Journal of Cell Science (J CELL SCI)

Publications in this journal

• Article: The molecular chaperone Hsp47 is essential for cartilage and endochondral bone formation

  Yusaku Masago, Akihiro Hosoya, Kunito Kawasaki, Shogo Kawano, Akira Nasu, Junya Toguchida, Katsumasa Fujita, Hiroaki Nakamura, Gen Kondoh, Kazuhiro Nagata
  [show abstract] [hide abstract]
  ABSTRACT: Heat shock protein 47 kDa (Hsp47) is considered as a molecular chaperone essential for the correct folding of type I and type IV procollagen in the ER. However, the function of Hsp47 for other types of procollagen and its importance for chondrogenesis have never been elucidated. To examine the function of Hsp47 in cartilage formation and endochondral ossification, we conditionally inactivated the Hsp47 gene in chondrocytes using Hsp47 floxed mice and mice carrying a chondrocyte-specific Col2a1-Cre transgene. Hsp47 conditional null mutant mice died just before or shortly after birth, and exhibited severe generalized chondrodysplasia and bone deformities with lower levels of type II and type XI collagen. Second-harmonic generation (SHG) analysis and electron microscopy revealed the accumulation of misaligned type I collagen molecules in the intervertebral discs and a substantial decrease in type II collagen fibers, respectively. Whole-mount skeletal staining showed no calcified region in the vertebral bodies of sacral vertebrae, and revealed that the endochondral bones were severely twisted and shortened. These results demonstrate that Hsp47 is indispensable for well-organized cartilage and normal endochondral bone formation.
  Journal of Cell Science 03/2012; 125(Pt 5):1118-1128.
• Article: Differential effects of Sec61α-, Sec62- and Sec63-depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells

  Sorin Fedeles
  Journal of Cell Science 02/2012;
• Article: Contrasting roles of condensin I and II in mitotic chromosome formation

  Green LC, Kalitsis P, Chang TM, Cipetic M, Kim JH, Marshall O, Turnbull L, Whitchurch CB, Vagnarelli P, Samejima K, Earnshaw WC, Choo KH, Hudson DF
  Journal of Cell Science 02/2012;
• Article: Trans-synaptic interaction between ICAM-5 and 1 integrin regulates neuronal synapse formation.

  Ning, Tian, Smirnov, Vihinen, Llano, Vick, Davis, R. L, Rivera, Gahmberg C. G
  Journal of Cell Science 01/2012;
• Article: Regulation of sphingolipid synthesis through Orm1 and Orm2 in yeast

  Ming Liu, Chunjuan Huang, Surendranath R Polu, Roger Schneiter, Amy Chang
  [show abstract] [hide abstract]
  ABSTRACT: Sphingolipids are crucial components of membranes, and sphingolipid metabolites serve as signaling molecules. Yeast Orm1 and Orm2 belong to a conserved family of ER membrane proteins that regulate serine palmitoyltransferase, which catalyzes the first and rate-limiting step in sphingolipid synthesis. We now show that sphingolipid synthesis through Orm1 is a target of TOR signaling, which regulates cell growth in response to nutritional signals. Orm1 phosphorylation is dependent on the Tap42–phosphatase complex, which acts downstream of TOR protein kinase complex 1. In temperature-sensitive tap42-11 cells, impaired Orm1 phosphorylation occurs concomitantly with reduced sphingolipid synthesis. A second mechanism for regulating sphingolipid synthesis is through control of Orm2 protein level. The Orm2 protein level responds to ER stress conditions, increasing when cells are treated with tunicamycin or DTT, agents that induce the unfolded protein response (UPR). The sphingolipid intermediates (long chain base and ceramide) are decreased when ORM2 is overexpressed, suggesting that sphingolipid synthesis is repressed under ER stress conditions. Finally, in the absence of the Orms, the UPR is constitutively activated. Lipid dysregulation in the absence of the Orms might signal to the ER from the plasma membrane because UPR activation is dependent on a cell surface sensor and the mitogen-activated protein kinase (MAPK) cell wall integrity pathway. Thus, sphingolipid synthesis and the UPR are coordinately regulated. Introduction Membrane biogenesis during cell growth and proliferation involves coordination of protein and lipid synthesis. In response to nutritional conditions, protein synthesis is modulated by multiple signaling pathways (Smets et al., 2010). One of these is target of rapamycin (TOR), a conserved protein kinase complex that regulates growth in response to nutrients and stresses. The unfolded protein response (UPR) pathway also plays a role in membrane biogenesis by adjusting the capacity of the endoplasmic reticulum (ER) to handle the load of newly synthesized proteins (Cox et al., 1997; Ron and Walter, 2007; Rutkowski and Hegde, 2010). The ER also serves as the initiation site for synthesis of the major lipid components of membranes. Transcriptional regulation serves as a major mechanism for controlling phospholipid and sterol synthesis (Nohturfft and Zhang, 2009). Insight into regulation of sphingolipid synthesis has come from recent discovery of the conserved ORMDL family of ER membrane proteins (Breslow et al., 2010; Han et al., 2010). Sphingolipids are crucial structural components of membranes and contribute to key physical properties (Breslow and Weissman, 2010). Sphingolipid metabolites also have important signaling functions. The ORMDL proteins regulate sphingolipid synthesis by physically associating with serine palmitoyltransferase (SPT), which catalyzes the first and rate-limiting step of sphingolipid synthesis. SPT mediates production of long chain bases from the condensation of serine and palmitoyl CoA (Funato et al., 2002). Ceramide is then generated upon addition of a second long chain fatty acid to a long chain base. Mature sphingolipids are made in the Golgi upon transport of ceramide from the ER. The ORMDL proteins repress SPT activity; in the absence of ORMDL regulation, SPT activity becomes hyperactive and long chain base accumulates (Breslow et al., 2010; Han et al., 2010). In yeast, SPT activity is regulated by Orm1 and Orm2, and phosphorylation of the Orm proteins adjusts SPT activity to maintain sphingolipid homeostasis (Breslow et al., 2010). In cells depleted of Orm1 and Orm2 (orm1D orm2D cells), dysregulation of sphingolipid synthesis results in pleiotropic phenotypes, including impaired growth and constitutive activation of the UPR (Han et al., 2010). As a first step to understanding how sphingolipid synthesis is regulated in response to growth conditions, we have identified the TOR signaling pathway as one regulatory component controlling sphingolipid synthesis through Orm1. In this study, we show that Orm1 phosphorylation and sphingolipid synthesis are dependent on the Sit4–Tap42 complex, a downstream target of the rapamycin-sensitive TOR protein kinase complex, TORC1. Another mechanism for regulating sphingolipid synthesis is by changing the amount of Orm2 protein. Orm2 protein level is increased by agents that cause ER stress, and sphingolipid synthesis is repressed upon ORM2 overexpression.
  Journal of Cell Science 01/2011; 125:2428-2435.
• Article: Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I.

  Nobuaki R Kudo, Martin Anger, Antoine H F M Peters, Olaf Stemmann, Hans-Christian Theussl, Wolfgang Helmhart, Hiromi Kudo, Christa Heyting, Kim Nasmyth
  [show abstract] [hide abstract]
  ABSTRACT: Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females.
  Journal of Cell Science 09/2009; 122(Pt 15):2686-98.
• Article: Integrins mediate their unconventional, mechanical-stress-induced secretion via RhoA and PINCH in Drosophila.

  Hans Schotman, Leena Karhinen, Catherine Rabouille
  [show abstract] [hide abstract]
  ABSTRACT: During the epithelium remodelling such as the flattening of the Drosophila follicular epithelium, the alpha-integrin subunits are unconventionally secreted through a dGRASP-dependent route that is built de novo. The biogenetic process starts with the upregulation of a small subset of targeted mRNAs, including dgrasp. Here, we show that dgrasp mRNA upregulation is triggered by the tension of the underlying oocyte and by applied external forces at the basal side of the follicular epithelium. We show that integrins are also involved in dgrasp mRNA upregulation and the epithelium remodelling. Tension leads to the recruitment of RhoA to the plasma membrane, where it participates in its remodelling. The LIM protein PINCH can cycle to the nucleus and is involved in dgrasp mRNA upregulation. We propose that integrins are involved in triggering the biogenesis of their own unconventional secretion route that they use to strengthen adhesion and ensure epithelial integrity at the next stages of development, perhaps by acting as mechanosensors of the underlying tension through RhoA and PINCH.
  Journal of Cell Science 09/2009; 122(Pt 15):2662-72.
• Article: Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation.

  Stanley S Stylli, T T I Stacey, Anne M Verhagen, San San Xu, Ian Pass, Sara A Courtneidge, Peter Lock
  [show abstract] [hide abstract]
  ABSTRACT: Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation.
  Journal of Cell Science 09/2009; 122(Pt 15):2727-40.
• Article: G2 histone methylation is required for the proper segregation of chromosomes.

  Ryan Heit, Jerome B Rattner, Gordon K T Chan, Michael J Hendzel
  [show abstract] [hide abstract]
  ABSTRACT: Trimethylation of lysine 9 on histone H3 (H3K9me3) is known both to be necessary for proper chromosome segregation and to increase in late G2. We investigated the role of late G2 methylation, specifically in mitotic progression, by inhibiting methylation for 2 hours prior to mitosis using the general methylation inhibitor adenosine dialdehyde (AdOx). AdOx inhibits all methylation events within the cell but, by shortening the treatment length to 2 hours and studying mitotic cells, the only methylation events that are affected are those that occur in late G2. We discovered that methylation events in this time period are crucial for proper mitosis. Mis-segregation of chromosomes is observed with AdOx treatment. Through studies of histone modifications, we have found that inhibiting late G2 methylation affects trimethylation of H3K9 and H4K20. The mitotic checkpoint is active and many kinetochore proteins localize properly, however, pericentric chromatin in these cells is found to be less compact (dense). The reduced integrity of pericentric heterochromatin might be responsible for a noted loss of tension at the centromere in AdOx-treated cells and activation of the spindle assembly checkpoint. We postulate that late G2 methylation is necessary for proper pericentric heterochromatin formation. The results suggest that a reduction in heterochromatin integrity might interfere both with microtubule attachment to chromosomes and with the proper sensing of tension from correct microtubule-kinetochore connections, either of which will result in activation of the mitotic checkpoint.
  Journal of Cell Science 09/2009; 122(Pt 16):2957-68.
• Article: Phosphorylation of histone H3 at Thr3 is part of a combinatorial pattern that marks and configures mitotic chromatin.

  Yolanda Markaki, Anastasia Christogianni, Anastasia S Politou, Spyros D Georgatos
  [show abstract] [hide abstract]
  ABSTRACT: We have previously shown that histone H3 is transiently phosphorylated at Thr3 during mitosis. Extending these studies, we now report that phosphorylated Thr3 is always in cis to trimethylated Lys4 and dimethylated Arg8, forming a new type of combinatorial modification, which we have termed PMM. PMM-marked chromatin emerges at multiple, peripheral sites of the prophase nucleus, then forms distinct clusters at the centric regions of metaphase chromosomes, and finally spreads (as it wanes) to the distal areas of segregating chromatids. The characteristic prophase pattern can be reproduced by expressing ectopically the kinase haspin at interphase, suggesting that the formation of the PMM signature does not require a pre-existing mitotic environment. On the other hand, the ;dissolution' and displacement of PMM clusters from a centric to distal position can be induced by partial dephosphorylation or chromosome unravelling, indicating that these changes reflect the regulated grouping and scrambling of PMM subdomains during cell division. Formation of PMM is prevented by haspin knockdown and leads to delayed exit from mitosis. However, PMM-negative cells do not exhibit major chromosomal defects, suggesting that the local structures formed by PMM chromatin may serve as a ;licensing system' that allows quick clearance through the metaphase-anaphase checkpoint.
  Journal of Cell Science 09/2009; 122(Pt 16):2809-19.
• Article: Opposing effects of Ndel1 and alpha1 or alpha2 on cytoplasmic dynein through competitive binding to Lis1.

  Chong Ding, Xujun Liang, Li Ma, Xiaobing Yuan, Xueliang Zhu
  [show abstract] [hide abstract]
  ABSTRACT: Lis1 is an essential protein whose insufficiency causes aberrant neuronal positioning during neocortical development. It is believed to regulate both cytoplasmic dynein, a microtubule minus-end-directed motor, through direct interaction, and platelet-activating factor acetylhydrolase (PAF-AH) Ib by complexing with the catalytic subunits alpha1 and alpha2. Although alpha1 and alpha2 are highly expressed in brain, their deficiencies fail to cause brain abnormality. Here, we show that overexpression of alpha2 or alpha1 results in inactivation of dynein characterized by Golgi and endosome dispersion and mitotic delay. Further overexpression of Lis1 or Ndel1, a Lis1- and dynein-binding protein that is also crucial for dynein function, restored Golgi and endosome distribution. Biochemical assays showed that alpha1 and especially alpha2, were able to compete against Ndel1 and dynein for Lis1 binding in a dose-dependent manner. Overexpression of alpha2 in developing rat brain repressed the radial migration of neurons and mitotic progression of neuroprogenitors. By contrast, a Lis1-binding-defective point mutant, alpha2(E39D), was ineffective in the above assays. These results indicate an antagonistic effect of alpha1, alpha2 and Ndel1 for Lis1 binding, probably to modulate dynein functions in vivo. They also help to explain why brain development is particularly sensitive to a decrease in Lis1 levels.
  Journal of Cell Science 09/2009; 122(Pt 16):2820-7.
• Article: FAM29A, a target of Plk1 regulation, controls the partitioning of NEDD1 between the mitotic spindle and the centrosomes.

  Hui Zhu, Kayleen Fang, Guowei Fang
  [show abstract] [hide abstract]
  ABSTRACT: We previously showed that FAM29A, a spindle-associated protein, promotes microtubule-dependent microtubule amplification through its interaction with and recruitment of NEDD1, the targeting subunit of the gamma-tubulin ring complex. We report here that FAM29A is regulated by Plk1, a kinase essential for spindle assembly and its bipolarity. Plk1, FAM29A and NEDD1 form three separate complexes in vivo, not one single complex. Plk1 recruits FAM29A to spindle microtubules, which, in turn, targets NEDD1 to the spindle. Plk1 also recruits NEDD1 to the centrosomes, probably through a Plk1-NEDD1 interaction, but this interaction does not contribute to targeting NEDD1 to the spindle. Altering intracellular levels of FAM29A changes the distribution of NEDD1 between the centrosomes and the spindle, indicating that FAM29A controls the partition of NEDD1 between these two mitotic structures. Thus, Plk1 promotes microtubule nucleation from the centrosomes through a FAM29A-independent pathway and from the spindle through a FAM29A-dependent pathway. FAM29A controls the relative contributions of these two pathways to microtubule polymerization during mitosis.
  Journal of Cell Science 09/2009; 122(Pt 15):2750-9.
• Article: FAK, PIP5KIgamma and gelsolin cooperatively mediate force-induced expression of alpha-smooth muscle actin.

  Matthew W C Chan, Pamma D Arora, Peter Bozavikov, Christopher A McCulloch
  [show abstract] [hide abstract]
  ABSTRACT: During the development of pressure-induced cardiac hypertrophy, fibroblasts are activated to become myofibroblasts, which exhibit actin-cytoskeletal remodeling and express alpha-smooth muscle actin (SMA; encoded by ACTA2). Currently, the mechanosensing signaling pathways that regulate SMA expression are not defined. Because focal-adhesion complexes are putative mechanosensing organelles, we examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin in the regulation of SMA expression. We subjected NIH3T3 cells to tensile forces (0.65 pN/mum(2)) by using collagen-coated magnetite beads attached to integrins. After stimulation by mechanical force, FAK and gelsolin were recruited to magnetite beads and there was increased phosphorylation of Tyr397FAK. Mechanical force enhanced SMA promoter activity by twofold; this increased activity was blocked by FAK knockdown using siRNA and by deletion of gelsolin. Force-induced nuclear translocation of MRTF-A, a transcriptional co-activator of SMA that is regulated by actin filaments, was also reduced by FAK knockdown. Phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)], which uncaps gelsolin from actin filaments, was enriched at sites of force application. Type-I phosphatidylinositol 4-phosphate 5 kinase-gamma (PIP5KIgamma), which generates PtdIns(4,5)P(2), associated with FAK and was required for force-mediated SMA-promoter activity and actin assembly. Catalytically inactive PIP5KIgamma inhibited force-induced phosphorylation of FAK at Tyr397. These data suggest a novel pathway in which mechanosensing by FAK regulates actin assembly via gelsolin and the activity of PIP5KIgamma; actin assembly in turn controls SMA expression via MRTF-A.
  Journal of Cell Science 09/2009; 122(Pt 15):2769-81.
• Article: Growth arrest induces primary-cilium formation and sensitizes IGF-1-receptor signaling during differentiation induction of 3T3-L1 preadipocytes.

  Di Zhu, Shuo Shi, Hongzhong Wang, Kan Liao
  [show abstract] [hide abstract]
  ABSTRACT: The first stage of 3T3-L1 adipocyte differentiation is growth arrest, which is achieved by contact inhibition at confluence. In growth-arrested confluent 3T3-L1 preadipocytes, alpha-tubulin acetylation and primary-cilium formation were induced. The blockade of primary-cilium formation by suppressing IFT88 or Kif3a inhibited 3T3-L1 adipocyte differentiation. IGF-1 (IGF-I)-receptor signaling, which is essential for differentiation induction, was sensitized by the formation of a primary cilium in confluent 3T3-L1 preadipocytes. The receptor located in primary cilium was more sensitive to insulin stimulation than that not located in cilia. During cilium formation, insulin receptor substrate 1 (IRS-1), one of the important downstream signaling molecules of the IGF-1 receptor, was recruited to the basal body at which it was phosphorylated on tyrosine by the receptor kinase in cilia. Akt-1, an important signal molecule of the IGF-1 receptor in adipocyte differentiation, was also activated at the basal body. These IGF-1-receptor signaling processes were all inhibited in IFT88- or Kif3a-knockdown cells. Thus, the primary cilium and its basal body formed an organized signaling pathway for the IGF-1 receptor to induce adipocyte differentiation in confluent 3T3-L1 preadipocytes.
  Journal of Cell Science 09/2009; 122(Pt 15):2760-8.
• Article: Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote.

  Sasa Stefanic, Laura Morf, Caroline Kulangara, Attila Regös, Sabrina Sonda, Elisabeth Schraner, Cornelia Spycher, Peter Wild, Adrian B Hehl
  [show abstract] [hide abstract]
  ABSTRACT: The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of ;naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.
  Journal of Cell Science 09/2009; 122(Pt 16):2846-56.