bluuebunny/crossref_metadata_2025 huggingface dataset:
The cadherin superfamily: diversity in form and function
Authors: Angst Brigitt D. , Marcozzi Cristiana , Magee Anthony I.
Published: April 2021

Over recent years cadherins have emerged as a growing superfamily of molecules, and a complex picture of their structure and their biological functions is becoming apparent. Variation in their extracellular region leads to the large potential for recognition properties of this superfamily. This is demonstrated strikingly by the recently discovered FYN-binding CNR-protocadherins; these exhibit alternative expression of the extracellular portion, which could lead to distinct cell recognition in different neuronal populations, whereas their cytoplasmic part, and therefore intracellular interactions, is constant. Diversity in the cytoplasmic moiety of the cadherins imparts specificity to their interactions with cytoplasmic components; for example, classical cadherins interact with catenins and the actin filament network, desmosomal cadherins interact with catenins and the intermediate filament system and CNR-cadherins interact with the SRC-family kinase FYN. Recent evidence suggests that CNR-cadherins, 7TM-cadherins and T-cadherin, which is tethered to the membrane by a GPI anchor, all localise to lipid rafts, specialised cell membrane domains rich in signalling molecules. Originally thought of as cell adhesion molecules, cadherin superfamily molecules are now known to be involved in many biological processes, such as cell recognition, cell signalling, cell communication, morphogenesis, angiogenesis and possibly even neurotransmission.
Segregation of nucleolar components coincides with caspase-3 activation in cisplatin-treated HeLa cells
Authors: Horký Marcel , Wurzer Gabriele , Kotala Vladimír , Anton Milan , Vojtěsěk Bólivoj , Vácha Jiří , Wesierska-Gadek Jozefa
Published: April 2021

We studied morphological changes of the nucleoli in HeLa cells treated with cisplatin and compared them with induction of markers of programmed cell death and TUNEL staining. We used different light microscopic nucleolar staining methods allowing us to visualize not only nucleolar proteins but also nucleolar RNA. Our results show predominantly compact, centrally localized nucleoli in intact control HeLa cells. In cisplatin-treated HeLa cells, we found an early onset of nucleolar segregation of proteins detected by argyrophilic nucleolar organizer regions and anti-nucleolar monoclonal antibody as well as an increased immunoreactivity for activated caspase-3 after 6 hours. Staining with Toluidine Blue and Methyl-green Pyronine revealed segregated nucleoli 12 hours after the treatment with cisplatin. TUNEL positivity in cisplatin-treated HeLa cells was accompanied by the aggregation of the argyrophilic proteins in the central portion of nucleus, disappearance of nucleolar RNA and shrinkage of the nucleus after 24 hours. Monitoring of the biochemical changes by immunoblotting revealed that activation of distinct caspases and degradation of their downstream protein substrates is executed in two phases. During an early apoptotic stage beginning 4.5 hours post treatment an activation of caspase-9 and caspase-3 was observed. This was accompanied by proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The caspase-9 activation seems to be mediated by recruitment by the activating factor Apaf-1 because the increased accumulation of Apaf-1 and cytochrome C in cytosol preceded the generation of mature caspase-9 form. A second phase of apoptosis occurring between 10 and 15 hours post treatment was characterized by degradation of other nucleolar and nuclear proteins such as nuclear lamins, topoisomerase I and B23.

In conclusion, remarkable segregation of nucleolar argyrophilic proteins, nucleolar RNA and a simultaneous activation of the cascade of caspases markedly preceded the TUNEL positivity in cisplatin-treated HeLa cells thereby substantiating the hypothesis that the nucleolus is a preferred target for caspase-3-dependent proteolysis in cisplatin-treated HeLa cells.
A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane
Authors: Milewski Michál I. , Mickle John E. , Forrest John K. , Stafford Diane M. , Moyer Bryan D. , Cheng Jie , Guggino William B. , Stanton Bruce A. , Cutting Garry R.
Published: April 2021

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370-1394 and 1404-1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.
Internalization of the M2 muscarinic acetylcholine receptor proceeds through an atypical pathway in HEK293 cells that is independent of clathrin and caveolae
Authors: Roseberry Aaron G. , Hosey M. Marlene
Published: April 2021

The M2 muscarinic acetylcholine receptor is a G-protein coupled receptor that undergoes agonist-induced internalization through an unidentified pathway that exhibits an atypical dependence on dynamin function in HEK293 cells. In this report we utilized several independent approaches to reveal that the internalization of the M2 muscarinic acetylcholine receptor did not utilize clathrin-coated pits or caveolae. However, we did observe that treatment with hypertonic sucrose, which is widely reported to specifically inhibit endocytosis through clathrin-coated pits, completely inhibited internalization of the M2 muscarinic acetylcholine receptor. Thus, the pathway that mediates the internalization of the M2 muscarinic acetylcholine receptor appears to be atypical in that it exhibits an unusual sensitivity to dynamin and is inhibited by hypertonic sucrose but lacks the involvement of clathrin and caveolae.
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

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

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

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

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

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

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 (>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

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

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

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

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

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

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

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.
Cholesterol requirement for cation-independent mannose 6-phosphate receptor exit from multivesicular late endosomes to the Golgi
Authors: Miwako Ishido , Yamamoto Akitsugu , Kitamura Toshio , Nagayama Kuniaki , Ohashi Masato
Published: April 2021

The regulation of endocytic traffic of receptors has central importance in the fine tuning of cell activities. Here, we provide evidence that cholesterol is required for the exit of cation-independent mannose 6-phosphate receptor (CI-MPR) from the endosomal carrier vesicle/multivesicular bodies (ECV/MVBs) to the Golgi. A previously established Chinese hamster ovary cell mutant, LEX2, exhibits arrested ECV/MVBs in which CI-MPR and lysosomal glycoprotein-B (lgp-B) are accumulated. The abnormal accumulation of CI-MPR within the ECV/MVBs in LEX2 cells was corrected in a post-translational manner by the supplementation of medium with cholesterol. Furthermore, it was shown that, by expression cloning using LEX2 mutant, the introduction of the NAD(P)H steroid dehydrogenase-like protein, an enzyme involved in the later stage of cholesterol biosynthesis, allows the exit of CI-MPR from the MVBs to the Golgi and reduces the number of arrested ECV/MVBs in LEX2 cells. The recovery of the exit transport of CI-MPR from the ECV/MVBs was associated with the restoration of the normal cellular free cholesterol level and segregation between CI-MPR and lgp-B, both of which had been localized at the internal small vesicles of the arrested ECV/MVBs. By contrast, the restoration of cholesterol failed to correct the defective processing of endocytosed LDL to a degradative compartment in LEX2 cells. These results suggest that cholesterol is required for ECV/MVB reorganization that drives the sorting/transport of materials destined for the Golgi out of the pathways towards lysosomes.
Roles of the N- and C-termini of GLUT4 in endocytosis
Authors: Al-Hasani Hadi , Kunamneni Raghu K. , Dawson Kevin , Hinck Cynthia S. , Müller-Wieland Dirk , Cushman Samuel W.
Published: April 2021

In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane. To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells. Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4. However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states. Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis. Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins μ1, μ2, and μ3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.
Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis
Authors: Raderschall Elke , Bazarov Alex , Cao Jiangping , Lurz Rudi , Smith Avril , Mann Wolfgang , Ropers Hans-Hilger , Sedivy John M. , Golub Efim I. , Fritz Eberhard , Haaf Thomas
Published: April 2021

After exposure of mammalian cells to DNA damage, the endogenous Rad51 recombination protein is concentrated in multiple discrete foci, which are thought to represent nuclear domains for recombinational DNA repair. Overexpressed Rad51 protein forms foci and higher-order nuclear structures, even in the absence of DNA damage, in cells that do not undergo DNA replication synthesis. This correlates with increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21. Following DNA damage, constitutively Rad51-overexpressing cells show reduced numbers of DNA breaks and chromatid-type chromosome aberrations and a greater resistance to apoptosis. In contrast, Rad51 antisense inhibition reduces p21 protein levels and sensitizes cells to etoposide treatment. Downregulation of p21 inhibits Rad51 foci formation in both normal and Rad51-overexpressing cells. Collectively, our results show that Rad51 expression, Rad51 foci formation and p21 expression are interrelated, suggesting a functional link between mammalian Rad51 protein and p21-mediated cell cycle regulation. This mechanism may contribute to a highly effective recombinational DNA repair in cell cycle-arrested cells and protection against DNA damage-induced apoptosis.
Angiopoietin 2 stimulates migration and tube-like structure formation of murine brain capillary endothelial cells through c-Fes and c-Fyn
Authors: Mochizuki Yasushi , Nakamura Takao , Kanetake Hiroshi , Kanda Shigeru
Published: April 2021

The angiopoietin (Ang)/Tie2 system is exclusively involved in vasculogenesis and angiogenesis. Ang2 is known to inhibit Ang1-mediated phosphorylation of Tie2 as well as cellular responses during embryonic development. Recent studies have demonstrated that Ang2 has angiogenic activities in adult tissues and cultured endothelial cells. In the present study, we examined the downstream signaling pathways involved in Ang2-mediated cellular responses by murine brain capillary cell line, IBE cells. Tie2 was tyrosine phoshorylated by Ang2. Ang2 showed no effect on proliferation, but stimulated chemotaxis and tube-like structure formation. Phosphoinositide 3-kinase (PI 3-kinase) was activated by Ang2 through c-Fes and was involved in chemotaxis toward Ang2. Ang2 also activated c-Fyn in IBE cells. Cells expressing kinase-inactive c-Fyn attenuated Ang2-induced tube formation, suggesting that c-Fyn was responsible for Ang-2-mediated tube formation. Collecting these data, Ang2 activates c-Fes and c-Fyn, leading to migration and tube formation by murine capillary endothelial cells.
The tyrosine motifs of Lamp 1 and LAP determine their direct and indirect targetting to lysosomes
Authors: Obermüller Stefanie , Kiecke Christina , von Figura Kurt , Höning Stefan
Published: April 2021

Lamp 1 and lysosomal acid phosphatase (LAP) are lysosomal membrane proteins that harbour a tyrosine-based sorting motif within their short cytoplasmic tails. Lamp 1 is delivered from the trans-Golgi network (TGN) via endosomes directly to lysosomes bypassing the plasma membrane, whereas LAP is indirectly transported to lysosomes and recycles between endosomes and the plasma membrane before being delivered to lysosomes.

By analysing truncated forms of LAP and chimeras in which the cytoplasmic tail or part of the cytoplasmic tails of LAP and Lamp 1 were exchanged, we were able to show that the YRHV tyrosine motif of LAP is necessary and sufficient to mediate recycling between endosomes and the plasma membrane. When peptides corresponding to the cytoplasmic tails of LAP and Lamp 1 and chimeric or mutant forms of these tails were assayed for in vitro binding of AP1 and AP2, we found that AP2 bound to LAP- and Lamp-1-derived peptides, whereas AP1 bound only to peptides containing the YQTI tyrosine motif of Lamp 1. Residues +2 and +3 of the tyrosine motif were critical for the differential binding of adaptors. LAP in which these residues (–HV) were substituted for those of Lamp 1 (–TI) was transported directly to lysosomes, whereas a chimera carrying the Lamp 1 tail in which residues +2 and +3 were substituted for those of LAP (–HV) gained the ability to recycle. In conclusion, the residues +2 and +3 of the tyrosine motifs determine the sorting of Lamp 1 and LAP in endosomes, mediating either the direct or the indirect pathway to lysosomes.
Essential functions of Sds22p in chromosome stability and nuclear localization of PP1
Authors: Peggie Mark W. , MacKelvie Sarah H. , Bloecher Andrew , Knatko Elena V. , Tatchell Kelly , Stark Michael J. R.
Published: April 2021

Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1C) and which has been proposed to regulate one or more functions of PP1C during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1C (Glc7p). Temperature-sensitive (Ts–) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts– mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37°C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts– growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts– growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p
Casein kinase I controls a late step in the endocytic trafficking of yeast uracil permease
Authors: Marchal Christelle , Dupré Sophie , Urban-Grimal Daniele
Published: April 2021

The modification of yeast uracil permease by phosphorylation at the plasma membrane is a key mechanism for regulating transporter endocytosis. Uracil permease is phosphorylated at several serine residues within a well characterized PEST sequence. The phosphorylation of these residues facilitates the ubiquitination and internalization of the permease. Following endocytosis, the permease is targeted to the lysosome/vacuole for proteolysis. We have shown that in casein kinase 1 (CK1)-deficient cells, the permease is poorly phosphorylated, poorly ubiquitinated and that Yck activity may play a direct role in phosphorylating the permease. We show here that CK1-deficient cells accumulated permease that was subjected to endocytosis in an internal compartment on its way to the vacuole. Uracil permease, produced as a fusion protein with green fluorescent protein in CK1-deficient cells, was detected in dots adjacent to the vacuole. These dots probably correspond to the late endosome/prevacuolar compartment because they were partially colocalized with the Pep12p marker. This accumulation was abolished by mutations affecting the adaptor-related complex, AP-3. The CPY and ALP pathways to the vacuole were both unaffected in CK1-deficient cells. Our analysis provides the first evidence that CK1 is important for the delivery of proteins to the vacuole after endocytosis.
Kinetochore localisation of the DNA damage response component 53BP1 during mitosis
Authors: Jullien Denis , Vagnarelli Paola , Earnshaw William C. , Adachi Yasuhisa
Published: April 2021

53BP1 is a vertebrate BRCT motif protein, originally described as a direct interactor of p53, which has recently been shown to be implicated in the early response to DNA damage. Upon DNA damage, 53BP1 re-localises to discrete nuclear foci that are thought to represent sites of DNA lesions and becomes hyperphosphorylated. Several observations suggest that 53BP1 is a direct substrate for the ataxia telangiectasia mutated (ATM) kinase. So far, 53BP1 behaviour during mitosis has not been reported in detail. We have examined 53BP1 subcellular distribution in mitotic cells using several antibodies against 53BP1, and ectopic expression of GFP-tagged 53BP1. We found that 53BP1 significantly colocalised with CENP-E to kinetochores. 53BP1 is loaded to kinetochores in prophase, before CENP-E, and is released by mid-anaphase. By expressing various GFP-tagged 53BP1 truncations, the kinetochore binding domain has been mapped to a 380 residue portion of the protein that excludes the nuclear localisation signal and the BRCT motifs. Like many kinetochore-associated proteins involved in mitotic checkpoint signalling, more 53BP1 appears to accumulate on the kinetochores of chromosomes not aligned on the metaphase plate. Finally, we show that 53BP1 is hyperphosphorylated in mitotic cells, and undergoes an even higher level of phosphorylation in response to spindle disruption with colcemid. Our data suggest that 53BP1 may have a role in checkpoint signalling during mitosis and provide the evidence that DNA damage response machinery and mitotic checkpoint may share common molecular components.
Dissection of HEF1-dependent functions in motility and transcriptional regulation
Authors: Fashena Sarah J. , Einarson Margret B. , O'Neill Geraldine M. , Patriotis Christos , Golemis Erica A.
Published: April 2021

Cas-family proteins have been implicated as signaling intermediaries in diverse processes including cellular attachment, motility, growth factor response, apoptosis and oncogenic transformation. The three defined Cas-family members (p130Cas, HEF1/Cas-L and Efs/Sin) are subject to multiple forms of regulation (including cell-cycle- and cell-attachment-mediated post-translational modification and cleavage) that complicate elucidation of the function of specific Cas proteins in defined biological processes. To explore the biological role of HEF1 further, we have developed a series of cell lines in which HEF1 production is regulated by an inducible promoter. In this system, HEF1 production rapidly induces changes in cellular morphology and motility, enhancing cell speed and haptotaxis towards fibronectin in a process partially dependent on intact ERK and p38 MAPK signaling pathways. Finally, cDNA expression array analysis and subsequent studies indicate that HEF1 production increases levels of mRNA transcripts encoding proteins that are associated with motility, cell transformation and invasiveness, including several metalloproteinases, MLCK, p160ROCK and ErbB2. Upregulation of such proteins suggests mechanisms through which misregulation of HEF1 may be involved in cancer progression.
Cdk5 regulates cell-matrix and cell-cell adhesion in lens epithelial cells
Authors: Negash Sewite , Wang Hwai-Shi , Gao Chun , Ledee Dolena , Zelenka Peggy
Published: April 2021

Cdk5 is a member of the cyclin-dependent kinase family, which is expressed predominantly in terminally differentiated neurons. Lower levels of Cdk5 are also found in a wide variety of cell types, including the lens. Although Cdk5 has been shown to play an important role in neuronal migration and neurite outgrowth, its function in non-neuronal cells is not known. Therefore, this study was undertaken to explore the role of Cdk5 in the lens. Results showed that, within the adult mouse lens, Cdk5 was localized to the cytoplasm,especially along the lateral membranes of differentiating primary fiber cells,which suggests a role in cell-cell adhesion. Staining at the tips of elongating fiber cells was also particularly strong, suggesting a role in cell-matrix adhesion. To examine the possible role of Cdk5 in lens epithelial cell adhesion, we stably transfected N/N1003A rabbit lens epithelial cells with cDNAs for Cdk5 or a dominant-negative mutation, Cdk5-T33. Attachment to a fibronectin matrix, as measured with substrate-coated cell adhesion strips,was increased by Cdk5 overexpression, while an equivalent overexpression of Cdk5-T33 had no effect. Cdk5 also increased the rate of cell attachment and spreading as measured by electric cell-substrate impedance sensing (ECIS). In addition, Cdk5 overexpression decreased cell-cell adhesion as measured by a cell aggregation assay. These findings suggest that Cdk5 plays a role in regulating both cell-matrix and cell-cell interactions in the lens.
Human cord blood-derived cells attain neuronal and glial features in vitro
Authors: Bużańska L. , Machaj E. K. , Zabłocka B. , Pojda Z. , Domańska-Janik K.
Published: April 2021

Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons,astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes — 11% of the total population of differentiating cells.
Genetic and morphological evidence for two parallel pathways of cell-cycle-coupled cytokinesis inDictyostelium
Authors: Nagasaki Akira , de Hostos Eugenio L. , Uyeda Taro Q. P.
Published: April 2021

Myosin-II-null cells of Dictyostelium discoideum cannot divide in suspension, consistent with the dogma that myosin II drives constriction of the cleavage furrow and, consequently, cytokinesis (cytokinesis A). Nonetheless, when grown on substrates, these cells exhibit efficient,cell-cycle-coupled division, suggesting that they possess a novel,myosin-II-independent, adhesion-dependent method of cytokinesis (cytokinesis B). Here we show that double mutants lacking myosin II and either AmiA or coronin, both of which are implicated in cytokinesis B, are incapable of cell-cycle-coupled cytokinesis. These double mutants multiplied mainly by cytokinesis C, a third, inefficient, method of cell division, which requires substrate adhesion and is independent of cell cycle progression. In contrast,double mutants lacking AmiA and coronin were no sicker than each of the single mutants, indicating that the severe defects of myosin II-/AmiA- or myosin II-/coronin-mutants are not simple additive effects of two mutations. We take this as genetic evidence for two parallel pathways both of which lead to cell-cycle-coupled cytokinesis. This conclusion is supported by differences in morphological changes during cytokinesis in the mutant cell lines.