bluuebunny/crossref_metadata_2025 huggingface dataset:
A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication
Authors: Starborg Maria , Brundell Eva , Gell Katarina , Larsson Catharina , White Irene , Daneholt Bertil , Höög Christer
Published: April 2021

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3-5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromo-somal localization of the P1 gene was determined by fluo-rescence in situ hybridization to region 6p12 in human metaphase chromosomes.
Phagocytosis of apoptotic bodies by liver endothelial cells
Authors: Dini Luciana , Lentini Alessandro , Diez Guzman Diez , Rocha Marian , Falasca Laura , Serafino Lucia , Vidal-Vanaclocha Fernando
Published: April 2021

Using electron microscopy and cytofluorimetry we studied the role of carbohydrate-specific recognition systems in the interaction of apoptotic bodies with normal and interleukin 1-activated sinusoidal endothelial cells. Microfluorimetric observation of liver tissue sections revealed octadecyl-rhodamine B-labelled apoptotic body binding to the sinu-soidal wall of mouse liver, when they were injected intra-portally. Plate-scanning cytofluorimetry demonstrated that about 20-25% of Acridine Orange-labelled apoptotic bodies could adhere specifically to cultured endothelial cells after 15 minutes of incubation. Adhesion increased to 30% when the cells were incubated for 60 minutes. Using a mixture of galactose/N-acetylglucosamine/mannose as competition solution apoptotic body adhesion was signifi-cantly reduced especially after longer times of incubation, when the percentage of inhibition reached 50%. Following 4 hours exposure of liver endothelial cells to 1 ng/ml human recombinant interleukin-1β adhesion markedly increased after 60 minutes of incubation, whereas the co-incubation of interleukin-1β with the inhibitors brings down the adhesion to basal values obtained in controls. Electron microscopic observation of the adhesion process showed that the number of endothelial cells binding apoptotic bodies gradually increased from low to high values with time. After 60 minutes of incubation, the majority of apoptotic bodies were seen inside phagosomes and only a few remained at the cell surface. Liver endothelial cells bound and endocytosed apoptotic bodies through carbohy-drate-specific receptors. Moreover, this scavenger action was interleukin-1 enhanced, thus suggesting its possible activation during inflammatory and immune processes.
αv and α3 integrin subunits are associated with myofibrils during myofibrillogenesis
Authors: McDonald K. A. , Lakonishok M. , Horwitz A. F.
Published: April 2021

The development of the myofibrillar apparatus in skeletal muscle is a process in which transmembrane linkages with adhesion molecules are implicated. Integrins are one class of transmembrane adhesion receptors which appear to mediate these interactions. Two prominent linkages are at the myotendinous junction (MTJ), which resides at the ends of the cell and connects myofibrils to the tendon, and the costameres, which encircle the girth of the cell and connect the Z-disks to the sarcolemma. In this study we report that the αv integrin subunit is a prominent component of the costamere. The αv subunit is present initially on developing myotubes in a diffuse staining pattern with some concentration along nascent myofibrils. However, it appears in a striated pattern at the costamere and inconsistently at the M-line following the striation of α-actinin and titin but before that of desmin. Its recruitment to preformed striation suggests that it is incorporated into a pre-existing structure. The presence of αv in the costamere points to a role in lateral myofibrillar anchorage. In addition, we find that the α3 subunit is transiently associated with myofibrils along portions of their lengths and at their ends during myofibrillogenesis. The α3 subunit staining shows a novel localization and junctional structure. As myofibrils become striated the α3 integrin dis-sociates from the localized pattern and becomes diffuse. This suggests a possible role in the stabilization of nascent myofibrils prior to striation. Antibody-induced perturba-tion of adhesion mediated by the integrin β1 subunit in developing myotubes inhibits assembly of the sarcomeric architecture. The observations presented here also con-tribute to an emerging theme, where different integrins occupy unique junctional regions on muscle.
Neutralisation of TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous rat wounds reduces scarring
Authors: Shah Mamta , Foreman David M. , Ferguson Mark W. J.
Published: April 2021

Exogenous addition of neutralising antibody to transforming growth factor-β1,2 to cutaneous wounds in adult rodents reduces scarring. Three isoforms of transforming growth factor-β (1, 2 and 3) have been identified in mammals. We investigated the isoform/isoforms of TGF-β responsible for cutaneous scarring by: (i) reducing specific endogenous TGF-β isoforms by exogenous injection of isoform specific neutralising antibodies; and (ii) increasing the level of specific TGF-β isoforms by exogenous infiltration into the wound margins. Exogenous addition of neutralising antibody to TGF-β1 plus neutralising antibody to TGF-β2 reduced the monocyte and macrophage profile, neovascu-larisation, fibronectin, collagen III and collagen I deposi-tion in the early stages of wound healing compared to control wounds. Treatment with neutralising antibodies to TGF-βs 1 and 2 markedly improved the architecture of the neodermis to resemble that of normal dermis and reduced scarring while the control wounds healed with scar formation. Exogenous addition of neutralising antibody to TGF-β1 alone also reduced the monocyte and macrophage profile, fibronectin, collagen III and collagen I deposition compared to control wounds. However, treatment with neutralising antibody to TGF-β1 alone only marginally reduced scarring. By contrast, wounds treated with neu-tralising antibody to TGF-β2 alone did not differ from control wounds. Interestingly, exogenous addition of the TGF-β3 peptide also reduced the monocyte and macrophage profile, fibronectin, collagen I and collagen III deposition in the early stages of wound healing and markedly improved the architecture of the neodermis and reduced scarring. By contrast, wounds treated with either TGF-β1 or with TGF-β2 had more extracellular matrix deposition in the early stages of wound healing but did not differ from control wounds in the final quality of scarring. This study clearly demonstrates isoform specific differ-ences in the role of TGF-βs in wound healing and cutaneous scarring. TGF-β1 and TGF-β2 are implicated in cutaneous scarring. This study also suggests a novel therapeutic use of exogenous recombinant, TGF-β3 as an anti-scarring agent.
Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos
Authors: Kellum Rebecca , Raff Jordan W. , Alberts Bruce M.
Published: April 2021

Heterochromatin protein 1 (HP1) was initially discovered as a protein that is associated with the heterochromatin at the chromocenter of polytene chromosomes in Drosophila larval salivary glands. In this paper we investigate the localization of heterochromatin protein 1 in the diploid nuclei of Drosophila embryos. We focus on its association with the interphase heterochromatin in fixed embryos before and during cycle 14, the developmental time at which heterochromatin becomes most conspicuous, and also follow its localization during mitosis. The GAGA transcription factor was recently shown to be localized at sequences within α-heterochromatin in pre-cycle 14 embryos, and an antibody against this protein serves as a convenient marker for these sequences.

We find an enrichment of heterochromatin protein 1 in the intensely DAPI-staining regions near the apical surface of nuclear cycle 10 embryos. At this stage GAGA factor is localized into punctate structures in this same region. This enrichment for HP1 is markedly increased during nuclear cycle 14.

Surprisingly, whereas GAGA factor retains its association with the heterochromatin throughout the cell cycle, a significant fraction of HP1 is dispersed throughout the spindle around the segregating chromosomes during mitosis. This dispersed pool of heterochromatin protein 1 was observed during mitosis in both early and late Drosophila embryos and in an analysis of a bacterially produced 6× histidine-heterochromatin protein 1 fusion protein injected into living Drosophila embryos. When Drosophila tissue culture cells were prepared by a method which removes soluble protein and avoids fixation of the mitotic chromosomes, an enrichment for heterochromatin protein 1 in the heterochromatin of the chromosomes was discovered also.
The expression of megalin (gp330) and LRP diverges during F9 cell differentiation
Authors: Czekay Ralf-Peter , Orlando Robert A. , Woodward Luann , Adamson Eileen D. , Farquhar Marilyn G.
Published: April 2021

The receptor-associated protein, RAP, is a chaperonin-like molecule that binds to two members of the low density lipoprotein receptor (LDLR) superfamily – megalin (gp330) and the LDL receptor-related protein (LRP). In F9 embryonal carcinoma cells, expression of RAP mRNA increases when differentiation is induced with retinoic acid and dibutyryl-cyclic AMP. We have investigated the expression of megalin and LRP and their interaction with RAP in F9 cells using biochemical and immunocytochemical methods. Both receptors are expressed in uninduced F9 cells, but only megalin co-precipitates with RAP. When F9 cells were induced to differentiate into parietal endoderm, the expression of megalin was dramatically increased. The expression of megalin exceeded that of LRP and RAP by an order of magnitude and both receptors co-precipitated with RAP. By immunoelectron microscopy, megalin and LRP were localized to clathrin-coated pits at the cell surface in both undifferentiated and differentiated F9 cells, whereas RAP was found mainly in the ER. A sizeable pool of LRP was also detected in the ER. When F9 cells were grown in suspension in the presence of RA and induced to develop into embryoid bodies, the expression of megalin and LRP segregated into different cell types: megalin was found in the outer epithelial layer and LRP in the stem cells of the inner core. Our results demonstrate that F9 cells induced to differentiate in monolayer culture express megalin, LRP and RAP, and RAP is capable of interacting simultaneously with both receptors. In embryoid bodies the expression of megalin and LRP diverges, and only megalin is expressed in the outer epithelial layer. The high levels of expression and cell specificity of megalin and LRP suggest important roles for these receptors in early embryonic development.
Import of microinjected proteins bearing the SKL peroxisomal targeting sequence into the peroxisomes of a human fibroblast cell line: evidence that virtually all peroxisomes are import-competent
Authors: Hill Paula E. , Walton Paul A.
Published: April 2021

Peroxisomes import virtually all of their membrane and matrix proteins post-translationally. It is presently unknown whether, in mammalian cells, their exists a pool of mature peroxisomes which have received their complement of proteins and are import-incompetent. Previous work has shown that fibroblasts are capable of importing microinjected peroxisomal proteins into peroxisomes. This report describes the import of a hybrid peroxisomal protein into virtually all peroxisomes of the microinjected cell. The peroxisomal import was uniform in both short and long incubations. Pretreatment of the cells with cycloheximide did not affect the import of the peroxisomal protein, nor was there any difference in the distribution of the imported protein. Sequential microinjection experiments demonstrated that peroxisomes that had imported luciferase were capable of importing another peroxisomal protein injected 24 hours later. These results suggest that, in fibroblasts, all peroxisomes have associated protein-import machinery; this evidence does not support the hypothesis that there exists a pool of import-incompetent peroxisomes.
Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells
Authors: Roobol Anne , Holmes Fiona E. , Hayes Nandini V. L. , Baines Anthony J. , Carden Martin J.
Published: April 2021

Chaperonins containing t-complex polypeptide-1 (CCT) are cytosolic molecular chaperone particles implicated especially in the biogenesis of cytoskeletal proteins by promoting the correct folding of the major ubiquitous cytoskeletal components, tubulin and actin. We have purified cytosolic chaperonins from the ND7/23 cell line, determined their subunit composition and examined changes in the intracellular locations of their components during differentiation of ND7/23 cells to a neuronal phenotype by using immunocytochemistry and immunoblots. Chaperonins containing the CCTα (TCP1? subunit enter neuritic processes and are particularly noticeable at the leading edge of growth cone-like structures where they co-localise with actin. Chaperonins containing three other components (CCTβ, ε and γ), however, remain predominantly restricted to perikaryal cytoplasm. These findings suggest a heterogeneous population of chaperonin particles within single differentiated ND7/23 cells and this may reflect specialisation of chaperonin function in different cytoplasmic compartments of a neurone. Further, since ribosomes do not enter neurites while CCTα-containing chaperonins do, the latter may play roles, subsequent to translation, which influence cytoskeletal elaboration during neuritogenesis.
Stationary organization of the actin cytoskeleton in Vallisneria: the role of stable microfilaments at the end walls
Authors: Ryu Jung-Hwa , Takagi Shingo , Nagai Reiko
Published: April 2021

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea, bundles of microfilaments (MFs) serve as tracks for the rotational streaming of the cytoplasm, which occurs along the two longer side walls and the two shorter end walls. The stationary organization of these bundles has been shown to depend on the association of the bundles with the plasma membrane at the end walls. To identify the sites of such association, the effects of cytochalasin B (CB) on the configuration of the bundles of MFs were examined. In the case of the side walls, MFs were completely disrupted after treatment with CB at 100 μg/ml for 24 hours. By contrast, in the case of the end walls, a number of partially disrupted MFs remained even after 48 hours of treatment. After removal of CB, a completely normal arrangement of bundles of MFs was once again evident within 24 hours after a rather complicated process of reassembly. When reassembly had been completed, the direction of cytoplasmic streaming was reversed only in a small fraction of the treated cells, suggesting that bundles of MFs are anchored and stabilized at the end walls of each cell and that the polarity of reorganized bundles and, therefore, the direction of the cytoplasmic streaming is determined in a manner that depends on the original polarity of MFs that remained in spite of the disruptive action of CB. By contrast, the direction of reinitiated cytoplasmic streaming was reversed in 50% of cells in which the bundles of MFs had been completely disrupted by exogenously applied trypsin prior treatment with CB. The results confirm that protease-sensitive anchoring of microfilament bundles at the end walls is crucial for maintenance of the unique, stationary organization of the tracks for cytoplasmic streaming in these cells.
Saccharomyces cerevisiae Apl2p, a homologue of the mammalian clathrin AP β subunit, plays a role in clathrin-dependent Golgi functions
Authors: Rad Massoud Ramezani , Phan Huan L. , Kirchrath L. , Tan Philip K. , Kirchhausen Tomas , Hollenberg Cornelius P. , Payne Gregory S.
Published: April 2021

Clathrin-coated vesicles mediate selective intracellular protein traffic from the plasma membrane and the trans-Golgi network. At these sites, clathrin-associated protein (AP) complexes have been implicated in both clathrin coat assembly and collection of cargo into nascent vesicles. We have found a gene on yeast chromosome XI that encodes a homologue of the mammalian AP β subunits. Disruptions of this gene, APL2, and a previously identified β homologue, APL1, have been engineered in cells expressing wild-type (CHC1) or temperature sensitive (chc1-ts) alleles of the clathrin heavy chain gene. APL1 or APL2 disruptions (apl1Δ or apl2Δ) yield no discernable phenotypes in CHC1 strains, indicating that the Apl proteins are not essential for clathrin function. However, the apl2Δ, but not the apl1Δ, allele enhances the growth and α-factor pheromone maturation defects of chc1-ts cells. Disruption of APL2 also partially suppresses the vacuolar sorting defect that occurs in chc1-ts cells immediately after imposition of the non-permissive temperature. These Golgi-specific effects of apl2Δ in chc1-ts cells provide evidence that Apl2p is a component of an AP complex that interacts with clathrin at the Golgi apparatus.
Integrin α2 I-domain is a binding site for collagens
Authors: Tuckwell Danny , Calderwood David A. , Green Linda J. , Humphries Martin J.
Published: April 2021

Integrins α1β1 and α2β1 are major cellular receptors for collagens. The α1 and α2 subunits contain a ∼200 amino acid inserted domain (I-domain) in their N-terminal region and, because of the homology between the I-domains and the collagen-binding A-domains of von Willebrand factor, it has been suggested that the I-domains might mediate the collagen-binding functions of α1β1 and α2β1. In order to fully investigate this hypothesis, we have generated recombinant human α2 I-domain (rα2I) by reverse transcriptase-polymerase chain reaction/bacterial expression and tested its ability to mediate the collagen-binding functions of α2β1. Rα2I binds specifically to type I collagen in a concentration-dependent manner: binding is cation dependent and, like the complete receptor, is supported by magnesium and manganese ions but not by calcium ions. Rα2I is recognised by anti-functional anti-α2 monoclonal antibodies 6F1, 5E8 and P1E6 in capture ELISAs, and anti-functional antibodies inhibited rα2I-collagen binding. In addition, rα2I inhibits cell spreading on collagen. Rα2I is therefore a collagen-binding domain and can account for many of the collagen-binding functions of integrin α2β1. We have also determined the collagen specificity of rα2I and found that it binds types I, II and XI collagen.
Temporal control of gap junction assembly in preimplantation mouse embryos
Authors: Valdimarsson Gunnar , Kidder Gerald M.
Published: April 2021

The de novo assembly of gap junctions during compaction in the 8-cell stage of mouse development is a temporally regulated event. We have performed experiments designed to explore the relationship between this event and DNA replication in the second, third, and fourth cell cycles after fertilization. Inhibition of DNA synthesis by continuous treatment with the DNA synthesis inhibitor, aphidicolin, during the third and fourth cell cycles had no effect on the establishment of gap junctional coupling during compaction. However, a delay of 10 hours in DNA synthesis during the second cell cycle caused by a transient aphidicolin treatment resulted in the failure of gap junctional coupling at the time of compaction. Thus the timing of establishment of gap junctional coupling, like the timing of compaction itself, is linked to DNA replication in the 2-cell stage. Immunofluorescence analysis showed that the failure of gap junctional coupling after aphidicolin treatment in the 2-cell stage is correlated with the failure of nascent connexin43 to be inserted into plasma membranes. We propose that the developmental ‘clock’ that controls gap junction assembly is set in motion by events surrounding the second cycle of DNA replication, and that this ‘clock’ ultimately controls the post-translational processing of connexin43.
A simple epithelial cell line (MDCK) shows heterogeneity of desmoglein isoforms, one resembling pemphigus vulgaris antigen
Authors: José Vilela Marcelo , Hashimoto Takashi , Nishikawa Takeji , North Alison J. , Garrod David
Published: April 2021

The epidermal blistering desease, pemphigus vulgaris (PV), is caused by circulating autoantibodies that react with a desmosomal glycoprotein desmoglein (Dsg3). This antigen is expressed only in stratified epithelial tissues. Here we show that the simple epithelial canine kidney cell line, MDCK, expresses at least two desmoglein isoforms recognised by different monoclonal antibodies. One of these isoforms is a 130×103Mr polypeptide that is recognised by both PV autoantisera and a monoclonal antibody reactive with a cytoplasmic domain of human Dsg3. Antibodies in PV sera bind to the surface of MDCK cells but do not cause loss of intercellular adhesion. This is the first demonstration of the expression of a polypeptide related to human PV antigen by a simple epithelial cell type.
Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5′ regulatory region
Authors: Chang Kin-Chow , Fernandes Kenneth , Dauncey Margaret J.
Published: April 2021

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5′ end cDNA and 5′ regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (−1007 to −828 and −455 to −101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3′ region, extending from the transcriptional start site to part of intron 2, must be present in all the DNA constructs used.
Analysis of the γ-tubulin sequences: Implications for the functional properties of γ-tubulin
Authors: Burns Roy G.
Published: April 2021

γ-Tubulin was initially identified as a suppressor of an Aspergillus β-tubulin mutation (Oakley and Oakley, 1989). Highly homologous genes have since been identified in a variety of other eucaryotes (see Tables 1 and 2), and immunofluorescence studies have confirmed that γ-tubulin is expressed in a wide variety of eucaryotic cells. These observations indicate that γ-tubulin is functionally important, and this has been confirmed by mutational analysis (Oakley et al., 1990; Horio and Oakley, 1994) and by the elegant demonstration that the human γ-tubulin gene product supports the growth and division of Schizosaccharomyces pombe (Horio and Oakley, 1994).
Ubiquitin-activating enzyme, e1, is phosphorylated in mammalian cells by the protein kinase cdc2
Authors: Nagai Yukiko , Kaneda Sumiko , Nomura Kunihiko , Yasuda Hideyo , Seno Takeshi , Yamao Fumiaki
Published: April 2021

The ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 was found to be phosphorylated in cells of a mouse mammary carcinoma cell line, FM3A. Peptide mapping of trypsin digests of labeled E1 indicated that two oligopeptides were mainly phosphorylated in vivo. The same oligopeptides were also labeled in vitro on Cdc2 kinase-mediated phosphorylation of E1, affinity-purified from the same cell line. The Cdc2 kinase is a key enzyme playing a pivotal role in G2/M transition in the cell cycle. The phosphorylation of one of the two oligopeptides was prominent at the G2/M phase of the cell cycle, and dependent upon the Cdc2 kinase activity in vivo since it was significantly reduced in tsFT210, a mutant cell line deficient in Cdc2 kinase. Mutation analysis indicated that the serine residue at the fourth position of the E1 enzyme was a phosphorylation site of Cdc2 kinase. These findings suggest that E1 is a target of Cdc2 kinase in the cell, implying that the ubiquitin system may be dynamically involved in cell cycle control through phosphorylation of this key enzyme.
α3aβ1 integrin localizes to focal contacts in response to diverse extracellular matrix proteins
Authors: DiPersio C. Michael , Shah Sejal , Hynes Richard O.
Published: April 2021

In vitro binding assays and inhibition of cell adhesion with monoclonal antibodies have implicated the integrin α3β1 as a receptor for a variety of extracellular ligands. However, reports of α3β1-ligand interactions are inconsistent, and transfection studies have suggested that α3β1 is not sufficient for cell attachment to ligands other than kalinin/laminin 5. We used immunofluorescence to study subcellular localization of the α3A cytoplasmic domain variant in different cultured cell types. Using standard fixation and permeabilization methods, antibodies specific for α3A stained most cell types in a diffuse pattern, consistent with previous reports. Surprisingly, however, chemical cross-linking of integrins to the extracellular matrix and extraction of the cytoskeleton prior to immuno-fluorescence revealed α3A in focal contacts of most cells tested, suggesting that the cytoplasmic domain was concealed in intact focal contacts by cytoskeletal or other cytoplasmic proteins. The α3A subunit localized to focal contacts in several cell types cultured on fibronectin, kalinin/laminin 5, EHS-laminin/laminin 1, type IV collagen, or vitronectin. In contrast, α5 and αV integrins were detected in focal contacts only in cells grown on their known ligands (fibronectin, and fibronectin or vitronectin, respectively). Therefore, our results show that α3Aβ1 responds to a broad spectrum of extracellular ligands. Time course comparisons of the recruitment of α subunits from different fibronectin receptors suggested that localization of α3Aβ1 to fibronectin-induced focal contacts was independent of the recruitment of α5 and α4 integrins. However, other studies have shown that α3Aβ1 does not mediate initial cell adhesion to many of the ligands that induced its focal contact localization, including fibronectin. Therefore, we suggest that α3Aβ1 may be a secondary receptor with post-cell-adhesion functions for a broad spectrum of extracellular matrices.
PP125FAK Tyrosine kinase activity is not required for the assembly of f-actin stress fibres and focal adhesions in cultured mouse aortic smooth muscle cells
Authors: Wilson Lynn , Carrier Martin J. , Kellie Stuart
Published: April 2021

The observed increase in phosphotyrosine content of focal adhesion-associated proteins, in response to integrin engagement, indicates a role for integrin-regulatable tyrosine kinase(s) in cytoskeletal re-organisation. The tyrosine kinase pp125FAK, by virtue of its focal adhesion localisation in fibroblasts, represents a prime candidate to perform this function. We have investigated whether pp125FAK performs a similar function in mouse aortic smooth muscle cells (MASMC). MASMC cultured for 16 hours exhibit F-actin stress fibres and focal adhesions. We have shown that vinculin, pp125FAK and tyrosine-phosphorylated proteins are localised in focal adhesions during this time period. MASMC, under these culture conditions exhibit elevated pp125FAK tyrosine kinase activity, as measured by an increased autophosphorylation potential. We investigated the development of F-actin stress fibres and focal adhesions in MASMC in response to adherence to fibronectin, conditions shown to promote cytoskeletal reorganisation in fibroblasts. Within 30 minutes, MASMC exhibited well-developed F-actin stress fibres and prominent focal adhesions which immunostained intensely for vinculin, pp125FAK and phosphotyrosine. Adherence to fibronectin has been reported to activate pp125FAK tyrosine kinase in fibroblasts, leading to the proposal that pp125FAK plays a critical role in focal adhesion formation. Therefore pp125FAK activation, in response to adherence to fibronectin, was investigated in MASMC. Anti-phospho-tyrosine immunoblotting and in vitro kinase assays of MASMC lysates have revealed that, under conditions which promote focal adhesion formation, pp125FAK remains inactive. Since overnight cultures of MASMC exhibited elevated pp125FAK tyrosine kinase activity, we investigated whether these cells deposit their own combination of extracellular matrix (ECM) molecules and/or secrete factors into their conditioned medium which are capable of activating pp125FAK tyrosine kinase. Our results indicate that MASMC-elaborated ECM, but not their conditioned medium, supported pp125FAK tyrosine kinase activation. Furthermore, MASMC exposed to MASMC-ECM displayed a poorly defined F-actin stress fibre network and rudimentary focal adhesions. Thus we have demonstrated the existence of two adhesion-mediated situations in MASMC; one in which fibronectin promotes cytoskeletal reorganisation in the absence of pp125FAK tyrosine kinase activity and the other in which cells adhering to MASMC-ECM display elevated pp125FAK tyrosine kinase activity in association with an impaired ability to promote F-actin stress fibre and focal adhesion formation. These results indicate that in MASMC, pp125FAK tyrosine kinase activity is not involved in F-actin stress fibre assembly and focal adhesion formation.
Mammary epithelial cell differentiation in vitro is regulated by an interplay of egf action and tenascin-c downregulation
Authors: Wirl Gerhard , Hermann Martin , Ekblom Peter , Fässler Reinhard
Published: April 2021

Expression of the extracellular matrix glycoprotein tenascin-C in the mammary gland is associated with cellular proliferation and cell motility during organogenesis and tumorigenesis. Because the source and the regulation of tenascin-C in these tissues are unclear, we have used tenascin-C cDNA, FITC-immunofluorescence and immuno-precipitation to examine tenascin-C expression of mammary epithelial cells. Using several mammary epithelial cell lines we could show that tenascin-C can be produced and secreted by epithelial cells. However it was found that tenascin-C synthesis was inversely correlated with the polarized epithelial phenotype. Among three mouse mammary epithelial cell clones, tenascin-C expression was most abundant in HC-11 cells, the least differentiated cell type. Expression levels were high during the growth phase but were nearly abolished when cells were grown to confluence and induced to express milk proteins. Downregulation of tenascin-C by EGF apparently commits HC-11 cells to respond to lactogenic hormones and conse-quently, hormone induced levels of β-casein mRNA decreased significantly when HC-11 cells were grown on a tenascin-C substrate. On the other hand, TGF-β, another growth factor involved in coordinated growth and differentiation of the mammary gland in vivo was found to be a very potent inducer of tenascin-C.

The generation of fully polarized and tight epithelium affected the levels of tenascin-C expression. In contrast to HC-11 cells, which do not form epithelial domes in vitro, highly polarized and dome forming EpH4 and Fos-ER cells nearly lacked tenascin-C. Similarly, induction of dome formation in the rat mammary stem cell line Rama 25 by the differentiation inducer dimethylsulfoxide caused a loss of TN-C-transcripts. The inability of Fos-ER cells to develop domes in the presence of soluble tenascin-C also suggests its interference with induction and maintenance of mammary epithelial cell differentiation.
Communication of post-golgi elements with early endocytic pathway: Regulation of endoproteolytic cleavage of semliki forest virus p62 precursor
Authors: Sariola Merja , Saraste Jaakko , Kuismanen Esa
Published: April 2021

A number of cellular proteins and viral spike proteins are cleaved at a basic recognition sequence. To characterize the membrane traffic step at which this proteolysis occurs we have studied the intracellular processing site of Semliki Forest virus (SFV) spike precursor p62 in BHK21 cells. The p62 is endoproteolytically cleaved at a tetrabasic Arg-His-Arg-Arg recognition sequence. Previously, it has been shown that the SFV p62 remains uncleaved when accumulated to the trans-Golgi network (TGN/20°C block site). We show here that exit from the trans-Golgi is required for the cleavage of p62. Proteolytic processing was inhibited in synchronized assays when the 20°C transport block was released in the presence of brefeldin A, energy inhibitors (azide and deoxyglucose; carbonyl cyanide m-chlorophenylhydrazone, CCCP) or an effector of trimeric G proteins, AlFn. Endocytosed antibodies against the SFV spike glycoproteins or antibodies against a peptide corresponding to the enzymatically active motif of furin inhibited cleavage of p62 at a post-TGN location. The results indicate a post-TGN communication step between exocytic and endocytic elements. Kinetic experiments suggested that this communication may involve an early compartment of the endocytic pathway.
Reassessment of the subcellular localization of p63
Authors: Schweizer Anja , Rohrer Jack , Slot Jan W. , Geuze Hans J. , Kornfeld Stuart
Published: April 2021

p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.
Epidermal growth factor induces rapid and transient association of phospholipase c-γ1 with egf-receptor and filamentous actin at membrane ruffles of a431 cells
Authors: Diakonova Maria , Payrastre Bernard , Velzen Agnes G. van , Hage Willem J. , Henegouwen Paul M. P. van Bergen en , Boonstra Johannes , Cremers Fons F. M. , Humbel Bruno M.
Published: April 2021

Addition of epidermal growth factor to A431 cells results in dramatic changes in cell morphology. Initially the cells form membrane ruffles accompanied by increased actin polymerization, followed by cell rounding. Activation of the tyrosine kinase of the receptor by binding epidermal growth factor leads also to phosphorylation and activation of phospholipase C-γ1, a key enzyme in the phosphoinositide pathway. In this study we have investigated the localization of phospholipase C-γ1 during cell activation by epidermal growth factor. It is shown that addition of the growth factor to A431 cells leads to a translocation of phospholipase C-γ1 from the cytosol to the membrane fraction. Interestingly, this relocation is exclusively directed to the membrane ruffles. Most of the phospholipase C-γ1 associates to the membrane and a small fraction to the underlying skeleton. Immunocytochemical studies demonstrated that phospholipase C-γ1 co-localizes with the epidermal growth factor receptor and also filamentous actin at the membrane ruffles. Moreover, using anti-phosphotyrosine antibodies we found that the membrane ruffles are significantly enriched in phosphotyrosyl proteins. Between 5 and 10 minutes after stimulation the membrane ruffles disappear and also the co-localization of phospholipase C-γ1 with the epidermal growth factor receptor and filamentous actin. These results support the notion that activation of A431 cells by epidermal growth factor leads to the formation of a signalling complex of its receptor, phospholipase C-γ1 and filamentous actin which is primarily localized at membrane ruffles.
A novel role for α3β1 integrins in extracellular matrix assembly
Authors: Wu Chuanyue , Chung Albert E. , McDonald John A.
Published: April 2021

To study the biological role of α3β1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human α3 integrin subunit in the α4, α5 integrin deficient CHO cell line B2. The expression of α3β1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the α3β1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of α3β1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of α3β1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to α5β1 integrins, α3β1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by α5β1, α3β1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of α3β1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.
Sperm exocytosis reconstructed in a cell-free system: Evidence for the involvement of phospholipase c and actin filaments in membrane fusion
Authors: Spungin Ben , Margalit Ilana , Breitbart Haim
Published: April 2021

We used a cell-free system to study membrane fusion during sperm exocytosis (acrosome reaction). Extracted bovine sperm plasma and outer acrosomal membranes were labeled with chlorophyll a or DCY, respectively. The occurrence of membrane fusion is indicated by the ability of the probes to diffuse from one membrane species to another which is revealed by resonance energy transfer between the two probes. We have previously shown using this system that the requirement of capacitation for sperm exocytosis is retained in cell-free membrane fusion, and that the pH and calcium dependence of the cell-free fusion mimics those of exocytosis in intact cells. In the present report we further characterize the fusion of sperm membranes which we observe in our assay. Phosphoproteins and phospholipases were found to be involved in the membrane fusion step of sperm exocytosis. Protein kinases, phosphatases, and Gi-like proteins, while involved in exocytosis in intact cells, are not involved specifically in the membrane fusion step of exocytosis. The role of membrane bound F-actin in regulating membrane fusion was also studied using fluorescently labeled phalloidin. The results show that cortical F-actin has two roles in regulating sperm exocytosis. One is to form a scaffolding to hold phospholipase C at the membrane. It also functions as a physical barrier to membrane fusion which is removed by the increases in intracellular calcium and pH which precede fusion.
Expression and localization of α-adaptin isoforms
Authors: Ball Catriona L. , Hunt Stephen P. , Robinson Margaret S.
Published: April 2021

There are two α-adaptin genes, αA and αC, which in brain encode proteins of of Mr 108×103 and 104×103, respectively. Although both mRNAs can be detected on northern blots of brain and liver, the higher molecular mass polypeptide can only be detected on western blots of brain. Here we explain these observations by showing that αA is alternatively spliced and that the protein product in most tissues is different from the one expressed in brain in that it is missing 21 amino acids within the hinge region, giving it a similar mobility to that of αC. Monospecific antibodies were raised against the various α-adaptin isoforms and used to compare their distribution in cells and tissues. Both αA and αC are co-assembled into the same coated pits, and the larger isoform of αA is co-assembled with the smaller isoforms of α-adaptin, both in cells that naturally express it and in transfected cells. Examination of brain and spinal cord sections, labelled either for the larger isoform of αA or for αC, reveals that that the two are to some extent differentially distributed, consistent with previous in situ hybridisation studies. This finding, combined with the observation that there is considerable variability in the relative expression of the two isoforms in different tissues, indicates that the two genes are switched on in response to different stimuli. Moreover, the larger isoform of αA appears to be more efficiently concentrated in the nerve terminals than αC, which is found not only at the terminals but also diffusely distributed in the cell bodies and dendrites. This suggests that αC may play more of a role in the recycling of membrane components throughout the cell.
A chromomeric model for nuclear and chromosome structure
Authors: Cook Peter R.
Published: April 2021

The basic structural elements of chromatin and chromosomes are reviewed. Then a model involving only three architectural motifs, nucleosomes, chromatin loops and transcription factories/chromomeres, is presented. Loops are tied through transcription factors and RNA polymerases to factories during interphase and to the remnants of those factories, chromomeres, during mitosis. On entry into mitosis, increased adhesiveness between nucleosomes and between factories drives a ‘sticky-end’ aggregation to the most compact and stable structure, a cylinder of nucleosomes around an axial chromomeric core.
Arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices
Authors: Robertson E. J. , Pyke K. A. , Leech R. M.
Published: April 2021

The arc6 (accumulation and replication of chloroplasts) mutant of Arabidopsis has only two greatly enlarged chloroplasts per mature leaf mesophyll cell compared with ninety chloroplasts per cell in the wild type. The mutation is a single nuclear gene and the plant phenotype is normal. Shoot and root apical meristems of arc6 plants have been examined to determine how early during plastid development the mutant arc6 phenotype can be recognised. In the cells of the arc6 apical meristem there are only two proplastids, which are larger than wild type with a highly variable morphology. In the cells of the leaf primordia where differentiation of proplastids to chloroplasts occurs arc6 plastids are larger and at a more advanced developmental stage than wild-type plastids. In arc6 root cells statoliths and other plastids also show grossly abnormal morphology and the statoliths are greatly increased in size. During arc6 stomatal guard cell development the perturbation in proplastid population dynamics affects plastid segregation and 30% of stomata lack plastids in one or both guard cells. Our evidence would suggest that ARC6 is expressed throughout the vegetative cells of the Arabidopsis seedling with major effects on both the proplastid phenotype and the proplastid population. ARC6 is the first gene to be identified in Arabidopsis which has a global effect on plastid development in cells arising from both the shoot and root meristems, and is of major importance in the nuclear control of plastid differentiation in higher plants.
Functional conservation of the cell cycle-regulating transcription factor DRTF1/E2F and its pathway of control in Drosophila melanogaster
Authors: Hao Xu Feng , Alphey Luke , Bandara Lasantha R. , Lam Eric W.-F. , Glover David , Thangue Nicholas B. La
Published: April 2021

The cellular transcription factor DRTF1/E2F is implicated in the control of early cell cycle progression due to its interaction with important regulators of cellular proliferation, such as pocket proteins (for example, the retinoblastoma tumour suppressor gene product), cyclins and cyclin- dependent kinase subunits. In mammalian cells DRTF1/E2F is a heterodimeric DNA binding activity which arises when a DP protein interacts with an E2F protein. Here, we report an analysis of DRTF1/E2F in Drosophila cells, and show that many features of the pathway which regulate its transcriptional activity are conserved in mammalian cells, such as the interaction with pocket proteins, binding to cyclin A and cdk2, and its modulation by viral oncoproteins. We show that a Drosophila DP protein which can interact co-operatively with E2F proteins is a physiological DNA binding component of Drosophila DRTF1/E2F. An analysis of the expression patterns of a Drosophila DP and E2F protein indicated that DmDP is developmentally regulated and in later embryonic stages preferentially expressed in proliferating cells. In contrast, the expression of DmE2F-1 in late stage embryos occurs in a restricted group of neural cells, whereas in early embryos it is widely expressed, but in a segmentally restricted fashion. Some aspects of the mechanisms which integrate early cell cycle progression with the transcription apparatus are thus conserved between Drosophila and mammalian cells. The distinct expression patterns of DmDP and DmE2F-1 suggest that the formation of DP/E2F heterodimers, and hence DRTF1/E2F, is subject to complex regulatory cues.