BBA - Molecular Cell Research (v.1853, #7)
Editorial Board (i).
Facilitation of Orai3 targeting and store-operated function by Orai1 by Dalia Alansary; Ivan Bogeski; Barbara A. Niemeyer (1541-1550).
Orai1 subunits interacting with STIM1 molecules comprise the major components responsible for calcium release-activated calcium (CRAC) channels. The homologs Orai2 and Orai3 yield smaller store-operated currents when overexpressed and are mostly unable to substitute Orai1. Orai3 subunits are also essential components of store independent channel complexes and also tune inhibition of ICRAC by reactive oxygen species. Here we use patch-clamp, microscopy, Ca2 +-imaging and biochemical experiments to investigate the interdependence of Orai2, Orai3 and Orai1. We demonstrate that store-operation and localization of Orai3 but not of Orai2 to STIM1 clusters in HEK cells or to the immunological synapse in T cells is facilitated by Orai1 while Orai3's store-independent activity remains unaffected. On the other hand, one Orai3 subunit confers redox-resistance to heteromeric channels. The inefficient store operation of Orai3 is partly due to the lack of three critical C-terminal residues, the insertion of which improves interaction with STIM1 and abrogates Orai3's dependence on Orai1. Our results suggest that Orai3 down-tunes efficient STIM1 gating when in a heteromeric complex with Orai1.Display Omitted
Keywords: Immunological synapse; Clustering; Concatenated channel; CRAC; STIM1; Stoichiometry;
The non-glycosylated isoform of MIC26 is a constituent of the mammalian MICOS complex and promotes formation of crista junctions by Sebastian Koob; Miguel Barrera; Ruchika Anand; Andreas S. Reichert (1551-1563).
Mitochondrial membrane architecture is important for organelle function. Alterations thereof are linked to a number of human disorders including diabetes and cardiomyopathy. The MICOS complex was recently reported to be a central player determining cristae structure and formation of crista junctions. Here we investigated the functional role of MIC26, a lipoprotein formerly termed APOO. Its levels are increased in diabetic heart tissue and in blood plasma of patients suffering from acute coronary syndrome. We demonstrate that human MIC26 exists in three distinct forms: (1) a glycosylated and secreted 55 kDa protein, (2) an ER/Golgi-resident form thereof, and (3) a non-glycosylated 22 kDa mitochondrial protein. The latter isoform spans the mitochondrial inner membrane and physically interacts with several MICOS complex subunits such as MIC60, MIC27, and MIC10. We further demonstrate that MIC26 and MIC27, a homologous protein formerly termed APOOL, regulate their levels in an antagonistic manner. Both proteins are positively correlated with the levels of MIC10 as well as tafazzin, an enzyme required for cardiolipin remodeling. Overexpression of MIC26 induced fragmentation of mitochondria, promoted ROS formation and resulted in impaired mitochondrial respiration. Downregulation of MIC26 induced a decrease in mitochondrial oxygen consumption, whereas mitochondrial network morphology and ROS levels remained unaffected. MIC26 depletion led to alterations in mitochondrial ultrastructure and caused a significant reduction in the number of crista junctions. In summary, we show that the human apolipoprotein MIC26 is a bona fide subunit of the MICOS complex and that MIC26 is linked to cardiolipin metabolism and promotes crista junction formation.
Keywords: Mitochondria; Crista junctions; MICOS complex; Apolipoproteins; Cardiolipin metabolism; Arteriosclerosis;
Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA by Johanna Ohnheiser; Eva Ferlemann; Astrid Haas; Jan P. Müller; Eugen Werwein; Olesja Fehler; Abhiruchi Biyanee; Karl-Heinz Klempnauer (1564-1573).
The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs.Display Omitted
Keywords: Pdcd4; Hipk2; HIPK1; Translation; Autoregulation;
Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis by François Singh; Anne-Laure Charles; Anna-Isabel Schlagowski; Jamal Bouitbir; Annalisa Bonifacio; François Piquard; Stephan Krähenbühl; Bernard Geny; Joffrey Zoll (1574-1585).
Even though oxidative stress damage from excessive production of ROS is a well known phenomenon, the impact of reductive stress remains poorly understood. This study tested the hypothesis that cellular reductive stress could lead to mitochondrial malfunction, triggering a mitochondrial hormesis (mitohormesis) phenomenon able to protect mitochondria from the deleterious effects of statins. We performed several in vitro experiments on L6 myoblasts and studied the effects of N-acetylcysteine (NAC) at different exposure times. Direct NAC exposure (1 mM) led to reductive stress, impairing mitochondrial function by decreasing maximal mitochondrial respiration and increasing H2O2 production. After 24 h of incubation, the reactive oxygen species (ROS) production was increased. The resulting mitochondrial oxidation activated mitochondrial biogenesis pathways at the mRNA level. After one week of exposure, mitochondria were well-adapted as shown by the decrease of cellular ROS, the increase of mitochondrial content, as well as of the antioxidant capacities. Atorvastatin (ATO) exposure (100 μM) for 24 h increased ROS levels, reduced the percentage of live cells, and increased the total percentage of apoptotic cells. NAC exposure during 3 days failed to protect cells from the deleterious effects of statins. On the other hand, NAC pretreatment during one week triggered mitochondrial hormesis and reduced the deleterious effect of statins. These results contribute to a better understanding of the redox-dependant pathways linked to mitochondria, showing that reductive stress could trigger mitochondrial hormesis phenomenon.
Keywords: Reductive stress; Mitohormesis; N-acetylcysteine; Myoblast; Apoptosis; Statin;
PKC theta and p38 MAPK activate the EBV lytic cycle through autophagy induction by Roberta Gonnella; Marisa Granato; Antonella Farina; Roberta Santarelli; Alberto Faggioni; Mara Cirone (1586-1595).
PKC activation by combining TPA with sodium butyrate (T/B) represents the most effective and widely used strategy to induce the Epstein–Barr virus (EBV) lytic cycle. The results obtained in this study show that novel PKCθ is involved in such process and that it acts through the activation of p38 MAPK and autophagy induction. Autophagy, a mechanism of cellular defense in stressful conditions, is manipulated by EBV to enhance viral replication. Besides promoting the EBV lytic cycle, the activation of p38 and autophagy resulted in a pro-survival effect, as indicated by p38 or ATG5 knocking down experiments. However, this pro-survival role was counteracted by a pro-death activity of PKCθ, due to the dephosphorylation of AKT. In conclusion, this study reports, for the first time, that T/B activates a PKCθ-p38 MAPK axis in EBV infected B cells, that promotes the viral lytic cycle and cell survival and dephosphorylates AKT, balancing cell life and cell death.
Keywords: EBV; Lytic cycle; PKCθ; Autophagy; LC3; p38 MAPK;
Iron for proliferation of cell lines and hematopoietic progenitors: Nailing down the intracellular functional iron concentration by Emmanuel Pourcelot; Marine Lénon; Nicolas Mobilia; Jean-Yves Cahn; Josiane Arnaud; Eric Fanchon; Jean-Marc Moulis; Pascal Mossuz (1596-1605).
Iron is an essential nutrient which must be provided in sufficient amounts to support growth of eukaryotic cells. All organisms devote specialized pathways to ensure proper delivery. Yet, a quantitative assessment of the intra-cellular iron concentration needed to allow the cell cycle to proceed in mammalian cells is missing. Starting from iron-depleted cell lines or primary hematopoietic progenitors prepared with clinically implemented iron chelators, replenishment via transferrin and other iron sources has been quantitatively monitored through the main endogenous markers of the cellular iron status, namely proteins involved in the uptake (transferrin receptor), the storage (ferritin), and the sensing (Iron Regulatory Proteins) of iron. When correlated with measurements of iron concentrations and indicators of growth, this minimally intrusive approach provided an unprecedented estimate of the intracellular iron concentration acting upon iron-centered regulatory pathways. The data were analyzed with the help of a previously developed theoretical treatment of cellular iron regulation. The minimal cellular iron concentration required for cell division was named functional iron concentration (FIC) to distinguish it from previous estimates of the cellular labile iron. The FIC falls in the low nanomolar range for all studied cells, including hematopoietic progenitors. These data shed new light on basic aspects of cellular iron homeostasis by demonstrating that sensing and regulation of iron occur well below the concentrations requiring storage or becoming noxious in pathological conditions. The quantitative assessment provided here is relevant for monitoring treatments of conditions in which iron provision must be controlled to avoid unwanted cellular proliferation.Display Omitted
Keywords: Iron homeostasis; Post-transcriptional regulation; Leukemia; Cellular growth; Cell cycle; Modeling;
Rotenone inhibits primary murine myotube formation via Raf-1 and ROCK2 by Sander Grefte; Jori A.L. Wagenaars; Renate Jansen; Peter H.G.M. Willems; Werner J.H. Koopman (1606-1614).
Rotenone (ROT) is a widely used inhibitor of complex I (CI), the first complex of the mitochondrial oxidative phosphorylation (OXPHOS) system. However, particularly at high concentrations ROT was also described to display off-target effects. Here we studied how ROT affected in vitro primary murine myotube formation. We demonstrate that myotube formation is specifically inhibited by ROT (10–100 nM), but not by piericidin A (PA; 100 nM), another CI inhibitor. At 100 nM, both ROT and PA fully blocked myoblast oxygen consumption. Knock-down of Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) and, to a lesser extent ROCK1, prevented the ROT-induced inhibition of myotube formation. Moreover, the latter was reversed by inhibiting Raf-1 activity. In contrast, ROT-induced inhibition of myotube formation was not prevented by knock-down of RhoA. Taken together, our results support a model in which ROT reduces primary myotube formation independent of its inhibitory effect on CI-driven mitochondrial ATP production, but via a mechanism primarily involving the Raf-1/ROCK2 pathway.
Keywords: Fusion index; GW5074; U0126; Rotenone; Piericidin A; Rho-GTPase;
Enhanced amino acid utilization sustains growth of cells lacking Snf1/AMPK by Raffaele Nicastro; Farida Tripodi; Cinzia Guzzi; Veronica Reghellin; Sakda Khoomrung; Claudia Capusoni; Concetta Compagno; Cristina Airoldi; Jens Nielsen; Lilia Alberghina; Paola Coccetti (1615-1625).
The metabolism of proliferating cells shows common features even in evolutionary distant organisms such as mammals and yeasts, for example the requirement for anabolic processes under tight control of signaling pathways. Analysis of the rewiring of metabolism, which occurs following the dysregulation of signaling pathways, provides new knowledge about the mechanisms underlying cell proliferation.The key energy regulator in yeast Snf1 and its mammalian ortholog AMPK have earlier been shown to have similar functions at glucose limited conditions and here we show that they also have analogies when grown with glucose excess. We show that loss of Snf1 in cells growing in 2% glucose induces an extensive transcriptional reprogramming, enhances glycolytic activity, fatty acid accumulation and reliance on amino acid utilization for growth. Strikingly, we demonstrate that Snf1/AMPK-deficient cells remodel their metabolism fueling mitochondria and show glucose and amino acids addiction, a typical hallmark of cancer cells.Display Omitted
Keywords: Saccharomyces cerevisiae; Glucose; Budding yeast; Metabolism; Respiration; Gene chip;
p70 S6-kinase mediates the cooperation between Akt1 and Mek1 pathways in fibroblast-mediated extracellular matrix remodeling by Anna Goc; Harika Sabbineni; Maha Abdalla; Payaningal R. Somanath (1626-1635).
Previous studies have demonstrated both synergistic and opposing effects of Akt and Mek1/2 in various cell functions and disease states. Furthermore, Akt has been reported to inhibit and activate cRaf/Mek pathway, suggesting that their mutual interaction and cooperation may be cell type, stimuli and/or context specific. While PI3-kinase/Akt and cRaf/Mek pathways have been implicated in the regulation of extracellular matrix (ECM) remodeling, mutual interactions between these two pathways and their specific contributions to the events leading to ECM synthesis and assembly is not clear. We investigated the specific role of Akt1 and Mek1 in ECM synthesis and assembly by NIH 3T3 fibroblasts and how these effects were reconciled to mediate overall ECM remodeling. Our study identified that cooperation between Akt1 and Mek1 is necessary to mediate ECM synthesis. Whereas Akt1 activation resulted in Mek1 activation as evidenced by increased ERK1/2 phosphorylation, Mek1 inhibition using U0126 or DN-Mek1 resulted in enhanced Akt1 phosphorylation. Interestingly, both Akt1 and Mek1 activities were needed for the synthesis and assembly of ECM. The effect of Akt1 and Mek1 on ECM synthesis was reconciled through the activation of p70 S6-kinase via phosphorylation at T421/S424 and S411, respectively. Furthermore, Akt1 and Mek1 cooperated in mediating ECM assembly via activation of integrin β1. Together, we show for the first time that Akt1 and Mek1 pathways cooperate in the regulation of ECM remodeling by the fibroblasts.
Keywords: Akt1; Mek1; mTOR; p70 S6-kinase; Extracellular matrix; Integrin;
N-linked Glycosylation of human SLC1A5 (ASCT2) transporter is critical for trafficking to membrane by Lara Console; Mariafrancesca Scalise; Zlatina Tarmakova; Imogen R. Coe; Cesare Indiveri (1636-1645).
The human amino acid transporter SLC1A5 (ASCT2) contains two N-glycosylation sites (N163 and N212) located in the large extracellular loop. In the homology structural model of ASCT2 these Asn residues are extracellularly exposed. Mutants of the two Asn exhibited altered electrophoretic mobility. N163Q and N212Q displayed multiple bands with apparent molecular masses from 80 kDa to 50 kDa. N163/212Q displayed a single band of 50 kDa corresponding to the unglycosylated protein. The presence in membrane of WT and mutants was evaluated by protein biotinylation assay followed by immunoblotting. The double mutation significantly impaired the presence of the protein in membrane, without impairment in protein synthesis. [3H]glutamine transport was measured in cells transiently transfected with the WT or mutants. N163/212Q exhibited a strongly reduced transport activity correlating with reduced surface expression. The same proteins extracted from cells and reconstituted in liposomes showed comparable transport activities demonstrating that the intrinsic transport function of the mutants was not affected. The rate of endocytosis of ASCT2 was assayed by a reversible biotinylation strategy. N212Q and N163/212Q showed strongly increased rates of endocytosis respect to WT. ASCT2 stability was determined using cycloheximide. N163Q or N163/212Q showed a slightly or significantly lower stability with respect to WT. To assess trafficking to the membrane, a brefeldin-based assay, which caused retention of proteins in ER, was performed. One hour after brefeldin removal WT protein was localized to the plasma membrane while the double mutant was localized in the cytosol. The results demonstrate that N-glycosylation is critical for trafficking.
Keywords: Glutamine; Plasma membrane; Transport; Liposomes; Protein stability;
mTor mediates tau localization and secretion: Implication for Alzheimer's disease by Zhi Tang; Eniko Ioja; Erika Bereczki; Kjell Hultenby; Chunxia Li; Zhizhong Guan; Bengt Winblad; Jin-Jing Pei (1646-1657).
Abnormally hyperphosphorylated tau aggregates form paired helical filaments (PHFs) in neurofibrillary tangles, a key hallmark of Alzheimer's disease (AD) and other tauopathies. The cerebrospinal fluid (CSF) levels of soluble total tau and phospho-tau from clinically diagnosed AD patients are significantly higher compared with controls. Data from both in vitro and in vivo AD models have implied that an aberrant increase of mammalian target of rapamycin (mTor) signaling may be a causative factor for the formation of abnormally hyperphosphorylated tau. In the present study, we showed that in post-mortem human AD brain, tau was localized within different organelles (autophagic vacuoles, endoplasmic reticulum, Golgi complexes, and mitochondria). In human SH-SY5Y neuroblastoma cells stably carrying different genetic variants of mTor, we found a common link between the synthesis and distribution of intracellular tau. mTor overexpression or the lack of its expression was responsible for the altered balance of phosphorylated (p-)/-non phosphorylated (Np-) tau in the cytoplasm and different cellular compartments, which might facilitate tau deposition. Up-regulated mTor activity resulted in a significant increase in the amount of cytosolic tau as well as its re-localization to exocytotic vesicles that were not associated with exosomes. These results have implicated that mTor is involved in regulating tau distribution in subcellular organelles and in the initiation of tau secretion from cells to extracellular space.
Keywords: mTor; Tau secretion; Autophagy; Tau phosphorylation; Alzheimer's disease;
Emerging understanding of Bcl-2 biology: Implications for neoplastic progression and treatment by Cristina Correia; Sun-Hee Lee; X. Wei Meng; Nicole D. Vincelette; Katherine L.B. Knorr; Husheng Ding; Grzegorz S. Nowakowski; Haiming Dai; Scott H. Kaufmann (1658-1671).
Bcl-2, the founding member of a family of apoptotic regulators, was initially identified as the protein product of a gene that is translocated and overexpressed in greater than 85% of follicular lymphomas (FLs). Thirty years later we now understand that anti-apoptotic Bcl-2 family members modulate the intrinsic apoptotic pathway by binding and neutralizing the mitochondrial permeabilizers Bax and Bak as well as a variety of pro-apoptotic proteins, including the cellular stress sensors Bim, Bid, Puma, Bad, Bmf and Noxa. Despite extensive investigation of all of these proteins, important questions remain. For example, how Bax and Bak breach the outer mitochondrial membrane remains poorly understood. Likewise, how the functions of anti-apoptotic Bcl-2 family members such as eponymous Bcl-2 are affected by phosphorylation or cancer-associated mutations has been incompletely defined. Finally, whether Bcl-2 family members can be successfully targeted for therapeutic advantage is only now being investigated in the clinic. Here we review recent advances in understanding Bcl-2 family biology and biochemistry that begin to address these questions.
Keywords: Apoptosis; BH3 mimetic; Activation-induced cytidine deaminase; Mutation; Follicular lymphoma;
Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions by Maria Tsachaki; Julia Birk; Aurélie Egert; Alex Odermatt (1672-1682).
Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool.
Keywords: Endoplasmic reticulum; Redox sensor; Green-fluorescent protein; Live-imaging; Topology; 17β-hydroxysteroid dehydrogenase;
Recruitment and activation of SLK at the leading edge of migrating cells requires Src family kinase activity and the LIM-only protein 4 by Kyla D. Baron; Khalid Al-Zahrani; Jillian Conway; Cédrik Labrèche; Christopher J. Storbeck; Jane E. Visvader; Luc A. Sabourin (1683-1692).
The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover and is regulated by the LIM domain-binding proteins Ldb1 and Ldb2. These adapter proteins have been demonstrated to interact with LMO4 in the organization of transcriptional complexes. Therefore, we have assessed the ability of LMO4 to also interact and regulate SLK activity. Our data show that LMO4 can directly bind to SLK and activate its kinase activity in vitro and in vivo. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding and Cre-mediated deletion of LMO4 in conditional LMO4fl/fl fibroblasts inhibits cell migration and SLK activation. Deletion of LMO4 impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Supporting this, Src/Yes/Fyn-deficient cells (SYF) expressing very low levels of LMO4 do not recruit SLK to the leading edge. Re-expression of wildtype Myc–LMO4 in SYF cells, but not a mutant version, restores SLK localization and kinase activity. Overall, our data suggest that activation of SLK by haptotactic signals requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. Furthermore, this establishes a novel cytosolic role for the transcriptional co-activator LMO4.
Keywords: Ste20-like kinase; SLK; LMO4; Migration; c-Src; Ldb1;
Protein kinase CK2 potentiates translation efficiency by phosphorylating eIF3j at Ser127 by Christian Borgo; Cinzia Franchin; Valentina Salizzato; Luca Cesaro; Giorgio Arrigoni; Laura Matricardi; Lorenzo A. Pinna; Arianna Donella-Deana (1693-1701).
In eukaryotic protein synthesis the translation initiation factor 3 (eIF3) is a key player in the recruitment and assembly of the translation initiation machinery. Mammalian eIF3 consists of 13 subunits, including the loosely associated eIF3j subunit that plays a stabilizing role in the eIF3 complex formation and interaction with the 40S ribosomal subunit. By means of both co-immunoprecipitation and mass spectrometry analyses we demonstrate that the protein kinase CK2 interacts with and phosphorylates eIF3j at Ser127. Inhibition of CK2 activity by CX-4945 or down-regulation of the expression of CK2 catalytic subunit by siRNA cause the dissociation of j-subunit from the eIF3 complex as judged from glycerol gradient sedimentation. This finding proves that CK2-phosphorylation of eIF3j is a prerequisite for its association with the eIF3 complex. Expression of Ser127Ala-eIF3j mutant impairs both the interaction of mutated j-subunit with the other eIF3 subunits and the overall protein synthesis. Taken together our data demonstrate that CK2-phosphorylation of eIF3j at Ser127 promotes the assembly of the eIF3 complex, a crucial step in the activation of the translation initiation machinery.
Keywords: eIF3j phosphorylation; Protein kinase CK2; eIF3 complex; eIF3 assembly; Translation initiation activation;
Cell biology of yeast zygotes, from genesis to budding by Alan M. Tartakoff (1702-1714).
The zygote is the essential intermediate that allows interchange of nuclear, mitochondrial and cytosolic determinants between cells. Zygote formation in Saccharomyces cerevisiae is accomplished by mechanisms that are not characteristic of mitotic cells. These include shifting the axis of growth away from classical cortical landmarks, dramatically reorganizing the cell cortex, remodeling the cell wall in preparation for cell fusion, fusing with an adjacent partner, accomplishing nuclear fusion, orchestrating two steps of septin morphogenesis that account for a delay in fusion of mitochondria, and implementing new norms for bud site selection. This essay emphasizes the sequence of dependent relationships that account for this progression from cell encounters through zygote budding. It briefly summarizes classical studies of signal transduction and polarity specification and then focuses on downstream events.
Keywords: S. cerevisiae; Zygote; Polarity; Cell wall; Membrane fusion; Budding;
Role of actin filaments in fusopod formation and osteoclastogenesis by Yongqiang Wang; Patricia Joyce Brooks; Janet Jinyoung Jang; Alexandra Shade Silver; Pamma D. Arora; Christopher A. McCulloch; Michael Glogauer (1715-1724).
Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width > 2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell–cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.
Keywords: Rac1; Actin filaments; Fusopods; Osteoclast fusion;
5′-AMP-activated protein kinase alpha regulates stress granule biogenesis by Hicham Mahboubi; Ramla Barisé; Ursula Stochaj (1725-1737).
Stress granule (SG) assembly represents a conserved eukaryotic defense strategy against various insults. Although essential for the ability to cope with deleterious conditions, the signaling pathways controlling SG formation are not fully understood. The energy sensor AMP-activated protein kinase (AMPK) is critical for the cellular stress response. Human cells produce two AMPK catalytic α-subunits with not only partially overlapping, but also unique functions. Here, we provide direct support for structural and functional links between AMPK-α isoforms and SGs. As such, several stressors promote SG association of AMPK-α2, but not AMPK-α1. Multiple lines of evidence link AMPK activity to SG biogenesis. First, pharmacological kinase inhibition interfered with SG formation. Second, AMPK-α knockdown combined with in-depth quantitative SG analysis revealed isoform-specific changes of SG characteristics. Third, overexpression of mutant α-subunits further substantiated that AMPK regulates SG parameters. Finally, we identified the SG-nucleating protein G3BP1 as an AMPK-α2 binding partner. This interaction is stimulated by stress and notably occurs in SGs. Collectively, our data define the master metabolic regulator AMPK as a novel SG constituent that also controls their biogenesis.Display Omitted
Keywords: 5′-AMP-activated protein kinase; Stress granules; Signaling; Oxidative stress; Apoptosis; Quantitative microscopy;
Presumed pseudokinase VRK3 functions as a BAF kinase by Choon-Ho Park; Hye Guk Ryu; Seong-Hoon Kim; Dohyun Lee; Haengjin Song; Kyong-Tai Kim (1738-1748).
Vaccinia-related kinase 3 (VRK3) is known as a pseudokinase that is catalytically inactive due to changes in motifs that are essential for kinase activity. Although VRK3 has been regarded as a genuine pseudokinase from structural and biochemical studies, recent reports suggest that VRK3 acts as an active kinase as well as a signaling scaffold in cells. Here, we demonstrate that VRK3 phosphorylates the nuclear envelope protein barrier-to-autointegration factor (BAF) on Ser4. Interestingly, VRK3 kinase activity is dependent upon its N-terminal regulatory region, which is excluded from the determination of its crystal structure. Furthermore, the kinase activity of VRK3 is involved in the regulation of the cell cycle. VRK3 expression levels increase during interphase, whereas VRK1 is enriched in late G2 and early M phase. Ectopic expression of VRK3 induces the translocation of BAF from the nucleus to the cytoplasm. In addition, depletion of VRK3 decreases the population of proliferating cells. These data suggest that VRK3-mediated phosphorylation of BAF may facilitate DNA replication or gene expression by facilitating the dissociation of nuclear envelope proteins and chromatin during interphase.
Keywords: Pseudokinase; Vaccinia-related kinase 3; Barrier-to-autointegration factor;
Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells by Euan Parnell; Andreas Koschinski; Manuela Zaccolo; Ryan T. Cameron; George S. Baillie; Gemma L. Baillie; Alison Porter; Stuart P. McElroy; Stephen J. Yarwood (1749-1758).
Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP.Display Omitted
Keywords: Cyclic AMP; EPAC1; Cytoskeleton; Cell morphology;