bluuebunny/crossref_metadata_2025 huggingface dataset:
Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation
Authors: Ochotorena Iciar L. , Hirata Dai , Kominami Kin-ichiro , Potashkin Judith , Sahin Fikret , Wentz-Hunter Kelly , Gould Kathleen L. , Sato Kazuhito , Yoshida Yasuko , Vardy Leah , Toda Takashi
Published: April 2021

Accurate chromosome segregation is dependent upon the integrity of mitotic spindles, which pull each pair of sister chromatids towards opposite poles. In this study, we have characterised fission yeast pop3-5235, a diploidising mutant that is impaired in genome stability. Pop3 is the same as Wat1, a conserved protein containing 7 WD repeats. Pop3/Wat1 has also been isolated from a two-hybrid screen as a binding partner to Prp2, the large subunit of the essential splicing factor U2AF. In wat1 mutants, the cellular amount of α-tubulin is decreased to very low levels, which results in compromised microtubules and spindles, consequently leading to unequal chromosome separation. Further analysis shows that, in spite of the binding between Wat1 and Prp2, Wat1 may not be involved directly in splicing reactions per se. Instead, we find that Wat1 is required for the maintenance of α-tubulin mRNA levels; moreover, transcript levels of genes other than the α-tubulin gene are also equally decreased in this mutant. Wild-type Wat1, but not the mutant protein, forms a large complex in the cell with several other proteins, suggesting that Wat1 functions as a structural linker in the complex. The results suggest that Wat1 plays a role in mRNA maturation as a coupling protein between splicing and synthesis and/or stabilisation.
Inhibition of PYK2-induced actin cytoskeleton reorganization, PYK2 autophosphorylation and focal adhesion targeting by FAK
Authors: Du Quan-Sheng , Ren Xiu-Rong , Xie Yi , Wang Qiang , Mei Lin , Xiong Wen-Cheng
Published: April 2021

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) are structurally related tyrosine kinases. They are implicated in regulating actin cytoskeleton organization, a process critical for cell migration, mitosis and tumor metastasis. In this paper, we demonstrate that, although both PYK2 and FAK were expressed and colocalized at focal adhesions in fibroblasts,microinjection of PYK2, but not FAK, in Swiss 3T3 fibroblastic cells led to reorganization of focal adhesions and cell rounding. PYK2-mediated actin cytoskeleton reorganization required the PYK2 N terminus, the focal adhesion targeting (FAT) domain, catalytic activity and autophosphorylation. Remarkably, FAK suppressed PYK2-mediated reorganization of focal adhesions and cell rounding. In addition, FAK inhibited PYK2 autophosphorylation and focal adhesion targeting, which might contribute to FAK-mediated suppression of PYK2's phenotypes. Further analyses demonstrated that the inhibition of PYK2 autophosphorylation required the FAK N terminus but not FAK tyrosine phosphorylation. The FAK FAT domain seemed to be critical for FAK-mediated suppression of PYK2 focal adhesion targeting. Taken together, these results demonstrate that FAK could inhibit PYK2 autophosphorylation, focal adhesion targeting and actin cytoskeleton reorganization, suggesting that the balance between FAK and PYK2 tyrosine kinases is important for regulating cellular morphology, cell migration and cell growth.
Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin
Authors: Akimov Sergey S. , Belkin Alexey M.
Published: April 2021

Assembly of fibronectin into a fibrillar matrix is critical for regulation of cell growth and migration, embryogenesis and wound healing. We have previously shown that cell-surface tissue transglutaminase serves as an integrin-binding adhesion coreceptor for fibronectin. Here we report that transglutaminase strongly promotes fibronectin assembly mediated byα5β1 integrin. This effect is independent from transglutaminase-mediated enzymatic crosslinking of fibronectin and separate from the ability of transglutaminase to stimulate cell spreading. Surface transglutaminase increases the binding of fibronectin to cells via interaction with its gelatin-binding domain that contains modules I6II1,2I7-9 and lacks integrin-binding motifs. The gelatin-binding fragment of fibronectin binds to surface transglutaminase on cells in suspension but does not interact with cell monolayers where surface transglutaminase is occupied by fibronectin. Surface transglutaminase colocalizes with growing fibronectin fibrils at early timepoints of matrix formation and remains codistributed with fibronectin matrices thereafter. The observed stimulation of matrix assembly by transglutaminase is blocked by the gelatin-binding fragment of fibronectin,but is not strongly perturbed by its N-terminal fragment consisting of modules I1-5. These results implicate an interaction between transglutaminase and the gelatin-binding domain of fibronectin in matrix assembly and suggest its role in initiation of fibrillogenesis. However,blocking antibodies against α5β1 integrin or the cell-binding fragment of fibronectin that contains modules III2-11 most strongly suppress matrix formation and abolish the effects of transglutaminase. Hence,transglutaminase cooperates with but can not substitute for α5β1 integrin in fibronectin assembly. Treatment of fibroblasts with transforming growth factor β (TGFβ) significantly increases surface expression of transglutaminase and its association with β1 integrins, but not withαVβ3 integrin. TGFβ enhances the binding of fibronectin to the cell surface and elevates matrix formation, whereas antibody against transglutaminase or the gelatin-binding fragment of fibronectin suppresses these effects, indicating an involvement of transglutaminase in TGFβ-dependent fibronectin assembly. Therefore, TGFβ-induced fibronectin matrix deposition during normal wound healing or fibrotic disorders may depend on upregulation of integrin-associated surface transglutaminase.
Role of clathrin in the regulated secretory pathway of pancreaticβ-cells
Authors: Molinete Miguel , Dupuis Stéphane , Brodsky Frances M. , Halban Philippe A.
Published: April 2021

The role of clathrin in the sorting of proinsulin to secretory granules,the formation of immature granules and their subsequent maturation is not known. To this end, primary rat pancreatic β-cells were infected with a recombinant adenovirus co-expressing the Hub fragment, a dominant-negative peptide of the clathrin heavy chain and enhanced green fluorescent protein(EGFP as a marker of infected cells). A population of cells expressing the highest levels of EGFP (and thus Hub) was obtained using a fluorescence-activated cell sorter (FACS). Control cells were infected with an adenovirus expressing EGFP alone. By immunofluorescence, control cells showed intense staining for both clathrin light chain and proinsulin in a perinuclear region. In cells expressing high levels of Hub, the clathrin light-chain signal was faint and diffuse in keeping with its displacement from membranes. There was, however, no detectable effect of Hub expression on proinsulin staining or disposition within the cell. Proinsulin sorting and conversion,and the fate (release and/or degradation) of insulin and C-peptide, was studied by pulse-chase and quantitative reverse phase HPLC. In both Hub-expressing and control cells, >99% of all newly synthesized proinsulin was sorted to the regulated pathway and there was no effect of Hub on proinsulin conversion to insulin. In presence of Hub there was, however, a significant increase in the percentage of C-peptide truncated to des-(27-31)-C-peptide at early times of chase as well as more extensive degradation of C-peptide thereafter. It is concluded that clathrin is not implicated in the sorting or processing of proinsulin or in regulated exocytosis of secretory granules. These results confirm a role for clathrin in the removal of proteases from maturing granules, thus explaining the increased truncation and degradation of C-peptide in cells expressing Hub.
CDK1-mediated phosphorylation of the RIIα regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIα from centrosomes at mitosis
Authors: Carlson Cathrine R. , Witczak Oliwia , Vossebein Lutz , Labbé Jean-Claude , Skålhegg Bjørn S. , Keryer Guy , Herberg Friedrich W. , Collas Philippe , Taskén Kjetil
Published: April 2021

Protein kinase A regulatory subunit RIIα is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34cdc2 kinase (CDK1) has been shown to phosphorylate RIIα on T54 and this has been proposed to alter the subcellular localization of RIIα. We have made stable transfectants from an RIIα-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIα (RIIα(T54E)). When expressed, RIIα detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIα(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIα is solubilized from particular cell fractions and changes affinity for AKAP450 in the presence of CDK1. D and V mutations of T54 also reduce affinity for the N-terminal RII-binding domain of AKAP450, whereas small neutral residues do not change affinity detected by surface plasmon resonance. In addition, only RIIα(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIα(T54E) transfectant is poorer and occurs at a lower frequency than that of RIIα transfectants. Our results suggest that T54 phosphorylation of RIIα by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.
Regulation of anchoring of the RIIα regulatory subunit of PKA to AKAP95 by threonine phosphorylation of RIIα: implications for chromosome dynamics at mitosis
Authors: Landsverk Helga B. , Carlson Cathrine R. , Steen Rikke L. , Vossebein Lutz , Herberg Friedrich W. , Taskén Kjetil , Collas Philippe
Published: April 2021

CDK1 phosphorylates the A-kinase regulatory subunit RIIα on threonine 54 (T54) at mitosis, an event proposed to alter the subcellular localization of RIIα. Using an RIIα-deficient leukemic cell line (Reh) and stably transfected Reh cell clones expressing wild-type RIIα or an RIIα(T54E) mutant, we show that RIIα associates with chromatin-bound A-kinase anchoring protein AKAP95 at mitosis and that this interaction involves phosphorylation of RIIα on T54. During interphase, both RIIα and RIIα(T54E) exhibit a centrosome-Golgi localization, whereas AKAP95 is intranuclear. At mitosis and in a mitotic extract, most RIIα, but not RIIα(T54E), co-fractionates with chromatin, onto which it associates with AKAP95. This correlates with T54 phosphorylation of RIIα. Disrupting AKAP95-RIIα anchoring or depleting RIIα from the mitotic extract promotes premature chromatin decondensation. In a nuclear reconstitution assay that mimics mitotic nuclear reformation, RIIα is threonine dephosphorylated and dissociates from AKAP95 prior to assembly of nuclear membranes. Lastly, the Reh cell line exhibits premature chromatin decondensation in vitro, which can be rescued by addition of wild-type RIIα or an RIIα(T54D) mutant, but not RIIα(T54E, A, L or V) mutants. Our results suggest that CDK1-mediated T54 phosphorylation of RIIα constitutes a molecular switch controlling anchoring of RIIα to chromatin-bound AKAP95, where the PKA-AKAP95 complex participates in remodeling chromatin during mitosis.
Positive regulation of cell-cell and cell-substrate adhesion by protein kinase A
Authors: Whittard John D. , Akiyama Steven K.
Published: April 2021

Integrin receptor activation is an important regulatory mechanism for cell-substrate and cell-cell adhesion. In this study, we explore a signaling pathway activated by mAb 12G10, an antibody that can activate β1 integrins and induce integrin-mediated cell-cell and cell-substrate adhesion. We have found that the cAMP-dependent protein kinase (PKA) is required for both mAb 12G10-induced cell-cell and cell-substrate adhesion of HT-1080 cells. Binding of mAb 12G10 to β1 integrins stimulates an increase in intracellular cAMP levels and PKA activity, and a concomitant shift in the localization of the PKA type II regulatory subunits from the cytoplasm to areas where integrins expressing the 12G10 epitope are located. MAb 12G10-induced cell-cell adhesion was mimicked by a combination of clustering β1 integrins and elevating PKA activity with Sp-adenosine-3′,5′-cyclic monophosphorothioate or forskolin. We also show that two processes required for HT-1080 cell-cell adhesion, integrin clustering and F-actin polymerization are both dependent on PKA. Taken together, our data suggest that PKA plays a key role in the signaling pathway, resulting from activation of β1 integrins, and that this enzyme may be required for upregulation of cell-substrate and cell-cell adhesion.
Nuclear membrane protein LAP2β mediates transcriptional repression alone and together with its binding partner GCL (germ-cell-less)
Authors: Nili Einav , Cojocaru Gady S. , Kalma Yael , Ginsberg Doron , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Berger Raanan , Shaklai Sigal , Amariglio Ninette , Brok-Simoni Frida , Simon Amos J. , Rechavi Gideon
Published: April 2021

LAP2β is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, we have cloned a novel LAP2β-binding protein, mGCL, which contains a BTB/POZ domain and is the mouse homologue of the Drosophila germ-cell-less (GCL) protein. In Drosophila embryos, GCL was shown to be essential for germ cell formation and was localized to the nuclear envelope. Here, we show that, in mammalian cells, GCL is co-localized with LAP2β to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. Recently, mGCL was found to interact with the DP3α component of the E2F transcription factor. This interaction reduced the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. We demonstrate here that LAP2β is also capable of reducing the transcriptional activity of the E2F-DP complex and that it is more potent than mGCL in doing so. Co-expression of both LAP2β and mGCL with the E2F-DP complex resulted in a reduced transcriptional activity equal to that exerted by the pRb protein.
Syntaxin 1A is delivered to the apical and basolateral domains of epithelial cells: the role of munc-18 proteins
Authors: Rowe Joanna , Calegari Federico , Taverna Elena , Longhi Renato , Rosa Patrizia
Published: April 2021

SNARE (Soluble N-ethyl-maleimide sensitive factor Attachment protein Receptor) proteins assemble in tight core complexes, which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and are distributed in distinct subcellular compartments. The molecular mechanisms underlying sorting of SNAREs to their physiological sites of action are still poorly understood. Here have we analyzed the transport of syntaxin1A in epithelial cells. In line with previous data we found that syntaxin1A is not transported to the plasma membrane, but rather is retained intracellularly when overexpressed in MDCK and Caco-2 cells. Its delivery to the cell surface is recovered after munc-18-1 cotransfection. Furthermore, overexpression of the ubiquitous isoform of munc-18, munc-18-2, is also capable of rescuing the transport of the t-SNARE. The interaction between syntaxin 1A and munc-18 occurs in the biosynthetic pathway and is required to promote the exit of the t-SNARE from the Golgi complex. This enabled us to investigate the targeting of syntaxin1A in polarized cells. Confocal analysis of polarized monolayers demonstrates that syntaxin1A is delivered to both the apical and basolateral domains independently of the munc-18 proteins used in the cotranfection experiments. In search of the mechanisms underlying syntaxin 1A sorting to the cell surface, we found that a portion of the protein is included in non-ionic detergent insoluble complexes. Our results indicate that the munc-18 proteins represent limiting but essential factors in the transport of syntaxin1A from the Golgi complex to the epithelial cell surface. They also suggest the presence of codominant apical and basolateral sorting signals in the syntaxin1A sequence.
Inactivation of the checkpoint kinase Cds1 is dependent on cyclin B-Cdc2 kinase activation at the meiotic G2/M-phase transition inXenopusoocytes
Authors: Gotoh Tetsuya , Ohsumi Keita , Matsui Tomoko , Takisawa Haruhiko , Kishimoto Takeo
Published: April 2021

Checkpoint controls ensure chromosomal integrity through the cell cycle. Chk1 and Cds1/Chk2 are effector kinases in the G2-phase checkpoint activated by damaged or unreplicated DNA, and they prevent entry into M-phase through inhibition of cyclin B-Cdc2 kinase activation. However, little is known about how the effector kinases are regulated when the checkpoint is attenuated. Recent studies indicate that Chk1 is also involved in the physiological G2-phase arrest of immature Xenopus oocytes via direct phosphorylation and inhibition of Cdc25C, the activator of cyclin B-Cdc2 kinase. Bearing in mind the overlapping functions of Chk1 and Cds1, here we have studied the involvement of Xenopus Cds1 (XCds1) in the G2/M-phase transition of immature oocytes and the regulation of its activity during this period. Protein levels of XCds1 remained constant throughout oocyte maturation and early embryonic development. The levels of XCds1 kinase activity were high in immature oocytes and decreased at the meiotic G2/M-phase transition. Consistently, when overexpressed in immature oocytes, wild-type, but not kinase-deficient, XCds1 significantly delayed entry into M-phase after progesterone treatment. The inactivation of XCds1 depended on the activation of cyclin B-Cdc2 kinase, but not MAP kinase. Although XCds1 was not directly inactivated by cyclin B-Cdc2 kinase in vitro, XCds1 was inactivated by overexpression of cyclin B, which induces the activation of cyclin B-Cdc2 kinase without progesterone. Thus, the present study is the first indication of Cds1 activity in cells that are physiologically arrested at G2-phase, and of its downregulation at entry into M-phase.
Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane
Authors: Flesch Frits M. , Brouwers Jos F. H. M. , Nievelstein Patricia F. E. M. , Verkleij Arie J. , van Golde Lambert M. G. , Colenbrander Ben , Gadella Barend M.
Published: April 2021

Mammalian sperm cells are activated prior to fertilization by high bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux. The role of bicarbonate and cholesterol acceptors on the cholesterol organization in the sperm plasma membrane was tested. Bicarbonate induced an albumin-independent change in lipid architecture that was detectable by an increase in merocyanine staining (due to protein kinase A-mediated phospholipid scrambling). The response was limited to a subpopulation of viable sperm cells that were sorted from the non-responding subpopulation by flow cytometry. The responding cells had reduced cholesterol levels (30% reduction) compared with non-responding cells. The subpopulation differences were caused by variable efficiencies in epididymal maturation as judged by cell morphology. Membrane cholesterol organization was observed with filipin, which labeled the entire sperm surface of non-stimulated and non-responding cells, but labeled only the apical surface area of bicarbonate-responding cells. Addition of albumin caused cholesterol efflux, but only in bicarbonate-responding cells that exhibited virtually no filipin labeling in the sperm head area. Albumin had no effect on other lipid components, and no affinity for cholesterol in the absence of bicarbonate. Therefore, bicarbonate induces first a lateral redistribution in the low cholesterol containing spermatozoa, which in turn facilitates cholesterol extraction by albumin. A model is proposed in which phospholipid scrambling induces the formation of an apical membrane raft in the sperm head surface that enables albumin mediated efflux of cholesterol.
Protein phosphatase 2A binds to the cytoplasmic tail of carboxypeptidase D and regulates post-trans-Golgi network trafficking
Authors: Varlamov Oleg , Kalinina Elena , Che Fa-Yun , Fricker Lloyd D.
Published: April 2021

Carboxypeptidase D (CPD) is a transmembrane protein that processes proteins in the trans-Golgi network (TGN). A 20-residue region within the cytoplasmic tail of CPD binds protein phosphatase 2A (PP2A). PP2A also binds to the cytoplasmic tails of other secretory pathway proteins: peptidylglycine-α-amidating mono-oxygenase, the cation-independent mannose-6-phosphate receptor and TGN38. The CPD tail is phosphorylated on Thr residues in the AtT-20 cell line. The CPD tail can also be phosphorylated by purified protein kinase A, protein kinase C and casein kinase II. Both the in vitro and the in vivo phosphorylated CPD tail can be dephosphorylated by purified PP2A. The binding of CPD tail peptide to PP2A does not influence phosphatase activity.

The rate of transport of CPD from the TGN to the cell surface of AtT-20 cells is decreased 45% by okadaic acid, a PP2A inhibitor. Microinjection of the CPD tail into AtT-20 cells inhibits the transition of CPD from endosomal compartments to the TGN. However, okadaic acid does not affect the rate of budding of CPD from the TGN into nascent vesicles or the rate of uptake from the cell surface into endosomal compartments. These results are consistent with the model that PP2A is involved in the trafficking of proteins between a TGN recycling loop and a cell-surface recycling loop, but is not involved in the individual recycling loops.
Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization
Authors: Seipel Katja , O’Brien Stephen P. , Iannotti Elizabeth , Medley Quintus G. , Streuli Michel
Published: April 2021

Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis. This actin remodeling is regulated in part by Rho family GTPases. Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases. Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development. Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance. Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies. Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II. Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies. Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin. Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events. Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B. We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.
Structural models for the self-assembly and microtubule interactions of γ-, δ- and ε-tubulin
Authors: Inclán Yuki F. , Nogales Eva
Published: April 2021

αβ-tubulin heterodimers self-assemble to form microtubules nucleated by γ-tubulin in the cell. γ-tubulin is believed to recruit the αβ-tubulin dimers that form the minus ends of microtubules, but the molecular mechanism of this action remains a matter of heated controversy. Still less is known about the function and molecular interactions of δ-tubulin and ε-tubulin. δ-tubulin may seed the formation of the C triplet tubules in the basal bodies of Chlamydomonas and ε-tubulin is known to localize to the centrosome in a cell cycle-dependent manner. Using the structure of αβ tubulin as a model, we have analyzed the sequences of γ-, δ- and ε-tubulin in regions corresponding to different polymerization interfaces in the tubulin αβ dimer. The sequence comparisons sometimes show clear conservation, pointing to similar types of contacts being functionally important for the new tubulin considered. Conversely, certain surfaces show marked differences that rule out equivalent interactions for non-microtubular tubulins. This sequence/structure analysis has led us to structural models of how these special tubulins may be involved in protein-protein contacts that affect microtubule self-assembly. δ-tubulin most likely interacts longitudinally with α-tubulin at the minus ends of microtubules, while ε-tubulin most likely binds to the plus end of β-tubulin. Conservation of key residues in γ-tubulin suggests that it is capable of longitudinal self-assembly. The implications for the protofilament and template models of nucleation are considered.
The flightless I protein colocalizes with actin- and microtubule-based structures in motile Swiss 3T3 fibroblasts: evidence for the involvement of PI 3-kinase and Ras-related small GTPases
Authors: Davy Deborah A. , Campbell Hugh D. , Fountain Shelley , Jong Danielle de , Crouch Michael F.
Published: April 2021

The flightless I protein contains an actin-binding domain with homology to the gelsolin family and is likely to be involved in actin cytoskeletal rearrangements. It has been suggested that this protein is involved in linking the cytoskeletal network with signal transduction pathways. We have developed antibodies directed toward the leucine rich repeat and gelsolin-like domains of the human and mouse homologues of flightless I that specifically recognize expressed and endogenous forms of the protein. We have also constructed a flightless I-enhanced green fluorescent fusion vector and used this to examine the localization of the expressed protein in Swiss 3T3 fibroblasts. The flightless I protein localizes predominantly to the nucleus and translocates to the cytoplasm following serum stimulation. In cells stimulated to migrate, the flightless I protein colocalizes with β-tubulin- and actin-based structures. Members of the small GTPase family, also implicated in cytoskeletal control, were found to colocalize with flightless I in migrating Swiss 3T3 fibroblasts. LY294002, a specific inhibitor of PI 3-kinase, inhibits the translocation of flightless I to actin-based structures. Our results suggest that PI 3-kinase and the small GTPases, Ras, RhoA and Cdc42 may be part of a common functional pathway involved in Fliih-mediated cytoskeletal regulation. Functionally, we suggest that flightless I may act to prepare actin filaments or provide factors required for cytoskeletal rearrangements necessary for cell migration and/or adhesion.
Anomalous apical plasma membrane phenotype in CK8-deficient mice indicates a novel role for intermediate filaments in the polarization of simple epithelia
Authors: Ameen Nadia A. , Figueroa Yolanda , Salas Pedro J. I.
Published: April 2021

Previous results from our laboratory have indicated a requirement for CK intermediate filaments (IF) for the organization of the apical domain in polarized epithelial cells in culture. The results seemed to be challenged by the phenotype of cytokeratin (CK) 8-deficient mice, which comprises only colorectal hyperplasia, female sterility and a weaker hepatocyte integrity. In this work localization with anti-CK antibodies indicated that many Ck8−/− epithelia still form IF in CK8-deficient mice, perhaps because of the expression of the promiscuous CK7. In the small intestine, only villus enterocytes lacked IFs. These cells appeared to lose syntaxin 3, and three apical membrane proteins (alkaline phosphatase, sucrase isomaltase and cystic fibrosis transmembrane conductance regulator) as they progressed along the villus. At the distal third of the villi, γ-tubulin was found scattered within the cytoplasm of enterocytes, in contrast to its normal sub-apical localization, and the microtubules were disorganized. These results could not be attributed to increased numbers of apoptotic or necrotic cells. The only other cell type we found without IFs in CK8 null mice, the hepatocyte, displayed increased basolateral levels of one apical marker (HA4), indicating a correlation between the lack of intermediate filaments and an apical domain phenotype. These data suggest a novel function for intermediate filaments organizing the apical pole of simple polarized epithelial cells.
Where it all starts: eukaryotic origins of DNA replication
Authors: Bielinsky Anja-Katrin , Gerbi Susan A.
Published: April 2021

Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery. During the past decade much progress has been made in identifying replication origins in eukaryotic genomes. More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely. Beyond giving us the precise positions of start sites, the application of RIP mapping in yeast and human cells has revealed a single, defined start point at which replication initiates, a scenario very reminiscent of transcription initiation. More importantly, studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Therefore, in our pursuit of identifying ORC-binding sites in higher eukaryotes, RIP mapping may lead the way.
Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice
Authors: Gustafsson Erika , Brakebusch Cord , Hietanen Kristiina , Fässler Reinhard
Published: April 2021

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12-20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.
Protective role of Bcl2 in metabolic oxidative stress-induced cell death
Authors: Lee Yong J. , Chen Jenn C. , Amoscato Andrew A. , Bennouna Jaafar , Spitz Douglas R. , Suntharalingam Mohan , Rhee Juong G.
Published: April 2021

Previous studies have shown that overexpression of Bcl2 protects cells from glucose deprivation-induced cell death in multidrug-resistant human breast carcinoma, MCF-7/ADR cells. In this study, we further investigated the protective role of Bcl2 in glucose deprivation-induced cytotoxicity. Although Bcl2 did not prevent a 3.2-fold increase in the level of hydroperoxide during glucose deprivation, it led to a compartmentalization of hydroperoxide molecules in the mitochondria. It also inhibited glucose deprivation-induced cytochrome c release from the mitochondria. It is possible that overexpression of Bcl2 prevents glucose deprivation-induced ceramide generation, probably by preventing the leakage of hydroperoxide from the mitochondria. We also observed that glucose deprivation induced a sixfold increase in oxidized glutathione content, as well as in thiol precursor content. Overexpression of Bcl2 suppressed an increase in oxidized glutathione content and thiol precursor content. Our results indicate that Bcl2 protects cells from metabolic oxidative stress-induced damage by inhibiting the leakage of hydroperoxide from the mitochondria and subsequently preventing ceramide generation. Preventing ceramide generation inhibits the signal transduction pathway and results in the suppression of cytochrome c release from the mitochondria.
Plakophilin 1 interferes with plakoglobin binding to desmoplakin, yet together with plakoglobin promotes clustering of desmosomal plaque complexes at cell-cell borders
Authors: Bornslaeger Elayne A. , Godsel Lisa M. , Corcoran Connie M. , Park Jung K. , Hatzfeld Mechthild , Kowalczyk Andrew P. , Green Kathleen J.
Published: April 2021

Desmosomes are adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. These junctions play an important role in providing strength to tissues that experience mechanical stress such as heart and epidermis. The basic structural elements of desmosomes are similar to those of the better-characterized adherens junctions, which anchor actin-containing microfilaments to cadherins at the plasma membrane. This linkage of actin to classic cadherins is thought to occur through an indirect mechanism requiring the associated proteins, α- and β-catenin. In the case of desmosomes, both linear and lateral interactions have been proposed as playing an important role in formation of the plaque and linkage to the cytoskeleton. However, the precise nature of these interactions and how they cooperate in desmosome assembly are poorly understood.

Here we employ a reconstitution system to examine the assembly of macromolecular complexes from components found in desmosomes of the differentiated layers of complex tissues. We demonstrate the existence of a Triton-soluble complex of proteins containing full length desmoplakin (DP), the arm protein plakoglobin, and the cytoplasmic domain of the desmosomal cadherin, desmoglein 1 (Dsg1). In addition, full length DP, but not an N-terminal plakoglobin binding domain of DP, co-immunoprecipitated with the Dsg1 tail in the absence of plakoglobin in HT1080 cells. The relative roles of the arm proteins plakoglobin and plakophilin 1 (PKP1) were also investigated. Our results suggest that, in the Triton soluble pool, PKP1 interferes with binding of plakoglobin to full length DP when these proteins are co-expressed. Nevertheless, both plakoglobin and PKP1 are required for the formation of clustered structures containing DP and the Dsg1 tail that ultrastructurally appear similar to desmosomal plaques found in the epidermis. These findings suggest that more than one armadillo family member is required for normal assembly and clustering of the desmosomal plaque in the upper layers of the epidermis.
Cellular membranes that undergo cyclic changes in tension: Direct measurement of force generation by an in vitro contractile vacuole of Paramecium multimicronucleatum
Authors: Tani Tomomi , Allen Richard D. , Naitoh Yutaka
Published: April 2021

The contractile vacuole of the fresh water protozoan Paramecium is a membrane-bound vesicle that expels excess cytosolic water, acquired osmotically, through its periodic exocytotic activity. The in vitro contractile vacuole, isolated in a small amount of cytosol from the Paramecium cell and confined under mineral oil, showed periodic rounding and slackening at regular intervals for an extended time. The contractile vacuole rounded against the cytosol-mineral oil boundary tension. The tension at the surface of the contractile vacuole is, therefore, assumed to increase during the rounding phase. We first estimated the tension relative to the boundary tension from the degree of compression of the contractile vacuole by the boundary. We then determined the absolute value for the tension at the surface of the contractile vacuole from the degree of bending of an elastic carbon fiber microcantilever (8 μm thick; 2 mm long), whose free end was placed at the surface of an in vitro contractile vacuole. The tension was found to increase to its maximum value of approximately 5 mN m−1 when the contractile vacuole rounded. This value was more than 35 times higher than that for the slackened contractile vacuole. Electron micrographs of conventional thin sections of chemically fixed in vitro contractile vacuoles as well as those of in vivo contractile vacuoles obtained from rapid frozen and cryosubstituted cells revealed the lack of any ultrastructural evidence for the presence of a fibrous network system surrounding the contractile vacuole. Thus we conclude that the mechanism(s) by which tension is developed at the surface of the contractile vacuole membrane resides in the contractile vacuole membrane itself. We propose a hypothesis that periodic changes in the spontaneous curvature of the contractile vacuole’s lipid bilayer membrane is involved in the periodic development of higher contractile vacuole membrane tension. The isolated CV promises to be an excellent model system for understanding the molecular mechanisms of the dynamics of biological membrane.
Kendrin/pericentrin-B, a centrosome protein with homology to pericentrin that complexes with PCM-1
Authors: Li Qingqin , Hansen Darcy , Killilea Alison , Joshi Harish C. , Palazzo R. E. , Balczon Ron
Published: April 2021

The centrosome is responsible for nucleating microtubules and performing other cellular roles. To define the organization of the centrosome more completely, a human anti-centrosome serum was used to screen a human cDNA library, and a cDNA encoding a >350 kDa centrosome protein was identified. Sequence analyses revealed that this novel centrosome protein contains two coiled-coil domains bounded by non-coiled regions. The N-terminal region of the protein, named pericentrin-B, shares 61% identity (75% similarity) with pericentrin, suggesting an evolutionary relationship between these proteins. Antibodies against pericentrin-B stain centrosomes at all stages of the cell cycle, and pericentrin-B remains associated with centrosomes following microtubule depolymerization. Immunodepletion of neither pericentrin-B nor PCM-1 from cellular extracts inhibited the ability of salt-stripped centrosomes to recover microtubule nucleation potential, demonstrating that neither protein plays a key role in microtubule nucleation processes. Moreover, the binding of both PCM-1 and pericentrin-B with salt-stripped centrosomes required intact microtubules, demonstrating that the association of PCM-1 and pericentrin-B with centrosomes is a late event in the centrosome maturation process. Finally, pericentrin-B and PCM-1 coimmunoprecipitate, suggesting that PCM-1 and pericentrin-B form a functional complex in cells. This observation may help to explain the generation of anti-centrosome autoantibodies in certain autoimmune patients and may be important for centrosome function.
The NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the β3 integrin A isoform
Authors: Gawaz Meinrad , Besta Felicitas , Ylänne Jari , Knorr Thomas , Dierks Henning , Böhm Thomas , Kolanus Waldemar
Published: April 2021

β3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three β3 integrin isoforms (β3-A, -B, -C) have been described so far. Surface expression of β3 integrins is dynamically regulated through internalization of β3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of β3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of β3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the β3-A chimera had strongly reduced cell surface expression compared with the corresponding β3-B, or β3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at β3-A 756-759 is critical for plasma membrane expression of β3-A. Furthermore, delivery of β3-A to the cell surface was specifically modulated by the cytoplasmic protein β3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of β3-endonexin, which does not interact with the β3 tail, acted as a dominant negative inhibitor of β3-A-internalization and enhanced steady-state surface expression of the β3-A-chimera. Furthermore, anti-β3 antibody-induced internalization of the native β3 integrin (αIIbβ3) was dramatically reduced for the Tyr759-Ala substitution mutant αIIbβ3 (Y759A) and expression of the long isoform of β3-endonexin substantially decreased the internalization of wild-type αIIbβ3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the β3 integrin A isoform and β3-endonexin appears to couple the β3-A isoform to a specific receptor-recycling pathway.
Distinct roles for dystroglycan, β1 integrin and perlecan in cell surface laminin organization
Authors: Henry Michael D. , Satz Jakob S. , Brakebusch Cord , Costell Mercedes , Gustafsson Erika , Fässler Reinhard , Campbell Kevin P.
Published: April 2021

Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. Here we show that DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, we define roles for other molecules in this process. In β1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that β1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas β1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell.
Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells
Authors: Föger Niko , Marhaba Rachid , Zöller Margot
Published: April 2021

T cell activation is accompanied by actin-mediated receptor clustering and reorganization of lipid rafts. It has been suggested that costimulatory molecules might be involved in these processes. We here provide evidence that engagement of the adhesion molecule CD44 initiates cytoskeletal rearrangement and membrane reorganization in T cells.

Cross-linking of CD44 on a T helper line was accompanied by adhesion, spreading and actin bundle formation. These processes were energy dependent and required an intact actin and microtubule system. They involved the small GTPase Rac as evidenced by the absence of spreading in cells overexpressing a dominant negative form of Rac. The CD44 initiated reorganization of the cytoskeleton was associated with the recruitment of CD44 and the associated tyrosine phosphokinases p56lck and p59fyn into glycolipid enriched membrane microdomains (GEM). We interpret the data in the sense that CD44 functions as a costimulatory molecule in T cell activation by inducing actin cytoskeletal rearrangements and membrane protein and lipid reorganization including its association with GEMs. Due to the association of CD44 with lck and fyn this colocalization with the TCR allows an abundant provision of these kinases, which are essential to initiate the TCR signaling cascade.
Inhibition of neuronal maturation in primary hippocampal neurons from tau deficient mice
Authors: Dawson Hana N. , Ferreira Adriana , Eyster Michele V. , Ghoshal Nupur , Binder Lester I. , Vitek Michael P.
Published: April 2021

Conflicting evidence supports a role for tau as an essential neuronal cytoskeletal protein or as a redundant protein whose function can be fulfilled by other microtubule-associated proteins. To investigate the function of tau in axonogenesis, we created tau deficient mice by disrupting the TAU gene. The engineered mice do not express the tau protein, appear physically normal and are able to reproduce. In contrast to a previously reported tau knockout mouse, embryonic hippocampal cultures from tau deficient mice show a significant delay in maturation as measured by axonal and neuritic extensions. The classic technique of selectively enhancing axonal growth by growth on laminin substrates failed to restore normal neuronal maturation of tau knockout neurons. By mating human TAU-gene transgenic and tau knockout mice, we reconstituted tau-deficient neurons with human tau proteins and restored a normal pattern of axonal growth and neuronal maturation. The ability of human tau proteins to rescue tau-deficient mouse neurons confirms that tau expression affects the rate of neurite extension.