Journal of Cell Science (J Cell Sci)

Publications in this journal

• Article: Signaling Mechanisms of STIM1 in the Activation of Store- Independent LRC Channels

  Xuexin Zhang, José C. González-Cobos, Rainer Schindl, Martin Muik, Brian Ruhle, Rajender K. Motiani, Jonathan M. Bisaillon, Wei Zhang†, Marc Fahrner, Khalid Matrougui, Margarida Barroso, Christoph Romanin2 & Mohamed Trebak
  Journal of Cell Science 05/2013;
• Article: Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte.

  Caroline M Dalton, John Carroll
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  ABSTRACT: A fundamental rule of cell division is that daughter cells inherit half the DNA complement and an appropriate proportion of cellular organelles. The highly asymmetric cell divisions of female meiosis present a different challenge because one of the daughters, the polar body, is destined to degenerate, putting at risk essential maternally-inherited organelles such as mitochondria. We have therefore investigated mitochondrial inheritance during the meiotic divisions of the mouse oocyte. We find that mitochondria are aggregated around the spindle by a dynein-mediated mechanism during meiosis I, and migrate together with the spindle towards the oocyte cortex. However at cell division they are not equally segregated and move instead towards the oocyte-directed spindle pole and are excluded from the polar body. We show that this asymmetrical inheritance in favour of the oocyte is not caused by bias in spindle itself but is dependent on an intact actin cytoskeleton, spindle-cortex proximity, and cell cycle progression. Thus, oocyte-biased inheritance of mitochondria is a variation on rules that normally govern organelle segregation at cell division, and ensures that essential maternally inherited mitochondria are retained to provide ATP for early mammalian development.
  Journal of Cell Science 05/2013;
• Article: Histone H3 phosphorylation and elimination of paternal X chromosomes at early cleavages in sciarid flies.

  M Carmen Escribá, Clara Goday
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  ABSTRACT: In sciarid flies (Diptera, Sciaridae), one or two paternally derived X chromosomes are discarded from the soma at early cleavages to determine the sex of the embryo (XX females, X0 males). X chromosome/s elimination is achieved by an abnormal anaphase segregation so that X sister chromatids do not reach the poles and are not included in the daughter nuclei. A cis-acting locus (CE) within the heterochromatin proximal to the centromere is known to regulate X chromosome elimination. By immunofluorescence analysis in early embryos from Sciara ocellaris and Sciara coprophila, we investigated histone H3 phosphorylation at Ser10, Ser28 and Thr3 prior to, and during the X elimination process. We found that the regular syncytial nuclear divisions are characterized by a gradual loss of H3S10 phosphorylation along the chromosome arms at anaphase. Importantly, the eliminating X chromosomes show a retardation in anaphase chromatid segregation and high levels of H3S10 phosphorylation in the chromosome arms. We provide here the first evidence linking the hyper-phosphorylated H3 status of the X chromosome with a delay in sister chromatid separation at anaphase. Our findings support the idea that the CE induces a deficiency in H3 dephosphorylation in the paternal X chromosomes to be eliminated.
  Journal of Cell Science 05/2013;
• Article: Pbx1 restrains myeloid maturation while preserving lymphoid potential in hematopoietic progenitors.

  Francesca Ficara, Laura Crisafulli, Chenwei Lin, Masayuki Iwasaki, Kevin S Smith, Luca Zammataro, Michael L Cleary
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  ABSTRACT: The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the Pbx1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that Pbx1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of Pbx1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways including reduced expression of Meis1, a Pbx1 dimerization partner, and its subordinate transcriptional program. Conversely, Pbx1 maintains lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of Pbx1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by Pbx1. These results demonstrate a role for Pbx1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.
  Journal of Cell Science 05/2013;
• Article: Novel roles for Nanos in neural cell fate determination revealed by studies in a cnidarian.

  Justyna Kanska, Uri Frank
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  ABSTRACT: Nanos is a pan-metazoan germline marker, important for germ cell development and maintenance. In flies, Nanos also acts in posterior and neural development, but these functions have not been demonstrated experimentally in other animals. Using the cnidarian Hydractinia we uncover novel roles for Nanos in neural cell fate determination. Ectopic expression of Nanos2 increased the numbers of embryonic stinging cell progenitors, but decreased the numbers of neurons. Downregulation of Nanos2 had the opposite effect. Furthermore, Nanos2 blocked maturation of committed, post-mitotic nematoblasts. Hence, Nanos2 acts as a switch between two differentiation pathways, increasing the numbers of nematoblasts at the expense of neuroblasts, but preventing nematocyte maturation. Nanos2 ectopic expression also caused patterning defects, but these were not associated with deregulation of Wnt signaling, showing that the basic anterior-posterior polarity remained intact, and suggesting that numerical imbalance between nematocytes and neurons may have caused these defects, affecting axial patterning only indirectly. We propose that Nanos' functions in germ cells and neural development are evolutionarily conserved, but its role in posterior patterning is an insect or arthropod innovation.
  Journal of Cell Science 05/2013;
• Article: Molecular Mechanisms of Tau Binding to Microtubule and its Role in Microtubule Dynamics in Live Cells.

  Gilles Breuzard, Pierre Hubert, Roqiya Nouar, Tiphany De Bessa, François Devred, Pascale Barbier, James N Sturgis, Vincent Peyrot
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  ABSTRACT: Despite extensive studies, molecular mechanisms of Tau binding to microtubule (MT) and its consequences on MT stability still remain misunderstood. It is especially true in cells where spatio-temporal distribution of Tau-MT interactions is unknown. Using Förster Resonance Energy Transfer (FRET), we showed that Tau-MT interaction was distributed along MTs in periodic hotspots of high and low FRET intensities. Fluorescence Recovery After Photobleaching (FRAP) revealed a two phase of Tau exchange with MTs as a rapid diffusion followed by a slower binding phase. A real-time FRET assay showed that high FRET occurred simultaneously with rescue and pause transitions at MT ends. To further explore the functional interaction of Tau to the MT, the binding of paclitaxel (PTX), tubulin acetylation induced by trichostatin A (TSA) and the expression of non-acetylable tubulin were used. With PTX and TSA, FRAP curves best fitted with a single phase with a long time constant, while with non-acetylable α-tubulin, curves best fitted a two phase recovery. Upon PTX and TSA incubations, quantities of high and low FRET hotspots decreased by up to 50% and no hotspot was observed during rescue and pause transitions. In the presence of non-acetylable α-tubulin, a 34% increase of low FRET hotspots was measured, and our real-time FRET assay revealed that low FRET hotspots appeared with MTs recovering growth. In conclusion, we have evidence by FRET and FRAP a discrete Tau-MT interaction where Tau could induce conformational changes of MTs, favoring recovery of MT self-assembly.
  Journal of Cell Science 05/2013;
• Article: Regulation of mTORC1 and its impact on gene expression at a glance.

  Mathieu Laplante, David M Sabatini
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  ABSTRACT: The mechanistic (or mammalian) target of rapamycin (mTOR) is a kinase that regulates key cellular functions linked to the promotion of cell growth and metabolism. This kinase, which is part of two protein complexes termed mTOR complex 1 (mTORC1) and 2 (mTORC2), has a fundamental role in coordinating anabolic and catabolic processes in response to growth factors and nutrients. Of the two mTOR complexes, mTORC1 is by far the best characterized. When active, mTORC1 triggers cell growth and proliferation by promoting protein synthesis, lipid biogenesis, and metabolism, and by reducing autophagy. The fact that mTORC1 deregulation is associated with several human diseases, such as type 2 diabetes, cancer, obesity and neurodegeneration, highlights its importance in the maintenance of cellular homeostasis. Over the last years, several groups observed that mTORC1 inhibition, in addition to reducing protein synthesis, deeply affects gene transcription. Here, we review the connections between mTORC1 and gene transcription by focusing on its impact in regulating the activation of specific transcription factors including including STAT3, SREBPs, PPARγ, PPARα, HIF1α, YY1-PGC1α and TFEB. We also discuss the importance of these transcription factors in mediating the effects of mTORC1 on various cellular processes in physiological and pathological contexts.
  Journal of Cell Science 05/2013;
• Article: Intermolecular disulfide bonds among nucleoporins regulate karyopherin-dependent nuclear transport.

  Shige H Yoshimura, Shotaro Otsuka, Masahiro Kumeta, Mariko Taga, Kunio Takeyasu
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  ABSTRACT: Disulfide (S-S) bonds play important roles in the regulation of protein function and cellular stress responses. In this study, we demonstrate that distinct sets of nucleoporins (Nups), components of the nuclear pore complex (NPC), form S-S bonds and regulate nuclear transport through the NPC. Kinetic analysis of importin β demonstrated that the permeability of the NPC was increased by dithiothreitol treatment and reduced by oxidative stress. The permeability of small proteins such as GFP was not affected by either oxidative stress or a reducing reagent. Immunoblot analysis revealed that the oxidative stress significantly induced S-S bond formation in Nups358, 155, 153, and 62 but not 88 and 160. The direct involvement of cysteine residues in the formation of S-S bonds was confirmed by mutating conserved cysteine residues in Nup62, which abolished the formation of S-S bonds and enhanced the permeability of the NPC. Knocking down Nup62 reduced the stress-inducible S-S bonds of Nup155, suggesting that Nups62 and 155 are covalently coupled via S-S bonds. From these results, we propose that the inner channel of the NPC is somehow insulated from the cytoplasm, and is more sensitive than the cytoplasm to the intracellular redox state.
  Journal of Cell Science 05/2013;
• Article: The composition and role of cross links in mechanoelectrical transduction in vertebrate sensory hair cells.

  Carole M Hackney, David N Furness
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  ABSTRACT: The key components of acousticolateralis systems (lateral line, hearing and balance) are sensory hair cells. At their apex, these cells have a bundle of specialized cellular protrusions, which are modified actin-containing microvilli, connected together by extracellular filaments called cross links. Stereociliary deflections open nonselective cation channels allowing ions from the extracellular environment into the cell, a process called mechanoelectrical transduction. This produces a receptor potential that causes the release of the excitatory neurotransmitter glutamate onto the terminals of the sensory nerve fibres, which connect to the cell base, causing nerve signals to be sent to the brain. Identification of the cellular mechanisms underlying mechanoelectrical transduction and of some of the proteins involved has been assisted by research into the genetics of deafness, molecular biology and mechanical measurements of function. It is thought that one type of cross link, the tip link, is composed of cadherin 23 and protocadherin 15, and gates the transduction channel when the bundle is deflected. Another type of link, called lateral (or horizontal) links, maintains optimal bundle cohesion and stiffness for transduction. This Commentary summarizes the information currently available about the structure, function and composition of the links and how they might be relevant to human hearing impairment.
  Journal of Cell Science 05/2013;
• Article: Human Cep192 and Cep152 cooperate in Plk4 recruitment and centriole duplication.

  Katharina F Sonnen, Anna-Maria Gabryjonczyk, Eduard Anselm, York-Dieter Stierhof, Erich A Nigg
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  ABSTRACT: Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, but the mechanism underlying its recruitment to mammalian centrioles is not understood. In flies, Plk4 recruitment depends on Asterless, whereas nematodes rely on a distinct protein, Spd-2. Here, we have explored the roles of two homologous mammalian proteins, Cep152 and Cep192, respectively, in the centriole recruitment of human Plk4. We demonstrate that Cep192 plays a key role in centrosome recruitment of both Cep152 and Plk4. Double-depletion of Cep192 and Cep152 completely abolishes Plk4 binding to centrioles as well as centriole duplication, indicating that the two proteins cooperate. Most importantly, we show that Cep192 binds Plk4 through an N-terminal extension that is specific to the largest isoform. The Plk4 binding regions of Cep192 and Cep152 (residues 190-240 and 1-46, respectively) are rich in negatively charged amino acids, suggesting that Plk4 localization to centrioles depends on electrostatic interactions with the positively charged polo-box domain. We conclude that cooperation between Cep192 and Cep152 is crucial for centriole recruitment of Plk4 and centriole duplication during the cell cycle.
  Journal of Cell Science 05/2013;
• Article: Evidence of a triosephosphate isomerase non-catalytic function critical to behavior and longevity.

  Bartholomew P Roland, Kimberly A Stuchul, Samantha B Larsen, Christopher G Amrich, Andrew P Vandemark, Alicia M Celotto, Michael J Palladino
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  ABSTRACT: Triosephosphate isomerase (TPI) is a glycolytic enzyme that converts dihydroxyacetone phosphate (DHAP) into glyceraldehyde-3-phosphate (GAP). Glycolytic enzyme dysfunction leads to metabolic diseases collectively known as glycolytic enzymopathies. Of these enzymopathies, TPI deficiency is unique in the severity of neurological symptoms. The Drosophila sugarkill mutant closely models TPI deficiency and encodes a protein prematurely degraded by the proteasome. This result led us to question whether enzyme catalytic activity was critical to the pathogenesis of TPI sugarkill neurological phenotypes. To study TPI deficiency in vivo we developed a genomic engineering system for the TPI locus that enables the efficient generation of novel TPI genetic variants. Using this system we demonstrate that TPI sugarkill can be genetically complemented by TPI encoding a catalytically inactive enzyme. Further, our results demonstrate a non-metabolic function for TPI, the loss of which contributes significantly to the neurological dysfunction in this animal model.
  Journal of Cell Science 05/2013;
• Article: The yeast cell cortical protein Num1 integrates mitochondrial dynamics into cellular architecture.

  Till Klecker, Dirk Scholz, Johannes Förtsch, Benedikt Westermann
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  ABSTRACT: During the cell cycle each organelle has to be faithfully partitioned to the daughter cells. However, the mechanisms controlling organellar inheritance remain poorly understood. We studied the contribution of the cell cortex protein, Num1, to mitochondrial partitioning in yeast. Live cell microscopy revealed that Num1 is required for attachment of mitochondria to the cell cortex and retention in mother cells. Visualization of anchoring sites by electron tomography revealed plasma membrane invaginations directly contacting the mitochondrial outer membrane. Expression of chimeric plasma membrane tethers rescued mitochondrial fission defects in Δnum1 and Δmdm36 mutants. These findings provide new insights into the coupling of mitochondrial dynamics, immobilization, and retention during inheritance.
  Journal of Cell Science 05/2013;
• Article: Germline deletion of Cetn1 causes infertility in male mice.

  Prachee Avasthi, Jan Frederik Scheel, Guoxin Ying, Jeanne M Frederick, Wolfgang Baehr, Uwe Wolfrum
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  ABSTRACT: Centrins are calmodulin-like Ca(2+)-binding proteins that can be found in all ciliated eukaryotic cells from yeast to mammals. Expressed in male germ cells and photoreceptors, centrin 1 (CETN1) resides in the photoreceptor transition zone and connecting cilium. To identify its function in mammals, we deleted Cetn1 by homologous recombination. Cetn1(-/-) mice were viable and showed no sign of retina degeneration suggesting that CETN1 is nonessential for photoreceptor ciliogenesis or structural maintenance. Phototransduction components localized normally to the Cetn1(-/-) photoreceptor outer segments, and loss of CETN1 had no effect on light-induced translocation of transducin to the inner segment. Although Cetn1(-/-) females and Cetn1(+/-) males had normal fertility, Cetn1(-/-) males were infertile. The Cetn1(-/-) testes size was normal, and spermatogonia as well as spermatocytes developed normally. However, spermatids lacked tails suggesting severe spermiogenesis defects at the late maturation phase of spermatozoa. Viable sperm cells were absent and the few surviving spermatozoa were malformed. Light and electron microscopy analyses of Cetn1(-/-) spermatids revealed failures in centriole rearrangement during basal body maturation and in the basal body-nucleus connection. These results confirm an essential role for CETN1 in late steps of spermiogenesis and spermatid maturation.
  Journal of Cell Science 05/2013;
• Article: Lamin aggregation is an early sensor of porphyria-induced liver injury.

  Amika Singla, Nicholas W Griggs, Raymond Kwan, Natasha T Snider, D Maitra, Stephen A Ernst, Harald Herrmann, M Bishr Omary
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  ABSTRACT: Oxidative liver injury during steatohepatitis results in aggregation and transglutaminase-2 (TG2)-mediated crosslinking of the keratin cytoplasmic intermediate filament proteins (IFs) to form Mallory-Denk body (MDB) inclusions. The effect of liver injury on lamin nuclear IFs is unknown, though lamin mutations in several human diseases result in lamin disorganization and nuclear shape changes. We tested the hypothesis that lamins undergo aggregation during oxidative liver injury using two MDB mouse models: (i) feeding the porphyrinogenic drug 3, 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and, (ii) mice that harbor a mutation in ferrochelatase (fch), which converts protoporphyrin-IX to heme. Dramatic aggregation of lamin A/C and B1 was noted in the livers of both models in association with changes in lamin organization and nuclear shape as determined by immunostaining and electron microscopy. The lamin aggregates sequester other nuclear proteins including transcription factors and ribosomal and nuclear pore components into high molecular weight complexes, as determined by mass-spectrometry and confirmed biochemically. Lamin aggregate formation is rapid and precedes keratin aggregation in fch livers, and is seen in liver explants of patients with alcoholic cirrhosis. Exposure of cultured cells to DDC, protoporphyrin-IX or N-methyl-protoporphyrin, or incubating purified lamins with protoporphyrin-IX also results in lamin aggregation. In contrast, lamin aggregation is ameliorated by TG2 inhibition. Therefore, lamin aggregation is an early sensor of porphyria-associated liver injury and may serve to buffer oxidative stress. The nuclear shape and lamin defects associated with porphyria phenocopy the changes seen in laminopathies and could result in transcriptional alterations due to sequestration of nuclear proteins.
  Journal of Cell Science 05/2013;
• Article: Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance.

  Chao Qu, Trisha Dwyer, Qiangqiang Shao, Tao Yang, Huai Huang, Guofa Liu
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  ABSTRACT: The coupling of axon guidance cues, such as netrin-1, to microtubule (MT) dynamics is essential for growth cone navigation in the developing nervous system. However, whether axon guidance signaling regulates MT dynamics directly or indirectly is unclear. Here, we report that TUBB3, the most dynamic β-tubulin isoform in neurons, directly interacts with the netrin receptor DCC, and that netrin-1 induces this interaction in primary neurons. TUBB3 colocalizes with DCC in the growth cones of primary neurons and MT dynamics is required for netrin-1-promoted association of TUBB3 with DCC. Netrin-1 not only increases cosedimentation of DCC with polymerized MT, but also promotes MT dynamics in the growth cone. Knocking down TUBB3 inhibits netrin-1-induced MT dynamics, axon outgrowth and attraction in vitro and causes defects in commissural axon projection in the embryo. These results indicate that TUBB3 directly links netrin signaling pathways to MT dynamics and plays an important role in guiding commissural axons in vivo.
  Journal of Cell Science 05/2013;
• Article: Antagonism and synergy between extracellular BMP modulators Tsg and BMPER to balance blood vessel formation.

  Jennifer Heinke, Maria Juschkat, Anne Charlet, Leonie Mnich, Thomas Helbing, Christoph Bode, Cam Patterson, Martin Moser
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  ABSTRACT: Growth and regeneration of blood vessels are crucial processes during embryonic development and in adult disease. Members of the bone morphogenetic protein (BMP) family are growth factors known to play a key role in vascular development. The BMP pathway is controlled by extracellular BMP modulators such as BMP endothelial cell precursor derived regulator (BMPER), which we reported previously to act proangiogenic on endothelial cells in a concentration-dependent manner. Here, we explore the function of other BMP modulators and especially Tsg on endothelial cell behaviour and compare them to BMPER. In matrigel assays BMP modulators Chordin and Noggin had no stimulatory effect; however Gremlin and Tsg enhanced human umbilical vein endothelial cell (HUVEC) sprouting. As Tsg displayed similar activation dynamics as BMPER, we further investigated the proangiogenic effect of Tsg on endothelial cells. Tsg enhanced endothelial cell ingrowth in the mouse matrigel plug assay as well as HUVEC sprouting, migration and proliferation in vitro dependent on Akt, Erk and Smad signalling pathway activation in a concentration-dependent manner. Surprisingly, silencing of Tsg also increased HUVEC sprouting, migration and proliferation, which is again associated with Akt, Erk and Smad signalling pathway activation. Furthermore, we reveal that Tsg and BMPER interfere with each other to enhance proangiogenic events. However, in vivo the presence of Tsg as well as of BMPER is mandatory for regular development of the zebrafish vasculature. Taken together, our results suggest that BMPER and Tsg maintain a fine-tuned equilibrium that controls BMP pathway activity and is necessary for vascular cell homeostasis.
  Journal of Cell Science 05/2013;
• Article: Phosphorylation of STIM1 at ERK1/2 target sites regulates the interaction with the microtubule plus-end binding protein EB1

  Eulalia Pozo-Guisado, Vanessa Casas-Rua, Patricia Tomas-Martin, Aida M. Lopez-Guerrero, Alberto Alvarez-Barrientos, Francisco Javier Martin-Romero
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  ABSTRACT: STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca2+ concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although one knows the molecular details for STIM1-SOCs binding, the regulation of SOCE remains largely unknown. A detailed list of phosphoresidues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phospho-specific antibodies, it is demonstrated here that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608, and Ser621 during Ca2+ store depletion, and that Ca2+ entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. While Ser to Ala mutation of residues 575, 608, and 621 showed a constitutive binding to EB1 even after Ca2+ store depletion, Ser to Glu mutation of these residues, to mimic the phosphorylation profile attained after store depletion, triggered full dissociation from EB1. Given that wild-type STIM1 and STIM1S575E/S608E/S621E activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca2+ store depletion, an event that is fully reverted by Ca2+ entry and store refilling.
  Journal of Cell Science 05/2013;
• Article: Apicobasal secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by distinct mechanisms.

  Hideki Yamamoto, Chihiro Awada, Hideaki Hanaki, Hiroshi Sakane, Ikuko Tsujimoto, Yuko Takahashi, Toshifumi Takao, Akira Kikuchi
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  ABSTRACT: Wnts are glycan- and lipid-modified morphogens that are important for cellular responses, but how Wnt is secreted in polarized epithelial cells remains unclear. Although Wntless (Wls) has been shown to interact with Wnts and support their secretion, the role of Wls in the sorting of Wnts to the final destination in polarized epithelial cells have not been clarified. Glycosylation was shown to be important for the sorting of some transmembrane and secreted proteins, but glycan profiles and their roles in the polarized secretion of Wnts are not known. Here we show the apicobasal secretion of Wnts is regulated by different mechanisms. Wnt11 and Wnt3a were secreted apically and basolaterally, respectively, in polarized epithelial cells. Wls was localized to the basolateral membrane. Mass-spectrometric analyses revealed that Wnt11 is modified with complex/hybrid-(Asn40), high-mannose-(Asn90), and high-mannose/hybrid-(Asn300) type glycans and that Wnt3a is modified with two high-mannose-type glycans (Asn87 and Asn298). Glycosylation processing at Asn40 and galectin-3 were required for the apical secretion of Wnt11, while clathrin and adaptor protein-1 were required for the basolateral secretion of Wnt3a. By the fusion of the Asn40 glycosylation site of Wnt11, Wnt3a was secreted apically. The recycling of Wls by AP-2 was necessary for the basolateral secretion of Wnt3a but not for the apical secretion of Wnt11. These results suggest that Wls has different roles on the polarized secretion of Wnt11 and Wnt3a and that glycosylation processing of Wnts decides their secretory routes.
  Journal of Cell Science 04/2013;