bluuebunny/crossref_metadata_2025 huggingface dataset:
A three-dimensional reconstruction study of the rough ER-Golgi interface in serial thin sections of the pancreatic acinar cell of the rat
Authors: Sesso Antonio , Paulo de Faria Flávio , Iwamura Edna Sadayo Miazato , Corrêa Hélio
Published: April 2021

Distinctive views of the tubulo-vesicular elements interposed between the endoplasmic reticulum (ER) and the Golgi apparatus were obtained in thin sections. The tubules that protrude from the transitional rough ER (tRER) are of dissimilar length. The numbers of tubules and of the nearby omega- and pear-shaped profiles decrease after fasting and are partially restored by refeeding. This formation is designated herein as the budding chamber of the tRER. Close to the budding chamber, clusters of 56 nm diameter vesicles are consistently observed. In some of the cells, convoluted tubules appear enmeshed with the presumptive transport vesicles of 56 nm diameter and with irregular, vesicular formations. Apparently structureless, electron-lucent ellipsoidal areas are found adjacent to these membranous elements. Serial and semi-serial sections show that the budding chamber, the sinuous tubules, the irregular vesicles, the structureless regions and the 56 nm vesicles fill tunnel-like spaces limited by the outermost Golgi cisterna (OGC) on one side and by the tRER on the other. Curved tubules appear to link the lumen of the OGC with that of smooth membranous occupants of these tunnel-like spaces. A presumptive luminal connection between these membranous occupants and the tubules of the budding chamber can also be seen. The predominant configuration of the OGC is that of a perforated, flat saccule. However, OGC regions exhibiting progressively lower densities of fenestrae, including smooth surfaced sectors eventually accumulating an intraluminal content are seen. Two such dilated, saccular portions of the OGC were analyzed through reconstruction of serial sections. Bundles of microtubules run closely apposed to the cis side of the OGC.
Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells
Authors: Vélez-Granell Carlos S. , Arias Ariel E. , Torres-Ruíz José A. , Bendayan Moïse
Published: April 2021

Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluo-rescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.
Adhesive properties and integrin expression profiles of two colonic cancer populations differing by their spreading on laminin
Authors: Simon-Assmann P. , Leberquier C. , Molto N. , Uezato T. , Bouziges F. , Kedinger M.
Published: April 2021

The mostly undifferentiated parental HT29 (HT29p) human colonic adenocarcinoma cell line and a differentiated subpopulation selected by the anti-cancer drug 5-fluorouracil (HT29-Fu) (Lesuffleur et al. (1991)Int. J. Cancer 49, 721-730) display strikingly different behavior when grown on laminin coatings: the former grows as aggregates while the latter grows as monolayers. In an attempt to explain this difference, we performed a comparative study of cell adhesion properties and of expression, involvement and localization of the α6, β1 and β4 subunits constituting the integrin family among the two cell populations.

HT29p and HT29-Fu cells exhibited a similar adhesion pattern to laminin and laminin fragments E8 and P1. In both cell lines, cell adhesion could be blocked at about 90% with anti-α6 subunit antibodies and around 30-50% with anti-β1 antibodies; no inhibition of the cell adhesion was obvious when using anti-β4 antibodies. Immunoprecipitations of iodinated membrane-solubilized proteins and immunoblotting experiments showed that all α6 chains expressed in both HT29p and HT29-Fu cell populations exist as α6β4 integrins; β1 subunits are associated with α2 and α3 chains. When HT29p or HT29-Fu cells were injected subcutaneously in nude mice, a similar expression pattern of α6, β4 and β1 integrin subunits was noticeable in the resulting tumors: α6 and β4 subunits were localized at the basal surface of the tumor cells facing the stromal elements, and to a lesser extent at the cell-cell contacts within the tumor-cell clumps; β1 subunits were mainly found within the cytoplasm of the tumor cells.

Despite these overall similarities among the two cell lines, the following changes could account for their different behavior on laminin: less proteolytic processing of the β4 integrin subunit occurred in HT29-Fu cells yielding peptidic fragments of 175 kDa, which are absent from the parental cells; the immunostaining pattern of the various subunits demonstrated a segregation of α6, β4 and β1 integrin subunits on the basal side of the HT29-Fu cells when cultured on laminin to the detriment of their lateral location, a phenomenon that was not obvious in the parental cells.

Altogether, these results suggest that the distinct behavior of the undifferentiated versus differentiated HT29 cell populations on laminin is not related to altered adhesion properties of the cells but rather to a deficient stabilization of the adhesion leading to cell spreading. This difference is highlighted by a variable basal segregation potential of the laminin-binding integrins, which could be due to an altered form of the β4 subunits.
Analysis of the Schizosaccharomyces pombe cyclin puc1: evidence for a role in cell cycle exit
Authors: Forsburg Susan L. , Nurse Paul
Published: April 2021

The puc1+ gene, encoding a G1-type cyclin from the fission yeast Schizosaccharomyces pombe, was originally isolated by complementation in the budding yeast Saccharomyces cerevisiae. Here, we report the molecular characterization of this gene and analyse its role in S. pombe. We fail to identify any function of this cyclin at the mitotic G1/S transition in S. pombe, but demonstrate that it does function in exit from the mitotic cycle. Expression of the puc1+ gene is increased during nitrogen starvation, and puc1 affects the timing of sexual development in response to starvation. Overexpression of the puc1 protein blocks sexual development, and rescues pat1ts cells, which would otherwise undergo a lethal meiosis. We conclude that puc1 contributes to negative regulation of the timing of sexual development in fission yeast, and functions at the transition between cycling and non-cycling cells.
Study of the higher eukaryotic gene function CDK2 using fission yeast
Authors: Paris Jeannie , Leplatois Pascal , Nurse Paul
Published: April 2021

In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis. In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2. Temperature-sensitive mutations in S. pombe cdc2 that arrest the cell cycle in both G1 and G2 phases are not complemented by CDK2. We have used S. pombe to investigate what functions CDK2 can perform. We found that overexpression of the human homologue (HsCDK2) caused cell cycle arrest in G2/M showing that HsCDK2 interfered with mitotic events. Xenopus CDK2 (XlCDK2) overexpression did not cause cell cycle arrest and could rescue the G1 block but not the G2 block of a cdc2-M26 ts strain. A mutant XlCDK2-R33, which is inactive as a kinase, failed to rescue the G1 block, suggesting that the protein kinase activity of CDK2 is required to enter the cell cycle in these circumstances.

We designed screens to select mutants that would require XlCDK2 expression for viability, hoping to isolate new gene functions interacting with, or that could be replaced by, XlCDK2 in G1, or new cdc2 mutants altered solely in their G1 role. From these screens several cell cycle mutants were selected that were XlCDK2-dependent. These were all cdc2 mutants altered only in their G2/M function. Therefore XlCDK2 can influence both the G1/S and G2/M transition points of cdc2 in S. pombe.
Cytoskeletal involvement in the modulation of cell-cell junctions by the protein kinase inhibitor H-7
Authors: Citi Sandra , Volberg Tova , Bershadsky Alexander D. , Denisenko Natasha , Geiger Benjamin
Published: April 2021

The protein kinase inhibitor H-7 has been shown to block junction dissociation induced by low extracellular calcium in Madin Darby canine kidney epithelial cells (S. Citi, J. Cell Biol. (1992) 117, 169-178). To understand the basis of this effect, we have examined how H-7 affects the organization of junctions and the actin cytoskeleton in different types of epithelial cells in culture. Immunofluorescence microscopy showed that H-7 confers Ca2+ independence on cultured epithelial lens cells, which lack tight junctions and desmosomes but have microfilament-associated adherens junctions. In these cells, H-7 did not protect N-cadherin from trypsin digestion at low extracellular calcium, suggesting that H-7 does not stabilize the ‘active’ cadherin conformation. In cultured Madin Darby canine kidney cells, H-7 partially prevented the fall in transepithelial resistance induced by cytochalasin D, either alone or in conjunction with calcium chelators. Double-immunofluorescence microscopy showed that H-7 inhibits both the fragmentation of labeling for the tight junction protein cingulin and the condensation of actin into cytoplasmic foci induced by cytochalasin D. Taken together, these observations indicate that H-7 inhibits junction dissociation by affecting the contractility of the adherens junction-associated microfilaments following treatment with calcium chelators or cytochalasin D.
Exogenous histone H1 injection into mitotic cells disrupts synchronous progression of mitotic events by delaying chromosome decondensation
Authors: Matsuoka Yosuke , Takechi Shinji , Nakayama Tatsuo , Yoneda Yoshihiro
Published: April 2021

At the end of open mitosis, chromosome decondensation, nuclear envelope re-formation and reassembly of interphase microtubules following mitotic spindle dissociation occur coordinately. To determine whether these events progress only synchronously in vivo, we delayed chromosome decondensation by injecting of exogenous proteins into the mitotic rat kangaroo kidney epithelium (PtK2) cells. When histone H1 purified from calf thymus was injected at prometaphase, chromosome condensation was prolonged for several hours, and sister chromatid separation and cytokinesis did not occur. However, interphase microtubules reassembled and lamin B-positive structures re-formed around the condensed chromosomes. Exactly the same results were obtained on injection of bacterially expressed H1. Kinetic experiments showed that there were two types of lamin B-positive structures. One type (type A) was stained uniformly with anti-lamin B antibodies. The other (type B) showed peripheral lamin B staining; that is, the normal interphase staining pattern, and was found to be competent for nuclear protein transport. As the chromosomes decondensed, the amount of type A decreased and that of type B increased. However, even cells containing highly condensed chromosomes had both type A and type B. From these results, we conclude that the re-formation of microtubules and reassembly of a nuclear transport-competent envelope do not depend on chromosome decondensation.
Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins
Authors: Milgram Sharon L. , Mains Richard E.
Published: April 2021

Vesicular transport within the secretory pathway can be arrested by incubating cells at 15°C or 20°C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble peptidylglycine α-amidating monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4°C, 15°C, 20°C or 37°C. The endoproteolytic processing of peptidylglycine α-amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble peptidylglycine α-amidating monooxygenase proteins was completely blocked by incubation of cells at 20°C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15°C and 20°C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20°C. In AtT-20 cells and neurointermediate pituitary cultures, generation of β-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20°C, while processing of β-lipotropin to β-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while peptidylglycine α-amidating monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.
Endocrine and mucous differentiation by a cloned human rectal adenocarcinoma cell line (HRA-19) in vitro: inhibition by TGF-β1
Authors: Kirkland Susan C. , Henderson Karen
Published: April 2021

Colorectal epithelium is composed of absorptive, mucous and endocrine cells, all of which are considered to arise from a common stem cell located in the crypt base. However, the factors controlling the commitment to differentiate are poorly understood. This is partly due to the lack of in vitro model systems for the study of differentiation in colorectal epithelium. The HRA-19 cell line, established from a human rectal adenocarcinoma, has been shown to have multipotential characteristics with cloned HRA-19 cells able to differentiate into absorptive, mucous and endocrine cells when grown as xenografts. The lack of such differentiated cells in HRA-19 monolayers in vitro suggests that differentiation is controlled by extracellular matrix, stromal cells and/or soluble factors. Such observations show that differentiation in HRA-19 cells can be controlled by extrinsic factors and therefore provide a model system for studying control of differentiation in colorectal epithelium. Unfortunately, the restriction of differentiation to xenografts of the cell line limits the degree to which this differentiation can be manipulated.

In this study, the possibility that HRA-19 cells could be induced to differentiate in vitro under appropriate conditions has been investigated. Endocrine and mucous cells were identified by immunocytochemistry with differentiation-related antibodies and histology of monolayers. Preconfluent HRA-19 cells grown in 10% foetal calf serum formed a well polarised monolayer with apical tight junctions and sparse microvilli, but cells with mucous or endocrine phenotypes were only very occasionally observed. However, endocrine and mucous cells could reproducibly be demonstrated in postconfluent monolayers grown in 1% foetal calf serum. The numbers of endocrine and mucous cells were quantified in monolayers, grown on coverslips, and this formed the basis for a differentiation assay. In this assay, TGF-β inhibited HRA-19 cell proliferation and also inhibited differentiation into both endocrine and mucous cells.

Therefore, HRA-19 cells provide a useful model system for identifying factors controlling differentiation in colorectal epithelial cells.
Cytoskeletal proteins of insect muscle: location of zeelins in Lethocerus flight and leg muscle
Authors: Ferguson C. , Lakey A. , Hutchings A. , Butcher G. W. , Leonard K. R. , Bullard B.
Published: April 2021

Asynchronous insect flight muscles produce oscillatory contractions and can contract at high frequency because they are activated by stretch as well as by Ca2+. Stretch activation depends on the high stiffness of the fibres and the regular structure of the filament lattice. Cytoskeletal proteins may be important in stabilising the lattice. Two proteins, zeelin 1 (35 kDa) and zeelin 2 (23 kDa), have been isolated from the cytoskeletal fraction of Lethocerusflight muscle. Both zeelins have multiple isoforms of the same molecular mass and different charge. Zeelin 1 forms micelles and zeelin 2 forms filaments when renatured in low ionic strength solutions. Filaments of zeelin 2 are ribbons 10 nm wide and 3 nm thick.

The position of zeelins in fibres from Lethocerusflight and leg muscle was determined by immunofluorescence and immunoelectron microscopy. Zeelin 1 is found in flight and leg fibres and zeelin 2 only in flight fibres. In flight myofibrils, both zeelins are in discrete regions of the A- band in each half sarcomere. Zeelin 1 is across the whole A-band in leg myofibrils. Zeelins are not in the Z-disc, as was thought previously, but migrate to the Z-disc in glycerinated fibres. Zeelins are associated with thick filaments and analysis of oblique sections showed that zeelin 1 is closer to the filament shaft than zeelin 2.

The antibody labelling pattern is consistent with zeelin molecules associated with myosin near the end of the rod region. Alternatively, the position of zeelins may be determined by other A-band proteins. There are about 2.0 to 2.5 moles of myosin per mole of each zeelin. The function of these cytoskeletal proteins may be to maintain the ordered structure of the thick filament.
Fission yeast protein kinase C gene homologues are required for protoplast regeneration: a functional link between cell wall formation and cell shape control
Authors: Kobori Hiromi , Toda Takashi , Yaguchi Hiroko , Toya Mika , Yanagida Mitsuhiro , Osumi Masako
Published: April 2021

Two novel protein kinase C (nPKC) gene homologues, pck1+and pck2+were isolated from the fission yeast Schizosaccharomyces pombe(Toda et al. (1993)EMBO J. 12, 1987). We examined the functional differences of pck1+and pck2+in cell wall formation and actin organization of S. pombe. Regenerating protoplasts of a wild-type strain, single gene disruptants of pck1+(Δpck1) and pck2+(Δpck2) were used as a simple model to examine the functional links between PKC, cell wall formation and actin organization. Protoplasts of the wild-type strain and those of Δpck1reverted to intact cells in osmotically stabilized liquid medium. A close spatial association between new cell wall formation and actin was observed in these two strains. In Δpck2, protoplasts did not revert to intact cells: (1) scarcely any new cell wall material was formed; (2) actin was not reorganized; and (3) nuclear division and an increase in the amount of cytoplasm were observed in the regenerating protoplasts. These findings demonstrate that the pck2+gene has a function essential for protoplast regeneration but the pck1+gene does not. Involvement of nPKCs in cell wall formation and actin organization was also clarified. The effect of staurosporine (a potent inhibitor of protein kinases) on regenerating protoplasts of the three strains confirmed the assumption that the pck2 protein is an in vivo target of staurosporine in the fission yeast.
Macrophage recruitment during limb development and wound healing in the embryonic and foetal mouse
Authors: Hopkinson-Woolley James , Hughes Derralynn , Gordon Siamon , Martin Paul
Published: April 2021

Macrophages play a pivotal role in the adult inflammatory response to wounding. They are directly responsible for cellular débridement and, by providing a source of growth factors and cytokines, they recruit other inflammatory and fibroblastic cells and influence cell proliferation and tissue remodelling. In this paper we investigate the role of macrophages in clearing areas of programmed cell death in the developing embryo and also their role in embryonic and foetal wound healing. Immunocytochemistry using the monocyte/macrophage-specific monoclonal antibody, F4/80, reveals a close association between areas of programmed cell death in the remodelling interdigital regions of the mouse footplate and of F4/80-positive cells, suggesting that monocyte-derived macrophages, and not locally recruited fibroblastic cells, as previously believed, are responsible for phagocytosing and clearing areas of interdigital apoptosis. Our studies of wound healing reveal that macrophages are not recruited to, and therefore cannot be playing an active role in the healing of, excisional wounds made in the mouse embryo at any stage up until E14.5. Beyond this transition stage we see a significant recruitment of macrophages within 12 hours of wounding. We find that macrophages can be attracted to wounds in earlier embryos if the wound results in significant cell death such as after burning.
p53 expression in cultured cells following radioisotope labelling
Authors: Dover Robin , Jayaram Yogi , Patel Kirtika , Chinery Rebecca
Published: April 2021

p53 inhibits division following cellular damage. Cultured cells were found to express p53 protein following pulse labelling with radioisotopes, even at low doses normally used for growth and metabolic labelling studies. Some stem cells are exquisitely sensitive to radiation and thus p53 may have evolved as a major regulator of stem cell function. Therefore anygenetic damage may be able to induce p53 expression, which in turn will affect the biochemical outcome of many experiments by both cell cycle arrest and other mechanisms. In some cases the use of radioisotopes may directly change the results of the experiment. This will require a careful re-evaluation of the current literature and experimental protocols utilising radioisotopes.
17-β-Estradiol induced alterations of cell-matrix and intercellular adhesions in a human mammary carcinoma cell line
Authors: DePasquale Joseph A. , Samsonoff William A. , Gierthy John F.
Published: April 2021

The MCF-7 human mammary carcinoma cell line undergoes morphological differentiation in vitro when treated with 17-β-estradiol. A prominent feature of this process is the postconfluent development of multicellular, three-dimensional nodules that rise above the surrounding monolayer. Formation of the nodules suggests that changes in cellular adhesion occur during this cellular overgrowth. Therefore changes in the distribution of cell-matrix and cell-cell adhesion plaque proteins were examined with respect to estradiol induction of nodule development. Estradiol treatment of the carcinoma cell line had the following effects: (1) vinculin- and talin-rich cell-matrix adhesion plaques were reduced in overall number and size in confluent and postconfluent cultures. No overt change in distribution or morphology of adhesion plaques was observed in subconfluent cultures. (2) Staining for vinculin was reduced in cell-cell adhesions situated at the apical region of subconfluent, confluent and postconfluent mono- layers. Staining for F-actin and plakoglobin was retained at this region in estradiol-induced cells. (3) vinculin was not detected in intercellular adhesions of nodule cells although intense labelling for both F-actin and plakoglobin was observed. In addition, in untreated monolayer cells, both F-actin and plakoglobin were concentrated in a subapical/basolateral location, as a vesicle-like pattern, which corresponded to intercellular spaces observed with phase-contrast microscopy. Treatment with estradiol caused the rearrangement of subapical/ basolateral F-actin and plakoglobin staining into a more uniform pattern. The findings of this study show that estradiol induces changes in both cell-matrix and cell-cell adhesions in an estrogen- responsive carcinoma cell line. The gradual loss of vinculin from cell-matrix and cell-cell adherens junctions of the monolayer could be a potential factor in the capacity of these cells to form multilayers or nodules in postconfluent growth. Furthermore, the development of the nodules in response to estradiol may provide a useful system in which to study steroid hormone regulation of adhesion and the cytoskeleton in responsive tumor cells.
Two proteins associated with secretory granule membranes identified in chicken regulated secretory cells
Authors: Jean-Léon Thomas , Stieber Anna , Gonatas Nicholas
Published: April 2021

Lately, we have identified two polypeptides of 92-94 kDa (GRL1) and 45-60 kDa (GRL2), expressed in cytoplasmic granules of chicken granulocytes and thrombocytes.

Here, we report that GRL1 and GRL2 are widely distributed in all exocrine and several endocrine cell types, but not in neurons of the central nervous system, during late stages of embryonic development, as well as in newly hatched and two-month-old chickens. Immunogold studies in ultrathin frozen sections of pancreatic acinar cells show that GRL1 and GRL2 are co-localized at the periphery of zymogen granules, in granules fused with apical acinar membranes and on apical membranes of acini, while the pregranular compartments of the secretory pathway are weakly or not labeled. Semiquantitative morphometric studies indicate that GRL1 and GRL2 are equally distributed in secretory granules.

A variety of physical and metabolic studies reveal that GRL2, a highly N-glycosylated polypeptide, is an intrinsic membrane protein, while GRL1 is a peripheral membrane polypeptide released by Na2CO3treatment of granulocyte membranes. In all hematopoietic, exocrine or endocrine cells examinated, GRL1 shows identical electrophoretic patterns, while GRL2 is identified as a diffuse band, at 40-65 kDa, in hematopoietic and pancreatic cells. Taken together, the morphological and biochemical studies indicate that GRL1 and GRL2 are components of the secretory granule membrane in chicken exocrine, endocrine and hemopoietic cell types.
The intracellular mobility of a viral membrane glycoprotein measured by confocal microscope fluorescence recovery after photobleaching
Authors: Storrie Brian , Pepperkok Rainer , Stelzer Ernst H. K. , Kreis Thomas E.
Published: April 2021

Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins. However, the application of FRAP to the study of intracellular membrane proteins has been hampered by the lack of specific probes and their physical inaccessibility in the cytoplasm. We have measured the mobility of a model transmembrane protein, the temperature-sensitive vesicular stomatitis viral membrane glycoprotein (ts-O45-G), in transit from the endoplasmic reticulum (ER) to the Golgi complex. ts-O45-G accumulates in the ER at nonpermissive temperature (39.5°C) and is transported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31°C). Rhodamine-labeled Fab fragments against a cyto-plasmic epitope of ts-O45-G (rh-P5D4-Fabs) were microin-jected into cells to visualize the intracellular viral membrane protein and to determine its mobility by FRAP with a confocal microscope. Moreover, we have measured the effects of microinjected antibodies against β-COP on the mobility of ts-O45-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-injected cells were bleached either following release of labeled ts-O45-G from the ER or upon its accumulation at 20°C in the trans-Golgi network (TGN). In contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to β-COP in vivo. The diffusion constant of mobile ts-O45-G under all conditions was ∼10×10−10cm2/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane protein and provide the first evidence that such a protein is highly mobile.
Modulation of tight junction structure in blood-brain barrier endothelial cells Effects of tissue culture, second messengers and cocultured astrocytes
Authors: Wolburg Hartwig , Neuhaus Jochen , Kniesel Uwe , Krauß Bodo , Schmid Eva-Maria , Öcalan Mücella , Farrell Catherine , Risau Werner
Published: April 2021

Tight junctions between endothelial cells of brain capillar- ies are the most important structural elements of the blood- brain barrier. Cultured brain endothelial cells are known to loose tight junction-dependent blood-brain barrier char- acteristics such as macromolecular impermeability and high electrical resistance. We have directly analyzed the structure and function of tight junctions in primary cultures of bovine brain endothelial cells using quantitative freeze-fracture electron microscopy, and ion and inulin permeability. The complexity of tight junctions, defined as the number of branch points per unit length of tight junc- tional strands, decreased 5 hours after culture but there- after remained almost constant. In contrast, the association of tight junction particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased continu- ously with a major drop between 16 hours and 24 hours. The complexity of tight junctions could be increased by elevation of intracellular cAMP levels while phorbol esters had the opposite effect. On the other hand, the P-face asso- ciation of tight junction particles was enhanced by elevation of cAMP levels and by coculture of endothelial cells with astrocytes or exposure to astrocyte-conditioned medium. The latter effect on P-face association was induced by astrocytes but not fibroblasts. Elevation of cAMP levels together with astrocyte-conditioned medium synergistically increased transendothelial electrical resis- tance and decreased inulin permeability of primary cultures, thus confirming the effects on tight junction structure and barrier function. P-face association of tight junction particles in brain endothelial cells may therefore be a critical feature of blood-brain barrier function that can be specifically modulated by astrocytes and cAMP levels. Our results suggest an important functional role for the cytoplasmic anchorage of tight junction particles for brain endothelial barrier function in particular and probably paracellular permeability in general.
Identification and characterization of tropomodulin and tropomyosin in the adult rat lens
Authors: Woo Mary K. , Fowler Velia M.
Published: April 2021

The lens fiber cells express all the major components of the erythrocyte membrane skeleton including spectrin, protein 4.1 and ankyrin. We have used immunoblot and immuno-precipitation analyses, as well as immunofluorescence localization to identify and characterize two additional components of the membrane skeleton in the rat lens: tropomyosin and the tropomyosin-binding protein tropomodulin. In the erythrocyte, tropomyosin and tropomodulin are proposed to stabilize and limit the lengths of the short actin filaments of the spectrin-actin network, thus influencing the organization and mechanical properties of the erythrocyte membrane skeleton.

Antibodies directed against erythrocyte tropomodulin specifically recognize a Mr43,000 polypeptide from rat lens that comigrates with erythrocyte tropomodulin on SDS-gels. A non-muscle isoform of tropomyosin is also present in the lens. This tropomyosin isoform migrates on SDS-gels with a Mrof ∼28,000 and is distinct from the two erythrocyte isoforms of tropomyosin (Mr27,000 and 29,000). Indirect immunofluorescence staining of 5 μm cryosections of adult rat lens reveals that both tropomodulin and tropomyosin colocalize with rhodamine phalloidin staining for actin filaments on fiber cell plasma membranes. Lens tropomodulin exhibits many characteristics that are similar to its erythrocyte counterpart. For example, lens tropomodulin binds tropomyosin in a solid-phase blot binding assay, and extraction experiments with Triton X-100, urea and NaOH show that the membrane-bound tropomodulin in the lens is a tightly associated peripheral membrane protein that is a component of the Triton-insoluble cytoskeleton. However, unlike the erythrocyte, there are approximately 2000 actin monomers per tropo-modulin in the lens. This differs greatly from the 16/1 ratio seen in the erythrocyte, where actin, tropomodulin and tropomyosin associate to form the short actin filaments in the membrane skeleton. In addition, we have identified a large soluble pool of actin and tropomodulin in the lens, which does not exist in erythrocytes. These differences imply the presence of distinct pools of cytoskeletal components in the lens. The membrane-bound portion of tropomodulin and tropomyosin may associate with that portion of lens actin that is associated with spectrin and protein 4.1 to form a membrane skeleton in the lens fiber cell, whereas the soluble pools of tropomodulin and actin may serve as precursors necessary for rapid assembly of the membrane skeleton during fiber cell elongation. The membrane skeleton may play a role in the maintenance of cell shape and deformability during visual accommodation as well as in the generation of plasma membrane domains during fiber cell differentiation.
Cell adhesion and gap junction formation in the early mouse embryo are induced prematurely by 6-DMAP in the absence of E-cadherin phosphorylation
Authors: Aghion Joël , Gueth-Hallonet Catherine , Antony Claude , Gros Daniel , Maro Bernard
Published: April 2021

Compaction of the mouse embryo, which takes place at the 8-cell stage, is dependent upon the adhesion molecule E-cadherin (uvomurulin), but does not require protein synthesis, suggesting that post-translational modification(s) is (are) implicated in the setting up of this phenomenon. The demonstration recently that E-cadherin is phosphorylated at the 8-cell stage just before compaction supports this theory. In this work we used 6-dimethylaminopurine, a serine-threonine kinase inhibitor, to investigate the role of protein phosphorylation in compaction of mouse embryos. 6-dimethylaminopurine is able to induce cell flattening and gap junction formation prematurely at the 4-cell stage; however, it does not induce cell surface polarization, as occurs during normal compaction. 6-dimethylaminopurine-induced premature flattening is inhibited when the embryos are cultured in the presence of an anti-E-cadherin antibody or without extra-cellular Ca2+, demonstrating that this process requires functional E-cadherin; whereas cell flattening and gap junction formation take place in the absence of E-cadherin phosphorylation, suggesting that its phosphorylation is not required normally for these events. The relationship between E-cadherin-mediated cell flattening and gap junction formation during compaction is discussed.
Expression of neural cell adhesion molecule (N-CAM) in rat islets and its role in islet cell type segregation
Authors: Cirulli V. , Baetens D. , Rutishauser U. , Halban P. A. , Orci L. , Rouiller D. G.
Published: April 2021

Endocrine cell types are non-randomly distributed within pancreatic islets of Langerhans. In the rat, insulin- secreting B-cells occupy the core of the islets and are sur- rounded by A-, D- and PP-cells, secreting glucagon, somatostatin and pancreatic polypeptide, respectively. Furthermore, dissociated islet cells have the ability in vitro to form aggregates with the same cell-type organization as native islets (pseudoislets). These observations suggest that a differential expression of cell adhesion molecules (CAMs) might characterize B- and non-B-cells (A-, D- and PP-cells), and be in part responsible for the establishment and main- tenance of islet architecture. Indirect immunofluorescence using antibodies against CAMs and islet hormones was performed on serial sections of the splenic and duodenal parts of the rat pancreas. Staining for the Ca2+-dependent CAM E-cadherin was detected on both exocrine and endocrine tissue and was uniform over the entire islet section, in both pancreatic regions. By contrast, staining for the Ca2+-independent neural CAM (N-CAM) was restricted to endocrine tissue and nerve endings. Further- more, N-CAM staining of endocrine cells was stronger in the islet periphery, a region composed mostly of non-B-cells. Serial sections demonstrate that cells staining strongly for N-CAM in the splenic part correspond to glucagon cells and in the duodenal part to pancreatic polypeptide cells. Within pseudoislets in vitro a stronger staining for N-CAM was also observed on peripheral cells, corresponding to non-B-cells.These data suggest that a dif- ference in the expression of N-CAM might play a role in the segregation between islet cells. To verify this hypothe- sis, isolated islet cells were allowed to form aggregates in culture, in the presence of non-immune or anti-N-CAM Fab fragments. Inhibition of N-CAM function by specific Fab fragments prevented the segregation between cell types that normally occurs within 5 days. In conclusion, higher levels of N-CAM characterize peripheral islets cells, irrespective of their secretory type, and anti-N-CAM Fab fragments prevent islet cell type segregation within pseudoislets in vitro. These results suggest that differences in N-CAM expression between cell types contribute to the characteristic distribution of cells within islets of Langer- hans.
Differential effects of glycosaminoglycans on neurite outgrowth from hippocampal and thalamic neurones
Authors: Fernaud-Espinosa I. , Nieto-Sampedro M. , Bovolenta P.
Published: April 2021

Chondroitin sulphate proteoglycans are expressed in a temporally restricted pattern from embryonic day 17 to postnatal day 0 in both the thalamus and the cortical subplate, to which thalamic neurones transiently project. To study whether chondroitin sulphate proteoglycans could be specifically involved in the modulation of thalamic axon outgrowth, we compared neurite outgrowth from cultured rat embryonic hippocampal and thalamic neurones, in the presence of chondroitin sulphate type C (isolated from shark cartilage) and chondroitin sulphate type B (dermatan sulphate; isolated from bovine mucosa). When added to the culture medium, both types of gly- cosaminoglycan lowered the adhesion to laminin and polylysine of both hippocampal and thalamic neurones. However, only chondroitin sulphate specifically modified the pattern of thalamic but not hippocampal neurone outgrowth, promoting axon growth. The morphological changes induced by chondroitin sulphate were concentra- tion dependent and correlated with the selective binding of chondroitin sulphate to the neuronal plasma membrane and its subsequent internalisation. Chondroitin sulphate loosely bound to the surface of hippocampal neurones, but was not internalised. These results indicate that proteogly- cans, and in particular the glycosaminoglycan component of these molecules, can differentially modulate neurite outgrowth, depending on their biochemical composition and on the type of neurones they bind to; this would be a possible mechanism of controlling axon guidance in vivo.
Apoptosis is induced in post-mitotic rat sympathetic neurons by arabinosides and topoisomerase II inhibitors in the presence of NGF
Authors: Tomkins Chris E. , Edwards Susan N. , Tolkovsky Aviva M.
Published: April 2021

Sympathetic neurons depend on nerve growth factor (NGF) for their survival and die by apoptosis when NGF is withdrawn, despite their post-mitotic state. Martin et al. (1990, J. Neurosci. 10, 184-193) showed that cytosine ara- binoside, but no other arabinofuranosyl nucleoside, could induce cell death in the presence of NGF and they suggested that it may block a critical step in the NGF-sig- nalling pathway. We show that cytosine arabinoside is not the only nucleoside capable of inducing apoptosis in sym- pathetic neurons in the presence of NGF. In newly isolated neurons from P0 rat pups cultured in the presence of NGF, all the arabinose nucleosides (adenine, cytosine, guanine and thymine) induce apoptosis at 10 μM when combined with 5-fluorodeoxyuridine treatment. Because 1-β-arabi- nofuranosylcytosine is associated with double-strand breaks and chromosomal abberrations, we examined whether topoisomerase II inhibitors, which also cause double-strand breaks by stabilising the enzyme-DNA ‘cleavable complex’, were capable of promoting apoptosis in these neurons. Although P0 rat neurons are strictly post- mitotic, topoisomerase II inhibitors teniposide and mitox- antrone induced them to die by apoptosis in the presence of NGF with the same apparent time-course as arabinose treatment or NGF withdrawal. By contrast, ICRF 193, a catalytic inhibitor of topoisomerase II, reduced the extent of apoptosis induced by mitoxantrone or teniposide by 80% if added simultaneously with the latter but by 2 hours it had no rescue effect, suggesting that topoisomerase II is highly active in these neurons. ICRF 193 also partially reduced the induction of fluorodeoxyuridine-dependent apoptosis by the arabinose nucleosides. These data suggest that induction of double-strand breaks in the DNA or NGF withdrawal activate similar apoptotic programmes in sym- pathetic neurons. We propose that treatment with FdUr results in dUTP incorporation in the neurons, that the presence of arabinose nucleosides causes disruption of the repair system that involves topoisomerase II and this causes double-strand breaks and thus apoptosis.
Kinesin-related proteins in eukaryotic flagella
Authors: Fox Laura A. , Sawin Kenneth E. , Sale Winfield S.
Published: April 2021

To identify kinesin-related proteins that are important for ciliary and eukaryotic flagellar functions, we used affinity-purified, polyclonal antibodies to synthetic peptides corresponding to conserved sequences in the motor domain of kinesin (Sawin et al. (1992)J. Cell Sci. 101, 303-313). Using immunoblot analysis, two antibodies to distinct sequences (LNLVDLAGSE, ‘LAGSE’ and, HIPYRESKLT, ‘HIPYR’) reveal a family of proteins in flagella and axonemes isolated from Chlamydomonas. Similar analysis of axonemes from mutant Chlamydomonas strains or fractionated axonemes indicates that none of the immunoreactive proteins are associated with dynein arm or spoke structures. In contrast, one protein, ∼110 kDa, is reduced in axonemes from mutant strains defective in the central pair apparatus. Immunoreactive proteins with masses of 96 and 97 kDa (the ‘97 kDa’ proteins) are selectively solubilized from isolated axonemes in 10 mM ATP. The 97 kDa proteins co-sediment in sucrose gradients at about 9 S and bind to axonemes or purified microtubules in a nucleotidedependent fashion characteristic of kinesin. These results reveal that flagella contain kinesin-related proteins, which may be involved in axonemal central pair function and flagellar motility, or directed transport involved in morphogenesis or mating responses in Chlamydomonas.
Brush border myosin-I microinjected into cultured cells is targeted to actin- containing surface structures
Authors: Footer Matthew , Bretscher Anthony
Published: April 2021

The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When intro- duced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the con- sequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associ- ated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I. As this same 30 nm separation is seen in microvilli of control cells and we see more prominent links in the myosin-I-injected cells, it seems likely that cultured cells contain an immunologically distinct, but functionally similar, myosin-I that links the microfilament core bundle to the plasma membrane.
Cyclic AMP-regulated AChR assembly is independent of AChR subunit phosphorylation by PKA
Authors: Jayawickreme Shiranthi P. , Green William N. , Claudio Toni
Published: April 2021

Forskolin treatment of cells expressing Torpedo acetyl- choline receptors leads to enhanced assembly efficiency of subunits, which correlates with increased phosphorylation of the γ subunit. To determine the role of the two potential protein kinase A sites of the γ subunit in receptor assembly, cell lines expressing different mutant receptors were estab- lished. Mouse fibroblast cell lines stably expressing wild- type Torpedo acetylcholine receptor α, β, δ subunits plus one of three γ subunit mutations (S353A, S354A, or S353,354A) were established to identify the protein kinase A phosphorylation sites of γ in vivo, and to determine if increased phosphorylation of the γ subunit leads to enhanced expression of receptors. We found that both serines (353, 354) in γ are phosphorylated in vivo by protein kinase A, however, phosphorylation of either or both of these sites does not lead to increased assembly efficiency. We established a cell line expressing α, β, and γ(S353,354A) subunits only (no δ), and found that the presence of δ (or its phosphorylation) is also not necessary for the observed stimulation by forskolin. αβγ, αγ, and βγ associations were stimulated by forskolin but αβ and αδ interactions were not. These data imply that the presence of γ is necessary for forskolin action. We postulate that forskolin may stimulate acetylcholine receptor expression through a cellular protein that is involved in the folding and/or assembly of protein complexes, and that forskolin may regulate the action of such a protein through phosphorylation.