bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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

ABSTRACT
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.