bluuebunny/crossref_metadata_2025_split huggingface dataset:
Na,K-ATPase in skeletal muscle: two populations of β-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules
Authors: Williams McRae W. , Resneck Wendy G. , Kaysser Tamma , Ursitti Jeanine A. , Birkenmeier Connie S. , Barker Jane E. , Bloch Robert J. 
Published: April 2021

ABSTRACT
We used immunological approaches to study the factors controlling the distribution of the Na,K-ATPase in fast twitch skeletal muscle of the rat. Both α subunits of the Na,K-ATPase colocalize with β-spectrin and ankyrin 3 in costameres, structures at the sarcolemma that lie over Z and M-lines and in longitudinal strands. In immunoprecipitates, the α1 and α2 subunits of the Na,K-ATPase as well as ankyrin 3 associate with β-spectrin/α-fodrin heteromers and with a pool of β-spectrin at the sarcolemma that does not contain α-fodrin. Myofibers of mutant mice lacking β-spectrin (ja/ja) have a more uniform distribution of both the α1 and α2 subunits of the Na,K-ATPase in the sarcolemma, supporting the idea that the rectilinear sarcomeric pattern assumed by the Na,K-ATPase in wild-type muscle requires β-spectrin. The Na,K-ATPase and β-spectrin are distributed normally in muscle fibers of the nb/nb mouse, which lacks ankyrin 1, suggesting that this isoform of ankyrin is not necessary to link the Na,K-ATPase to the spectrin-based membrane skeleton. In immunofluorescence and subcellular fractionation experiments, the α2 but not the α1 subunit of the Na,K-ATPase is present in transverse (t-) tubules. The α1 subunit of the pump is not detected in increased amounts in the t-tubules of muscle from the ja/ja mouse, however. Our results suggest that the spectrin-based membrane skeleton, including ankyrin 3, concentrates both isoforms of the Na,K-ATPase in costameres, but that it does not play a significant role in restricting the entry of the α1 subunit into the t-tubules.
The origin of annular junctions: a mechanism of gap junction internalization
Authors: Jordan Karen , Chodock Rochelle , Hand Art R. , Laird Dale W. 
Published: April 2021

ABSTRACT
Gap junctional intercellular communication is established when connexin proteins oligomerize into connexon hemichannels, which then pair at the cell surface with connexons from neighboring cells to form functional gap junction channels. Gap junction channels routinely cluster into gap junction plaques, which can exhibit dynamic characteristics while under the frequent processes of formation and removal from the cell surface. We have three lines of evidence to suggest that one mechanism of gap junction removal occurs when one of two contacting cells internalizes the gap junction contribution from both cells. First, in coculture experiments, green fluorescent protein-tagged connexin43 (Cx43-GFP) expressed in normal rat kidney (NRK) cells can be internalized into contacting cells that do not express Cx43-GFP, and the incidences of identifying these internalized structures increase in the presence of lysosomal inhibitors. Secondly, time-lapse imaging of live NRK cells revealed that large areas of gap junction plaques containing Cx43-GFP were internalized as vesicular-like structures into one of two adjacent cells. Finally, when live NRK cells that express endogenous Cx43 were microinjected with anti-Cx43 antibodies, antibody-tagged gap junctions were visualized in cells that contacted the microinjected cell within 3-6.5 hours. Together our results strongly suggest that one mechanism of gap junction removal from the cell surface involves a unique process in which the entire gap junction or a fragment of it is internalized into one of the two contacting cells as an annular junction.
Phagocytosis and the actin cytoskeleton
Authors: May Robin C. , Machesky Laura M. 
Published: April 2021

ABSTRACT
The process of engulfing a foreign particle – phagocytosis – is of fundamental importance for a wide diversity of organisms. From simple unicellular organisms that use phagocytosis to obtain their next meal, to complex metazoans in which phagocytic cells represent an essential branch of the immune system, evolution has armed cells with a fantastic repertoire of molecules that serve to bring about this complex event. Regardless of the organism or specific molecules concerned, however, all phagocytic processes are driven by a finely controlled rearrangement of the actin cytoskeleton. A variety of signals can converge to locally reorganise the actin cytoskeleton at a phagosome, and there are significant similarities and differences between different organisms and between different engulfment processes within the same organism. Recent advances have demonstrated the complexity of phagocytic signalling, such as the involvement of phosphoinostide lipids and multicomponent signalling complexes in transducing signals from phagocytic receptors to the cytoskeleton. Similarly, a wide diversity of ‘effector molecules’ are now implicated in actin-remodelling downstream of these receptors.
Paranemin and the organization of desmin filament networks
Authors: Schweitzer Susan C. , Klymkowsky Michael W. , Bellin Robert M. , Robson Richard M. , Capetanaki Yassemi , Evans Robert M. 
Published: April 2021

ABSTRACT
De novo expression of vimentin, GFAP or peripherin leads to the assembly of an extended intermediate filament network in intermediate filament-free SW13/cl.2 cells. Desmin, in contrast, does not form extended filament networks in either SW13/cl.2 or intermediate filament-free mouse fibroblasts. Rather, desmin formed short thickened filamentous structures and prominent spot-like cytoplasmic aggregates that were composed of densely packed 9-11 nm diameter filaments. Analysis of stably transfected cell lines indicates that the inability of desmin to form extended networks is not due to a difference in the level of transgene expression. Nestin, paranemin and synemin are large intermediate filament proteins that coassemble with desmin in muscle cells. Although each of these large intermediate filament proteins colocalized with desmin when coexpressed in SW-13 cells, expression of paranemin, but not synemin or nestin, led to the formation of an extended desmin network. A similar rescue of desmin network organization was observed when desmin was coexpressed with vimentin, which coassembles with desmin, or with keratins, which formed a distinct filament network. These studies demonstrate that desmin filaments differ in their organizational properties from the other vimentin-like intermediate filament proteins and appear to depend upon coassembly with paranemin, at least when they are expressed in non-muscle cells, in order to form an extended filament network.
Overexpression of the integrin-linked kinase mesenchymally transforms mammary epithelial cells
Authors: Somasiri Aruna , Howarth Andrew , Goswami Duna , Dedhar Shoukat , Roskelley Calvin D. 
Published: April 2021

ABSTRACT
Signals generated by the interaction of β1 integrins with laminin in the basement membrane contribute to mammary epithelial cell morphogenesis and differentiation. The integrin-linked kinase (ILK) is one of the signaling moieties that associates with the cytoplasmic domain of β1 integrin subunits with some specificity. Forced expression of a dominant negative, kinase-dead form of ILK subtly altered mouse mammary epithelial cell morphogenesis but it did not prevent differentiative milk protein expression. In contrast, forced overexpression of wild-type ILK strongly inhibited both morphogenesis and differentiation. Overexpression of wild-type ILK also caused the cells to lose the cell-cell adhesion molecule E-cadherin, become invasive, reorganize cortical actin into cytoplasmic stress fibers, and switch from an epithelial cytokeratin to a mesenchymal vimentin intermediate filament phenotype. Forced expression of E-cadherin in the latter mesenchymal cells rescued epithelial cytokeratin expression and it partially restored the ability of the cells to differentiate and undergo morphogenesis. These data demonstrate that ILK, which responds to interactions between cells and the extracellular matrix, induces a mesenchymal transformation in mammary epithelial cells, at least in part, by disrupting cell-cell junctions.
Cell-cell adhesion in human fibroblasts requires calcium signaling
Authors: Ko Kevin S. , Arora Pamela D. , Bhide Vinay , Chen Austin , McCulloch Christopher A. G. 
Published: April 2021

ABSTRACT
In connective tissues, intercellular adhesion is essential for tissue morphogenesis, development and wound healing. However, the signaling mechanisms initiated by cell-cell adhesion in fibroblasts and that regulate it are not known. In this study we tested the hypothesis that intracellular calcium signaling is required to mediate intercellular adhesion between fibroblasts. Fura-2 or fluo-3 labeled human fibroblasts were used to investigate calcium homeostasis during intercellular adhesion. After contact with suspended fibroblasts there was a rise in cytosolic free calcium ([Ca2+]i) and multiple calcium oscillations in substrate-attached cells. Antibodies against the extracellular but not the cytoplasmic domain of cadherin induced a similar calcium response, indicating that these responses were initiated by cadherin binding. As shown by the near-plasma membrane Ca2+ indicator (Fura-C18) and by confocal microscopy of fluo-3-loaded cells, [Ca2+]i transients probably originated at sites of cell-cell contact. Cell-cell adhesion was dependent on both calcium influx through membrane channels and release of Ca2+ from internal calcium stores, because the calcium channel inhibitor LaCl3 or pretreatment of cells with thapsigargin significantly inhibited (&gt;35%) cell-cell attachment. The [Ca2+]i changes induced by cell-cell adhesion were temporally correlated with increased recruitment of intercellular junctional proteins into the cytoskeleton and movement of GFP-actin to sites of cell-cell contact. [Ca2+]i responses induced by intercellular adhesion were essential for both junctional protein recruitment and the establishment of strong cell-cell contacts, as loading cells with BAPTA/AM significantly inhibited cell-cell adhesion and recruitment of cadherins and β-catenin to the actin cytoskeleton. Actin depolymerization by cytochalasin D dramatically reduced cell-cell adhesion and recruitment of cadherins and catenin to the actin cytoskeleton. These results demonstrate that cadherin-cadherin interaction induces [Ca2+]i transients during cell-cell adhesion in fibroblasts, and these calcium signals regulate cell-cell adhesion through remodeling of cortical actin and recruitment of cadherins and β-catenin into intercellular junctions.
Programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of acetylcholine
Authors: Nguyen Vu Thuong , Ndoye Assane , Hall Leon L. , Zia Shaheen , Arredondo Juan , Chernyavsky Alexander I. , Kist David A. , Zelickson Brian D. , Lawry Monica A. , Grando Sergei A. 
Published: April 2021

ABSTRACT
The programmed cell death of the stratified squamous epithelial cells comprising human epidermis culminates in abrupt transition of viable granular keratinocytes (KC) into dead corneocytes sloughed by the skin. The granular cell-corneocyte transition is associated with a loss in volume and dry cell weight but the mechanism for and biological significance of this form of keratinocyte apoptosis remain obscure. We show that terminally differentiated KC extrude into the intercellular spaces of living epidermis the cytoplasmic buds containing randomly congregated components of the cytosol as well as filaggrin, a precursor of the natural moisturizing factor. The discharge of secretory product is reminiscent of holocrine secretion, suggesting the term ‘apoptotic secretion’ for this novel, essential step in the process of cornification. The secretory product may become a part of the glycocalyx (a.k.a. ‘intercellular cement substance’ of epidermis) and serve as a humectant that counterbalances the osmotic pressure imposed by the natural moisturizing factor located in the stratum corneum comprised by corneocytes. The apoptotic secretion commences upon secretagouge action of acetylcholine which is synthesized and released by KC. A combination of a cholinergic nicotinic agonist and a muscarinic antagonist which increases intracellular calcium levels is required to trigger the apoptotic secretion. Analysis of the relative amounts of cholinergic enzymes and receptors expressed by KC capable of secretion and the pharmacological profiles of secretion regulation revealed an upward concentration gradient of free acetylcholine in epidermis which may provide for its unopposed secretagogue action via the m1 muscarinic and the α7, and α9 nicotinic receptor types expressed by KC at the latest stage of their development in the epidermis.
A common regulatory locus affects both HNF4/HNF1α pathway activation and sensitivity to LPS-mediated apoptosis in rat hepatoma cells
Authors: Bulla Gary A. , Givens Elena , Brown Stuart , Oladiran Bola , Kraus David 
Published: April 2021

ABSTRACT
Lipopolysaccharide (LPS) has been shown to protect certain cultured mammalian cells from undergoing programmed cell death (apoptosis) when exposed to tumor necrosis factor (TNF). However, LPS has also been reported to induce apoptosis in cultured endothelial cells, suggesting that apoptotic response mechanisms may be dependent upon cell type. In order to understand the influence of tissue-specific gene expression on apoptosis, we compared LPS-induced apoptosis in hepatoma cells with dedifferentiated hepatoma variant cells that have been selected for the loss of the liver-enriched HNF4/HNF1α transcriptional activation pathway. We report here that while human, rat and mouse hepatoma cell lines are resistant to LPS-mediated cell death, the HNF4−/HNF1α− rat hepatoma variant cells undergo rapid apoptosis (as determined by morphological analysis, DNA laddering and the TUNEL assay) upon exposure to LPS. Genetic rescue experiments show that restoration of the HNF4/HNF1α pathway via chromosome transfer render the hepatoma variant cells resistant to LPS-mediated apoptosis. However, the introduction of HNF1α alone failed to alter the apoptotic phenotype, suggesting that the defect(s) in the hepatoma variant cells that influence apoptotic responses lies upstream of HNF4/HNF1α expression. This study provides for the first time direct evidence of a common regulatory locus involved in activation of hepatic gene expression and sensitivity to LPS-mediated apoptosis.
Mutant cadherin affects epithelial morphogenesis and invasion, but not transformation
Authors: Troxell Megan L. , Loftus David J. , Nelson W. James , Marrs James A. 
Published: April 2021

ABSTRACT
MDCK cells were engineered to reversibly express mutant E-cadherin protein with a large extracellular deletion. Mutant cadherin overexpression reduced the expression of endogenous E-and K-cadherins in MDCK cells to negligible levels, resulting in decreased cell adhesion. Despite severe impairment of the cadherin adhesion system, cells overexpressing mutant E-cadherin formed fluid-filled cysts in collagen gel cultures and responded to hepatocyte growth factor/scatter factor (HGF/SF) that induced cellular extension formation with a frequency similar to that of control cysts. However, cells were shed from cyst walls into the lumen and into the collagen matrix prior to and during HGF/SF induced tubule extension.
Despite the propensity for cell dissociation, MDCK cells lacking cadherin adhesion molecules were not capable of anchorage-independent growth in soft agar and cell proliferation rate was not affected. Thus, cadherin loss does not induce transformation, despite inducing an invasive phenotype, a later stage of tumor progression. These experiments are especially relevant to tumor progression in cells with altered E-cadherin expression, particularly tumor samples with identified E-cadherin extracellular domain genomic mutations.
SH3 domains: complexity in moderation
Authors: Mayer Bruce J. 
Published: April 2021

ABSTRACT
The SH3 domain is perhaps the best-characterized member of the growing family of protein-interaction modules. By binding with moderate affinity and selectivity to proline-rich ligands, these domains play critical roles in a wide variety of biological processes ranging from regulation of enzymes by intramolecular interactions, increasing the local concentration or altering the subcellular localization of components of signaling pathways, and mediating the assembly of large multiprotein complexes. SH3 domains and their binding sites have cropped up in many hundreds of proteins in species from yeast to man, which suggests that they provide the cell with an especially handy and adaptable means of bringing proteins together. The wealth of genetic, biochemical and structural information available provides an intimate and detailed portrait of the domain, serving as a framework for understanding other modular protein-interaction domains. Processes regulated by SH3 domains also raise important questions about the nature of specificity and the overall logic governing networks of protein interactions.
Specific changes to the mechanism of cell locomotion induced by overexpression of β-actin
Authors: Peckham Michelle , Miller Gaynor , Zicha Daniel , Dunn Graham A. 
Published: April 2021

ABSTRACT
Overexpression of β-actin is known to alter cell morphology, though its effect on cell motility has not been documented previously. Here we show that overexpressing β-actin in myoblasts has striking effects on motility, increasing cell speed to almost double that of control cells. This occurs by increasing the areas of protrusion and retraction and is accompanied by raised levels of β-actin in the newly protruded regions. These regions of the cell margin, however, show decreased levels of polymerised actin, indicating that protrusion can outpace the rate of actin polymerisation in these cells. Moreover, the expression of β*-actin (a G244D mutant, which shows defective polymerisation in vitro) is equally effective at increasing speed and protrusion. Concomitant changes in actin binding proteins show no evidence of a consistent mechanism for increasing the rate of actin polymerisation in these actin overexpressing cells. The increase in motility is confined to poorly spread cells in both cases and the excess motility can be abolished by blocking myosin function with butanedione monoxime (BDM).
Our observations on normal myoblasts are consistent with the view that they protrude by the assembly and cross linking of actin filaments. In contrast, the additional motility shown by cells overexpressing β-actin appears not to result from an increase in the rate of actin polymerisation but to depend on myosin function. This suggests that the additional protrusion arises from a different mechanism. We discuss the possibility that it is related to retraction-induced protrusion in fibroblasts. In this phenomenon, a wave of increased protrusion follows a sudden collapse in cell spreading. This view could explain why it is only the additional motility that depends on spreading, and has implications for understanding the differences in locomotion that distinguish tissue cells from highly invasive cell types such as leucocytes and malignant cells.
Compartmentalization of Ras proteins
Authors: Prior Ian A. , Hancock John F. 
Published: April 2021

ABSTRACT
The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effectors and exchange factors.
RNA anchoring in the vegetal cortex of the Xenopus oocyte
Authors: Alarcón Vernadeth B. , Elinson Richard P. 
Published: April 2021

ABSTRACT
The body plan of the embryo is established by a polarized source of developmental information in the oocyte. The Xenopus laevis oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including Vg1 and Xwnt-11, which might function in body plan specification, and Xcat-2, which might function in germ cell development. To identify components of the RNA anchoring mechanism, we used the manually isolated vegetal cortex (IVC) to assay loss or change in spatial arrangement of mRNAs caused by disruption of cortical elements. The role of cytoskeleton in mRNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin microfilaments and cytokeratin filaments. Treatment of oocytes with cytochalasin B caused clumping of Vg1 and Xwnt-11 as revealed by in situ hybridization of the IVC, but did not cause their release, as confirmed by RT-PCR analysis. These mRNA clumps did not match the distribution of actin microfilament clumps, but were distributed similarly to the remnant cytokeratin filaments. Treatment of oocytes with monoclonal anti-cytokeratin antibody C11 released these mRNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. These results show that Vg1 and Xwnt-11 are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depend on an intact actin meshwork. Colcemid did not disrupt Vg1 and Xwnt-11 retention in the IVC, so anchoring of these mRNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased Vg1 and Xwnt-11. Loss of these mRNAs was due mainly to ribonuclease activity released from membrane components. However, when ribonuclease activity was suppressed under cold temperature, a higher amount of Vg1 and Xwnt-11 was recovered in the supernatant. This result suggested that a fraction of these mRNAs required membranes to be retained in the cortex. By contrast, Xcat-2 mRNA was neither released nor degraded following treatments with cytochalasin B, C11, colcemid and Triton X-100 under cold temperature, so no cortical element could be implicated in its anchoring.