro
Published: April 2021

Nicotiana tabacum pollen tubes contain two high molecular weight polypeptides (about 400 kDa), which are specifically expressed during pollen germination and pollen tube growth in BK medium. The high molecular weight doublet resembles the dynein heavy chains in some biochemical properties. Sedimentation profiles of pollen tube extracts show that the high molecular weight bands have sedimentation coefficients of 22 S and 12 S, respectively. ATPase assay of sedimentation fractions shows an activity ten times higher when stimulated by the presence of bovine brain microtubules in fractions containing the 22 S high molecular weight polypeptide. Both these high molecular weight polypeptides can bind microtubules in an ATP-dependent fashion. A mouse antiserum to a synthetic peptide reproducing the sequence of the most conserved ATP-binding site among dynein heavy chains recognized the two high molecular weight polypeptides. Therefore these polypeptides have sequences immunologically related to the ATP binding sites of dynein heavy chains.
The functional thrombin receptor is associated with the plasmalemma and a large endosomal network in cultured human umbilical vein endothelial cells
Authors: Horvat R. , Palade G. E.
Published: April 2021

The functional thrombin receptor, normally expressed by endothelial cells and platelets, is a member of the G protein-coupled, seven membrane-spanning-domain receptor family and is thought to be responsible for most, if not all, the cell stimulatory effects of thrombin. Upon binding, thrombin cleaves the receptor’s N-terminal ectodomain, unmasking a new N terminus, which by itself activates the receptor. Using antibodies to different domains of the human thrombin receptor, we have localized the receptor in cultured human umbilical vein endothelial cells by indirect immunofluorescence and immunoelectron microscopy. We found the receptor expressed on the plas-malemma of cultured endothelial cells in individual units rather than in clusters, at lower concentration than, and at different sites from, thrombomodulin. We also found the receptor associated with a distinct, intracellular, transfer-rin receptor-containing, tubulovesicular network. The thrombin receptor-positive structure spread from the per-inuclear region to the periphery of the cells, exhibiting a number of varicosities interconnected by branching tubular elements, strikingly similar to an image recently described for a continuous endosomal reticulum. Our results provide morphological evidence for the presence of the functional thrombin receptor at relative low density on the surface of cultured endothelial cells (compared to thrombomodulin) and in relatively large quantities inside the cells, associated with an endosomal compartment.
A high molecular mass protein isolated from chicken gizzard: its localization at the dense plaques and dense bodies of smooth muscle and the Z-disks of skeletal muscle
Authors: Terasaki Asako Goi , Nakagawa Hiroyuki , Kotani Eiji , Mori Hajime , Ohashi Kazuyo
Published: April 2021

We purified a 450 kDa protein from a low-salt alkaline extract of chicken gizzard smooth muscle. This high molecular mass protein could be extracted with the low-salt alkaline solution at 37°C but not at 4°C. The 450 kDa protein was isolated from the extract by ammonium sulfate fractionation and following sequential column chromatography using hydroxylapatite, DEAE-Cellulofine A-800m and phenyl-Sepharose CL-4B resins. The partially purified protein molecule resembled a flexible rod with a globular head and an irregular-shaped tail. Its length was approxi-mately 300 nm. The nucleotide sequence of the partial cDNA encoding this protein was determined and analyzed with a data base. The analysis showed that the protein revealed significant homology with the rod region of chicken filamin (57% homology in amino acid sequence). Immunoblot analysis showed that an affinity-purified antibody reacted exclusively with the 450 kDa protein band of smooth, skeletal and cardiac muscle tissues. By indirect immunofluorescence microscopy, we examined the local-ization of the 450 kDa protein in smooth and skeletal muscle cells. The affinity-purified antibody against the 450 kDa protein stained the dense plaques and dense bodies of smooth muscle, the peripheral region of Z-disks and the subsarcolemmal region of skeletal muscle. Immunoelectron microscopy confirmed the localization of the 450 kDa protein at the peripheral regions of the actin anchoring structures mentioned above. Judging from its amino acid sequence, molecular size, molecular shape, immunological reactivity and localization in muscle cells, the 450 kDa protein seemed to be a new component associated with the actin-anchoring structures of muscle tissues. We tenta-tively call this protein ‘fulcin’.