bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic rearrangement of the spectrin membrane skeleton during the generation of epithelial polarity in Drosophila
Authors: Thomas Claire M. , Williams Janice A. 
Published: April 2021

ABSTRACT
The origin of epithelial cell polarity during development is a fundamental problem in cell biology. Central to this process is the establishment of asymmetric membrane domains that will ultimately form the apical and basolateral surfaces. The spectrin-based membrane skeleton has long been thought to participate in the generation of this asymmetry. Drosophila melanogaster contains two known β-spectrin isoforms: a conventional β-spectrin chain, and the novel isoform βHeavy-spectrin. These two proteins are restricted to the basolateral and apical membrane domains, respectively. To assay for the emergence of membrane asymmetry, we have characterized the distribution of these two β-spectrins during the formation of the primary epithelium in the fly embryo. Our results show that the syncytial embryo contains a maternally established apical membrane skeleton containing βHeavy-spectrin into which the basolateral β-spectrin membrane skeleton is added. We have called this process basolateral interpolation. Although basolateral membrane skeleton addition begins during cellularization, it does not become fully established until the formation of a mature zonula adherens at mid to late gastrulation. The behavior of β-spectrin is consistent with a primary role in establishing and/or maintaining the basolateral domain while the behavior of βHeavy-spectrin suggests that its primary role is associated with a specialized DE-cadherin complex associated with the furrow canals and with the maturation of the zonula adherens. Thus, the apical spectrin membrane skeleton appears to play a distinct rather than analogous role to the basolateral spectrin membrane skeleton, during the emergence of cell polarity. We find that there are several parallels between our observations and previous studies on the establishment of primary epithelial polarity in vertebrates, suggesting that basolateral interpolation of the membrane skeleton may be a common mechanism in many organisms.
α3β1 integrin regulates epithelial cytoskeletal organization
Authors: Wang Zemin , Symons Jordan M. , Goldstein Stuart L. , McDonald Alice , Miner Jeffrey H. , Kreidberg Jordan A. 
Published: April 2021

ABSTRACT
Epithelial cell morphology and cytoskeletal organization are determined by interactions, with both adjacent cells and the extracellular matrix, which are mediated by integrins and cadherins. Little is known, however, of the relative contributions of integrins and cadherins to maintaining the sub-cortical cytoskeleton characteristic of epithelial cells. Since most studies that utilize integrin-blocking antibodies result in a loss of both cell-cell adhesion and sub-cortical cytoskeletal organization, it has been difficult to distinguish whether integrins and cadherins both mediate cytoskeletal assembly in epithelial cells. Therefore, cells derived from kidney collecting ducts of α3β1 integrin-deficient mice were used to examine the role of integrins in epithelial cell morphology and cytoskeletal organization. In primary cell culture, α3β1 integrin-deficient kidney collecting duct cells maintain cadherin-mediated cell-cell adhesions but fail to form the sub-cortical cytoskeleton that is characteristic of epithelial cells, and instead assemble actin stress fibers. Moreover, the cell-cell junctions in mutant cells were irregular, rather than being uniformly oriented perpendicular to the culture substrate. These results demonstrated that integrins have an primary and essential function in establishing and maintaining the sub-cortical cytoskeleton that is characteristic of epithelial cells. To further study the role of α3β1 integrin in establishing and maintaining cytoskeletal organization in tubular epithelial cells, we derived immortalized cell lines from wild-type and α3β1 integrin-deficient kidney collecting ducts that duplicated the cytoskeletal and cadherin organization observed in primary cells. E-cadherin and α- and β-catenin were complexed together in equal amounts in membranes of wild-type and α3β1 integrin-deficient cells. However, association of the cadherin:catenin complex with α-actinin was greatly decreased in mutant cells, indicating that integrin-mediated assembly of the sub-cortical cytoskeleton is essential for subsequent association of the cytoskeleton with the cadherin:catenin complex. These results present direct evidence for integrin:cadherin cross-regulation in which cadherin function is dependent on the presence of an integrin.
Activation of the small GTPase Cdc42 by the inflammatory cytokines TNFα and IL-1, and by the Epstein-Barr virus transforming protein LMP1
Authors: Puls Axel , Eliopoulos Aristides G. , Nobes Catherine D. , Bridges Tina , Young Lawrence S. , Hall Alan 
Published: April 2021

ABSTRACT
Cdc42, a Rho-family GTPase, has been implicated in several signal transduction pathways, including organization of the actin cytoskeleton, activation of the c-Jun N-terminal MAP kinase (JNK) and stimulation of the nuclear transcription factor kappa B (NFκB). We report here that exposure of fibroblasts to the inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) triggers the activation of Cdc42 leading first to filopodia formation and subsequently to Rac and Rho activation. Inhibition of Cdc42 completely suppresses cytokine-induced actin polymerization, but not activation of JNK or NFκB. The latent membrane protein 1 of Epstein-Barr virus, LMP1, is thought to mimic constitutively activated TNF family receptors. When expressed in fibroblasts, LMP1 stimulates Cdc42-dependent filopodia formation as well as JNK and NFκB activation. Using LMP1 mutants, we show that activation of Cdc42 and JNK/NFκB occur through distinct pathways and that Cdc42 activation is independent of LMP1’s interaction with TRADD and TRAF proteins.
Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/β-catenin
Authors: Hiscox Stephen , Jiang Wen G. 
Published: April 2021

ABSTRACT
Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. The function of these proteins is to link the plasma membrane to the actin cytoskeleton. Merlin is defective or absent in schwannomas and meningiomas and has been suggested to function as a tumour suppressor. In this study, we have examined the role of ezrin as a potential regulator of the adhesive and invasive behaviour of tumour cells. We have shown that following inhibition of ezrin expression in colo-rectal cancer cells using antisense oligonucleotides, these cells displayed a reduced cell-cell adhesiveness together with a gain in their motile and invasive behaviour. These cells also displayed increased spreading over matrix-coated surfaces. Immunofluorescence studies revealed that antisense-treated cells also displayed an increased staining of paxillin in areas representing focal adhesions. Furthermore, coprecipitation studies revealed an association of ezrin with E-cadherin and β-catenin. Induction of the phosphorylation of ezrin by orthovanadate and hepatocyte growth factor/scatter factor resulted in changes similar to those seen with antisense treatment, together with a marked decrease in the association of ezrin with both β-catenin and E-cadherin. It is concluded that ezrin regulates cell-cell and cell-matrix adhesion, by interacting with cell adhesion molecules E-cadherin and β-catenin, and may thus play an important role in the control of adhesion and invasiveness of cancer cells.
Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms
Authors: Valdenaire Olivier , Barret Alain , Schweizer Anja , Rohrbacher Elisabeth , Mongiat Françoise , Pinet Florence , Corvol Pierre , Tougard Claude 
Published: April 2021

ABSTRACT
Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting endothelins. Three ECE-1 isoforms, differing by their N-terminal cytoplasmic tails, are generated from a single gene. When expressed in CHO cells, they display comparable enzymatic activity but whereas ECE-1a is strongly expressed at the cell surface, ECE-1b is exclusively intracellular and ECE-1c presents an intermediate distribution. In the present study these different localizations were further described at the ultrastructural level, by electron microscope immunocytochemistry. To characterize the motifs responsible for the intracellular localization of ECE-1b we constructed chimeric proteins and point mutants. Two di-leucine-based motifs, contained in the N-terminal part of ECE-1b, were thus identified. One of these motifs (LV), displayed by both ECE-1b and ECE-1c, accounts for the reduced surface expression of ECE-1c as compared to ECE-1a. Mutation of both motifs (LL and LV) induces a very strong appearance of ECE-1b at the cell surface indicating that their presence in the N-terminal extremity of ECE-1b is critical for its exclusively intracellular localization.
The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression
Authors: Christopher Renee A. , Judge Stephen R. , Vincent Peter A. , Higgins Paul J. , McKeown-Longo Paula J. 
Published: April 2021

ABSTRACT
Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin’s amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by &gt;50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin’s amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.
Rapid recruitment of late endosomes and lysosomes in mouse macrophages ingesting Candida albicans
Authors: Káposzta Rita , Maródi László , Hollinshead Michael , Gordon Siamon , da Silva R. P. 
Published: April 2021

ABSTRACT
Candida albicans is an important opportunistic pathogen, whose interaction with cells of the immune system, in particular macrophages (MØ), is poorly understood. In order to learn more about the nature of the infectious mechanism, internalisation of Candida albicans was studied in mouse MØ by confocal immunofluorescence and electron microscopy in comparison with latex beads of similar size, which were coated with mannosyl-lipoarabinomannan (ManLAM) to target the MØ mannose receptor (MR). Uptake of Candida yeasts had characteristics of phagocytosis, required intact actin filaments, and depended on the activity of protein kinase C (PKC). Candida phagosomes rapidly attracted lysosome-associated membrane protein (Lamp)-rich vacuoles, indicative of fusion with late endosomes and lysosomes. Rapid recruitment of late endosomes and lysosomes could be observed regardless of heat-inactivation or serum-opsonisation of Candida, but did not follow binding of the mannosylated-beads to MØ, which suggest that this phenotype is not MR-specific. The yeasts developed germ tubes within phagolysosomes, distended their membranes and escaped, destroying the non-activated MØ. The filamentous form of Candida could penetrate intact MØ even when phagocytosis was blocked, and also attracted Lamp-rich organelles. Inhibition of lysosomal acidification and associated lysosomal fusion reduced germ tube formation of Candida within the phagolysosomes. These data suggest that rapid recruitment of late endocytic/ lysosomal compartments by internalising C. albicans favours survival and virulence of this pathogen.
Tensional forces in fibrillar extracellular matrices control directional capillary sprouting
Authors: Korff Thomas , Augustin Hellmut G. 
Published: April 2021

ABSTRACT
During angiogenesis, anastomosing capillary sprouts align to form complex three-dimensional networks of new blood vessels. Using an endothelial cell spheroid model that was developed to study endothelial cell differentiation processes, we have devised a novel collagen gel-based three-dimensional in vitro angiogenesis assay. In this assay, cell number-defined, gel-embedded endothelial cell spheroids act as a cellular delivery device, which serves as a focal starting point for the sprouting of lumenized capillary-like structures that can be induced to form complex anastomosing networks. Formation of capillary anastomoses is associated with tensional remodeling of the collagen matrix and directional sprouting of outgrowing capillaries towards each other. To analyze whether directional sprouting is dependent on cytokine gradients or on endothelial cell-derived tractional forces transduced through the extracellular matrix, we designed a matrix tension generator that enables the application of defined tensional forces on the extracellular matrix. Using this matrix tension generator, causal evidence is presented that tensional forces on a fibrillar extracellular matrix such as type I collagen, but not fibrin, are sufficient to guide directional outgrowth of endothelial cells. RGD peptides but not control RAD peptides disrupted the integrity of sprouting capillary-like structures and induced detachment of outgrowing endothelial cells cultured on top of collagen gels, but did not inhibit primary outgrowth of endothelial cells. The data establish the endothelial cell spheroid-based three-dimensional angiogenesis technique as a standardized, highly reproducible quantitative assay for in vitro angiogenesis studies and demonstrate that integrin-dependent matrix tensional forces control directional capillary sprouting and network formation.
Interaction between the Ret finger protein and the int-6 gene product and co-localisation into nuclear bodies
Authors: Morris-Desbois Christelle , Bochard Valérie , Reynaud Caroline , Jalinot Pierre 
Published: April 2021

ABSTRACT
The mouse int-6 gene was identified in mammary tumors as an integration site for the mouse mammary tumor virus. Its human counterpart encodes a product that interacts with the Tax viral oncoprotein of the human T cell leukaemia virus type 1. This interaction impedes the localisation of over-expressed Int-6 in nuclear bodies containing the promyelocytic leukaemia gene product (PML). In this study, Int-6 is characterised as a 52 kDa protein that is localised within nuclear bodies in primary lymphocytes. Screening of a human B cell cDNA library for proteins that interact with Int-6 led to isolation of four clones coding for the p110 subunit of eIF3, in accordance with previous detection of Int-6 in purified forms of this translation initiation factor. Another clone was interesting with respect to the subcellular localisation of Int-6. It encodes the Ret finger protein (Rfp) which interacts with PML and localises within a subset of PML nuclear bodies. The interaction of Rfp with Int-6 is mediated through a region in Rfp designated ‘Rfp domain’, distinct from that involved in the interaction with PML. Int-6 and Rfp are co-localised in certain PML nuclear bodies in lymphocytes and transfection studies in HeLa cells strongly suggest that Rfp triggers translocation of Int-6 to nuclear bodies.
Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation
Authors: Brouard Michel , Casado Mariano , Djelidi Sabri , Barrandon Yann , Farman Nicolette 
Published: April 2021

ABSTRACT
The amiloride-sensitive epithelial sodium channel (ENaC) is a main determinant of sodium absorption in renal and colonic epithelial cells. Surprisingly, it is also expressed in non-transporting epithelia such as the epidermis. To gain insight into the putative role of ENaC in keratinocytes, we have evaluated its expression in human skin and in cultured human keratinocytes. Our results indicate that (1) ENaC is expressed in the epidermis and in cultured keratinocytes, at the mRNA and at the protein levels, (2) the ratio of expression of the different ENaC subunits is drastically modified at the protein level during cell growth and differentiation, with a selective upregulation of the beta subunit, (3) no transepithelial sodium transport function is apparent in cultured keratinocytes, but patch-clamp recordings indicate the existence of functional sodium channels with properties similar to those of the cloned ENaC and (4) ENaC inhibition does not alter keratinocyte proliferation, but it significantly decreases the frequency of dome formation in confluent keratinocyte cultures. These results document for the first time the characteristics of ENaC subunit expression in human keratinocytes, and suggest that ENaC may be important during differentiation.
Chromosomes as well as chromosomal subdomains constitute distinct units in interphase nuclei
Authors: Visser Astrid E. , Aten Jacob A. 
Published: April 2021

ABSTRACT
Fluorescence in situ hybridization has demonstrated that chromosomes form individual territories in interphase nuclei. However, this technique is not suitable to determine whether territories are mutually exclusive or interwoven. This notion, however, is essential for understanding functional organizations in the cell nucleus. Here, we analyze boundary areas of individual chromosomes during interphase using a sensitive method based on replication labeling and immunocytochemistry. Thymidine analogues IdUrd and CldUrd were incorporated during S-phase into DNA of Chinese Hamster fibroblasts. Cells labeled with IdUrd were fused with cells labeled with CldUrd. Fused nuclei contained both IdUrd or CldUrd labeled chromosomes. Alternatively, the two labels were incorporated sequentially during successive S-phases and segregated to separate chromosomes by culturing the cells one more cell cycle. Metaphase spreads showed IdUrd-, CldUrd- and unlabeled chromosomes. Some chromatids were divided sharply in differently labeled subdomains by sister chromatid exchanges. With both methods, confocal imaging of interphase nuclei revealed labeled chromosomal domains containing fiber-like structures and unlabeled areas. At various sites, fiber-like structures were embedded in other territories. Even so, essentially no overlap between chromosome territories or between subdomains within a chromosome was observed. These observations indicate that chromosome territories and chromosomal subdomains in G1-phase are mutually exclusive at the resolution of the light microscope.
Cytoskeletal changes induced by GRAF, the

G

TPase

r

egulator

a

ssociated with

f

ocal adhesion kinase, are mediated by Rho
Authors: Taylor Joan M. , Macklem Marlene M. , Parsons J. Thomas 
Published: April 2021

ABSTRACT
Graf, the GTPase regulator associated with focal adhesion kinase was previously shown to have GAP activity for Rho A and Cdc42 in vitro (Hildebrand et al 1996 Mol. Cell Biol. 16: 3169-3178). In this study we sought to determine whether Graf acted at the level of Cdc42, Rho, or both in vivo and whether Graf was a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes were observed following injection of the Rho-inhibitor, C3 into these cells. The Graf response was dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) which block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, we demonstrate that Graf blocks sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results we examined the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types.
Overexpression of protein kinase C-α in the epidermis of transgenic mice results in striking alterations in phorbol ester-induced inflammation and COX-2, MIP-2 and TNF-α expression but not tumor promotion
Authors: Wang Hui Qin , Smart Robert C. 
Published: April 2021

ABSTRACT
Protein kinase Cα (PKCα) is one of six PKC isoforms expressed in keratinocytes of mouse epidermis. To gain an understanding of the role of epidermal PKCα, we have localized its expression to specific cells of normal mouse skin and examined the effect of keratin 5 (K5) promoter directed expression of PKCα in transgenic mice. In normal mouse skin, PKCα was extensively expressed in the outer root sheath (ORS) keratinocytes of the anagen hair follicle and weakly expressed in keratinocytes of interfollicular epidermis. K5-targeted expression of PKCα to epidermal basal keratinocytes and follicular ORS keratinocytes resulted in a tenfold increase in epidermal PKCα. K5-PKCα mice exhibited no abnormalities in keratinocyte growth and differentiation in the epidermis. However, a single topical treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a striking inflammatory response characterized by edema and extensive epidermal infiltration of neutrophils that formed intraepidermal microabscesses in the epidermis.
Compared to TPA-treated wild-type mice, the epidermis of TPA-treated K5-PKCα mice displayed increased expression of cyclooxygenase-2 (COX-2), the neutrophil chemotactic factor macrophage inflammatory protein-2 (MIP-2) mRNA and the proinflammatory cytokine TNFα mRNA but not IL-6 or IL-1α mRNA. To determine if K5-PKCα mice display an altered response to TPA-promotion, 7,12-dimethylbenz[a]anthracene-initiated K5-PKCα mice and wild-type mice were promoted with TPA. No differences in papilloma incidence or multiplicity were observed between K5-PKCα mice and wild-type littermates. These results demonstrate that the overexpression of PKCα in epidermis increases the expression of specific proinflammatory mediators and induces cutaneous inflammation but has little to no effect on epidermal differentiation, proliferation or TPA tumor promotion.