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1.  Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification 
Journal of cell science  2012;125(0 22):5353-5368.
Centrosome duplication is licensed by the disengagement, or ‘uncoupling’, of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the APC/C leading to securin degradation and release of active separase. APC/C activation during G2 arrest is dependent on Plk1-mediated degradation of the APC/C inhibitor, Emi1, but Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulate centriole disengagement in response to hydroxyurea or DNA damage-induced cell cycle arrest and this leads to centrosome amplification. However, the re-duplication of disengaged centrioles is dependent on Cdk2 activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Release from these arrests leads to mitotic entry but, due to the presence of disengaged and/or amplified centrosomes, formation of abnormal mitotic spindles that lead to chromosome missegregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability.
PMCID: PMC3939426  PMID: 22956538
2.  Activation-induced nuclear translocation of RING3 
Journal of cell science  2000;113(0 17):3085-3091.
RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serumstarved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2Fregulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.
PMCID: PMC3936601  PMID: 10934046
RING3; Nuclear translocation; Immunolocalisation; Confocal microscopy; Leukaemia
3.  Signaling through urokinase and urokinase receptor in lung cancer cells requires interactions with β1 integrins 
Journal of cell science  2008;121(0 22):3747-3756.
The urokinase receptor (uPAR) is upregulated upon tumor cell invasion and correlates with poor lung cancer survival. Although a cis-interaction with integrins has been ascribed to uPAR, whether this interaction alone is critical to urokinase (uPA)- and uPAR-dependent signaling and tumor promotion is unclear. Here we report the functional consequences of point mutations of uPAR (H249A-D262A) that eliminate β1 integrin interactions but maintain uPA binding, vitronectin attachment and association with αV integrins, caveolin and epidermal growth factor receptor. Disruption of uPAR interactions with β1 integrins recapitulated previously reported findings with β1-integrin-derived peptides that attenuated matrix-dependent ERK activation, MMP expression and in vitro migration by human lung adenocarcinoma cell lines. The uPAR mutant cells acquired enhanced capacity to adhere to vitronectin via uPAR–αVβ5-integrin, rather than through the uPAR–α3β1-integrin complex and they were unable to initiate uPA signaling to activate ERK, Akt or Stat1. In an orthotopic lung cancer model, uPAR mutant cells exhibited reduced tumor size compared with cells expressing wild-type uPAR. Taken together, the results indicate that uPAR–β1-integrin interactions are essential to signals induced by integrin matrix ligands or uPA that support lung cancer cell invasion in vitro and progression in vivo.
PMCID: PMC3903460  PMID: 18940913
Urokinase; Urokinase receptor; Integrin; Signaling; Lung cancer
4.  SARA, a FYVE domain protein, affects Rab5-mediated endocytosis 
Journal of cell science  2002;115(0 24):4755-4763.
Rab5, a member of the small GTPase family of proteins, is primarily localized on early endosomes and has been proposed to participate in the regulation of early endosome trafficking. It has been reported that phosphatidylinositol 3-kinases and FYVE domain proteins, such as EEA1, can be recruited onto early endosomes and act as Rab5 effectors. SARA (Smad anchor for receptor activation), also a FYVE domain protein, was initially isolated as a participant in signal transduction from the transforming growth factor β receptor. Overexpressed SARA has been found on EEA1-positive early endosomes. In this report, we show that endogenous SARA is present on early endosomes and overexpression of SARA causes endosomal enlargement. Functionally, SARA overexpression significantly delays the recycling of transferrin. The transferrin receptor distributed on the cell surfaces was also greatly reduced in cells overexpressing SARA. However, the internalization rate of transferrin is not affected by SARA overexpression. The morphological and functional alterations caused by SARA overexpression resemble those caused by overexpression of Rab5:GTP mutant Rab5Q79L. Finally, all SARA-mediated phenotypic changes can be counteracted by overexpression Rab5:GDP mutant Rab5S34N. These results collectively suggested that SARA plays an important functional role downstream of Rab5-regulated endosomal trafficking.
PMCID: PMC3899687  PMID: 12432064
Rab5; FYVE domain; SARA; Transferrin; EEA1; Endosome
5.  The evolutionarily conserved protein CG9186 is associated with lipid droplets, required for their positioning and for fat storage 
Journal of cell science  2013;126(0 10):10.1242/jcs.120493.
Lipid droplets (LDs) are specialized cell organelles for the storage of energy-rich lipids. Although lipid storage is a conserved feature of all cells and organisms, little is known about fundamental aspects of the cell biology of LDs, including their biogenesis, structural assembly and subcellular positioning, and the regulation of organismic energy homeostasis. We identified a novel LD-associated protein family, represented by the Drosophila protein CG9186 and its murine homolog MGI:1916082. In the absence of LDs, both proteins localize at the endoplasmic reticulum (ER). Upon lipid storage induction, they translocate to LDs using an evolutionarily conserved targeting mechanism that acts through a 60-amino-acid targeting motif in the center of the CG9186 protein. Overexpression of CG9186, and MGI:1916082, causes clustering of LDs in both tissue culture and salivary gland cells, whereas RNAi knockdown of CG9186 results in a reduction of LDs. Organismal RNAi knockdown of CG9186 results in a reduction in lipid storage levels of the fly. The results indicate that we identified the first members of a novel and evolutionarily conserved family of lipid storage regulators, which are also required to properly position LDs within cells.
PMCID: PMC3880856  PMID: 23525007
Lipid droplets; Organelle clustering; Organelle positioning; Lipid metabolism; Drosophila melanogaster
6.  LRP1 mediates Hedgehog-induced endocytosis of the GPC3–Hedgehog complex 
Journal of cell science  2012;125(0 14):10.1242/jcs.098889.
Glypican-3 (GPC3) is a heparan sulfate (HS) proteoglycan that is bound to the cell membrane through a glycosylphosphatidylinositol link. This glypican regulates embryonic growth by inhibiting the hedgehog (Hh) signaling pathway. GPC3 binds Hh and competes with Patched (Ptc), the Hh receptor, for Hh binding. The interaction of Hh with GPC3 triggers the endocytosis and degradation of the GPC3–Hh complex with the consequent reduction of Hh available for binding to Ptc. Currently, the molecular mechanisms by which the GPC3–Hh complex is internalized remains unknown. Here we show that the low-density-lipoprotein receptor-related protein-1 (LRP1) mediates the Hh-induced endocytosis of the GPC3–Hh complex, and that this endocytosis is necessary for the Hh-inhibitory activity of GPC3. Furthermore, we demonstrate that GPC3 binds through its HS chains to LRP1, and that this interaction causes the removal of GPC3 from the lipid rafts domains.
PMCID: PMC3843007  PMID: 22467855 CAMSID: cams3730
Glypican-3; Hedgehog; LRP1; Endocytosis
7.  Phosphorylation of Vascular Endothelial Cadherin Controls Lymphocyte Emigration 
Journal of cell science  2008;121(0 1):10.1242/jcs.022681.
Lymphocytes emigrate from the circulation to target tissues through the microvascular endothelial cell (EC) barrier. During paracellular transmigration cell-cell junctions have been proposed to disengage and provide homophilic and heterophilic interaction surfaces in a zip-like process. However, it is not known whether EC modulate junction proteins during this process. Here we show that tyrosine phosphorylation of adherens junction vascular endothelial cadherin (VEC) is required for successful transendothelial lymphocyte migration. We found that adhesion of lymphocytes or activation of the endothelial adhesion-receptor ICAM-1 led to tyrosine phosphorylation of VEC. Substitution of tyrosine to phenylalanine in VEC at position 645, 731 or 733 produced EC which were significantly less permissive to lymphocyte migration. We also found that these same tyrosines were involved in ICAM-1-dependent changes of VEC phosphorylation. ICAM-1 activation enhanced transendothelial permeability suggesting that junction disassembly occurred. In agreement the expression of Y645F, Y731F or Y733F VEC predominantly affected lymphocyte transmigration in paracellular areas. Taken together these results demonstrate that adherens junction phosphorylation constitutes a molecular endpoint of lymphocyte-induced vascular EC signaling and may be exploited as a new target of anti-inflammatory therapies.
PMCID: PMC3810954  PMID: 18096689
Lymphocyte Migration; VE-cadherin; Tyrosine Phosphorylation; Brain Endothelium; ICAM-1
8.  A complex containing LPP and α-Actinin mediates TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells 
Journal of cell science  2013;126(0 9):1981-1991.
Transforming Growth Factor β is a potent modifier of the malignant phenotype in ErbB2-expressing breast cancers. We demonstrate that epithelial-derived breast cancer cells, which undergo a TGFβ-induced EMT, engage signaling molecules that normally facilitate cellular migration and invasion of mesenchymal cells. We identify Lipoma Preferred Partner (LPP) as an indispensable regulator of TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We show that LPP re-localizes to focal adhesion complexes upon TGFβ stimulation and is a critical determinant in TGFβ-mediated focal adhesion turnover. Finally, we have determined that the interaction between LPP and α-Actinin, an actin cross-linking protein, is necessary for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. Thus, our data reveals that LPP, which is normally operative in cells of mesenchymal origin, can be co-opted by breast cancer cells during an EMT to promote their migration and invasion.
PMCID: PMC3791827  PMID: 23447672
breast cancer; ErbB2; TGFβ; EMT; LPP; migration; invasion
9.  Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates 
Journal of cell science  2012;126(0 2):580-592.
Aggregation of misfolded proteins and the associated loss of neurons are considered as a hallmark of numerous neurodegenerative diseases. Optineurin is present in protein inclusions observed in various neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, Parkinson’s disease, Creutzfeld-Jacob disease and Pick’s disease. Optineurin deletion mutations have also been described in ALS patients. However, the role of optineurin in mechanisms of protein aggregation remains unclear. In this report, we demonstrate that optineurin recognized various protein aggregates via its C-terminal coiled-coil domain in a ubiquitin-independent manner. We also show that optineurin depletion significantly increase protein aggregation in HeLa cells and morpholino-silencing of the optineurin ortholog in zebrafish causes the motor axonopathy phenotype similar to a zebrafish model of ALS. A more severe phenotype is observed when optineurin is depleted in zebrafish carrying ALS mutations. Furthermore, TANK1 binding kinase 1 (TBK1) is co-localized with optineurin on protein aggregates and is important in clearance of protein aggregates through the autophagy-lysosome pathway. TBK1 phosphorylates optineurin at position Ser-177 and regulates its ability to interact with autophagy modifiers. This study provides evidence for a ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates as well as additional relevance for TBK1 as an upstream regulator of the autophagic pathway.
PMCID: PMC3654196  PMID: 23178947
amyotrophic lateral sclerosis; Huntington disease; huntingtin; optineurin; phosphorylation; SOD1; TBK1; ubiquitin
10.  Analysis of Fyn function in hemostasis and αIIbβ3-integrin signaling 
Journal of cell science  2008;121(0 10):1641-1648.
Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signalling, and the β3 subunit of αIIbβ3 integrin has been shown to interact with multiple SFK members. Here, we analyzed the interactions and functional consequences of Fyn and Src binding to αIIbβ3. Fyn associated with the β3 subunit in resting and thrombin-aggregated platelets, whereas interaction between Src and αIIbβ3 was seen predominantly in resting but not in thrombin-aggregated platelets. We have also observed that Fyn but not Src localized to focal adhesions in CHO cells adherent to fibrinogen through αIIbβ3. On the basis of these differences, we wanted to determine the sequence requirements for the interaction of Fyn and Src within the β3-cytoplasmic domain. Whereas Src association required the C-terminal region of β3, Fyn continued to interact with mutants that could no longer associate with Src and that contained as few as 13 membrane-proximal amino acids of the β3-cytoplasmic tail. Using deletion mutants of β3-cytoplasmic tails expressed as GST-fusion proteins, we narrowed down the Fyn-binding site even further to the amino acid residues 721–725 (IHDRK) of the β3-cytoplasmic domain. On the basis of these observations, we explored whether Fyn−/− mice exhibited any abnormalities in hemostasis and platelet function. We found that Fyn−/− mice significantly differed in their second bleeding times compared with wild-type mice, and platelets from Fyn−/− mice exhibited delayed spreading on fibrinogen-coated surfaces. Using mutant forms of Fyn, it appears that its kinase activity is required for its localization to focal adhesions and to mediate αIIbβ3-dependent cell spreading. Our results suggest that Fyn and Src have distinct requirements for interaction with αIIbβ3; and, consequently, the two SFK can mediate different functional responses.
PMCID: PMC3711128  PMID: 18430780
Integrin; αIIbβ3; Src-family kinases
11.  Initiation and execution of lipotoxic ER stress in pancreatic β-cells 
Journal of cell science  2008;121(0 14):2308-2318.
Free fatty acids (FFA) cause apoptosis of pancreatic β-cells and might contribute to β-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary β-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca2+ stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor β-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary β-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced β-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca2+ depletion. The IRE1 and resulting JNK activation contribute to β-cell apoptosis. PERK activation by palmitate also contributes to β-cell apoptosis via CHOP.
PMCID: PMC3675788  PMID: 18559892
Pancreatic β-cell; Islet; Endoplasmic reticulum stress; Fatty acid; Oleate; Palmitate; Lipotoxicity; Apoptosis; Type 2 diabetes
12.  The endocannabinoid N-arachidonoyl glycine (NAGly) inhibits store-operated Ca2+ entry by abrogating STIM1/Orai1 interaction 
Journal of cell science  2012;126(0 4):879-888.
The endocannabiniod anandamide (AEA) and its derivate N-arachidonoyl glycine (NAGly) have a broad spectrum of physiological effects, which are induced by both binding to receptors and receptor-independent modulations of ion channels and transporters. The impact of AEA and NAGly on store-operated Ca2+ entry (SOCE), a ubiquitous Ca2+ entry pathway regulating multiple cellular functions, is unknown. Here we show that NAGly but not AEA reversibly hinders SOCE in a time- and concentration-dependent manner. The inhibitory effect of NAGly on SOCE was found in the human endothelial cell line EA.hy926, the rat pancreatic β-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca2+ depletion of the endoplasmic reticulum (ER), while it was not effective on Ca2+ entry via L-type voltage-gated Ca2+ channels. Enhanced Ca2+ entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, the stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly neither affected STIM1 oligomerization, nor STIM1 clustering, nor the co-localization of STIM1 with Orai1, which were induced by Ca2+ depletion of the ER. In contrast, independently from its slow depolarizing effect on mitochondria NAGly instantly and strongly diminished the interaction of STIM1 with Orai1, indicating that NAGly inhibits SOCE primarily by uncoupling STIM1 from Orai1. In summary, our findings unveiled the STIM1/Orai1-mediated SOCE machinery as a so far unknown molecular target of NAGly, which might have multiple implications in cell physiology.
PMCID: PMC3614444  PMID: 23239024
anandamide; calcium imaging; endocannabinoids; endothelial cells; fluorescent proteins; fluorescence microscopy; FRET; fura-2; INS-1 cells; NAGly
13.  Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked 
Journal of cell science  2012;126(0 2):473-483.
The endomembrane system of yeast contains different tail-anchored proteins that are posttranslationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment may be hazardous in the cytosol if membrane insertion fails resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function; promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis.
PMCID: PMC3613179  PMID: 23203805
chaperones; GET pathway; tail-anchored proteins; glucose starvation
14.  Changes in Association of the Xenopus Origin Recognition Complex with Chromatin On Licensing of Replication Origins 
Journal of cell science  1999;112(Pt 12):2011-2018.
During late mitosis and early G1, a series of proteins are assembled onto replication origins that results in them becoming “licensed” for replication in the subsequent S phase. In Xenopus this first involves the assembly onto chromatin of the Xenopus Origin Recognition complex XORC, and then XCdc6, and finally the RLF-M component of the replication licensing system. In this paper we examine changes in the way that XORC associates with chromatin in the Xenopus cell-free system as origins become licensed. Restricting the quantity of XORC on chromatin reduced the extent of replication as expected if a single molecule of XORC is sufficient to specify a single replication origin. During metaphase, XOrc1 associated only weakly with chromatin. In early interphase, XOrc1 formed a strong complex with chromatin, as evidenced by its resistance to elution by 200 mM salt, and this state persisted when XCdc6 was assembled onto the chromatin. As a consequence of origins becoming licensed the association of XOrc1 and XCdc6 with chromatin was destabilised, and XOrc1 became susceptible to removal from chromatin by exposure to either high salt or high Cdk levels. At this stage the essential function for XORC and XCdc6 in DNA replication had already been fulfilled. Since high Cdk levels are required for the initiation of DNA replication, this “licensing-dependent origin inactivation” may contribute to mechanisms that prevent re-licensing of replication origins once S phase has started.
PMCID: PMC3605702  PMID: 10341218
DNA Replication; Licensing Factor; ORC; Cdc6; RLF
15.  Specificity of RGS10A as a key component in the RANKL signaling mechanism for osteoclast differentiation 
Journal of cell science  2007;120(Pt 19):3362-3371.
Significant progress has been made in studies of the mechanisms by which RANKL induces terminal osteoclast differentiation. However, many crucial details in the RANKL-evoked signaling pathway for osteoclast differentiation remain to be defined. We characterized genes specifically expressed in osteoclasts by differential screening of a human osteoclastoma cDNA library, and found that the regulator of G-protein signaling 10A (RGS10A), but not the RGS10B isoform, was specifically expressed in human osteoclasts. The expression of RGS10A is also induced by RANKL in osteoclast precursors and is prominently expressed in mouse osteoclast-like cells. RGS10A silencing by RNA interference blocked intracellular [Ca2+]i oscillations, the expression of NFAT2, and osteoclast terminal differentiation in both bone marrow cells and osteoclast precursor cell lines. Reintroduction of RGS10A rescued the impaired osteoclast differentiation. RGS10A silencing also resulted in premature osteoclast apoptosis. RGS10A silencing affected the RANKL-[Ca2+]i oscillation-NFAT2 signaling pathway but not other RANKL-induced responses. Our data demonstrate that target components of RGS10A are distinct from those of RGS12 in the RANKL signaling mechanism. Our results thus show the specificity of RGS10A as a key component in the RANKL-evoked signaling pathway for osteoclast differentiation, which may present a promising target for therapeutic intervention.
PMCID: PMC3587975  PMID: 17881498
Differential screening; RGS10 RNA interference; [Ca2+]i oscillations; Osteoclast differentiation; RANKL signaling pathway
16.  Dissecting the role of PtdIns(4,5)P2 in endocytosis and recycling of the transferrin receptor 
Journal of cell science  2008;121(Pt 9):1488-1494.
Endocytosis and recycling of membrane proteins are key processes for nutrient uptake, receptor signaling and synaptic transmission. Different steps in these fission and fusion cycles have been proposed to be regulated by physiological changes in plasma membrane (PM) phosphatidylinositol (4,5)- bisphosphate [PtdIns(4,5)P2] concentration. Here, we use a chemical enzyme-translocation strategy to rapidly reduce PM PtdIns(4,5)P2 levels while monitoring clathrin-mediated endocytosis and recycling. PtdIns(4,5)P2 hydrolysis blocked transferrin receptor endocytosis and led to a marked increase in the concentration of transferrin receptors in the PM, suggesting that endocytosis is more sensitive to changes in PtdIns(4,5)P2 than recycling. Reduction of PM PtdIns(4,5)P2 levels led to a near complete dissociation of Adaptor protein 2 (AP-2) from the PM but had only a small effect on clathrin assembly. This argues that receptor-mediated PtdIns(4,5)P2 reduction preferentially suppresses AP-2-mediated targeting of cargo to endocytic sites rather than the assembly of clathrin coats or recycling of endocytic vesicles.
PMCID: PMC3579524  PMID: 18411250
Endocytosis; Phosphoinositides; Recycling
17.  Fusion-pore expansion during syncytium formation is restricted by an actin network 
Journal of cell science  2008;121(Pt 21):3619-3628.
Cell-cell fusion in animal development and in pathophysiology involves expansion of nascent fusion pores formed by protein fusogens to yield an open lumen of cell-size diameter. Here we explored the enlargement of micron-scale pores in syncytium formation, which was initiated by a well-characterized fusogen baculovirus gp64. Radial expansion of a single or, more often, of multiple fusion pores proceeds without loss of membrane material in the tight contact zone. Pore growth requires cell metabolism and is accompanied by a local disassembly of the actin cortex under the pores. Effects of actin-modifying agents indicate that the actin cortex slows down pore expansion. We propose that the growth of the strongly bent fusion-pore rim is restricted by a dynamic resistance of the actin network and driven by membrane-bending proteins that are involved in the generation of highly curved intracellular membrane compartments.
PMCID: PMC3552434  PMID: 18946025
Cell fusion; Syncytium formation; Fusion-pore expansion; Actin cytoskeleton; Membrane-bending proteins; Baculovirus gp64
18.  Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked 
Journal of Cell Science  2013;126(2):473-483.
The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis.
PMCID: PMC3613179  PMID: 23203805
Chaperones; GET pathway; Tail-anchored proteins; Glucose starvation
19.  tTorsinA binds the KASH domain of nesprins and participates in linkage between nuclear envelope and cytoskeleton 
Journal of cell science  2008;121(Pt 20):3476-3486.
A specific mutation (ΔE) in torsinA underlies most cases of the dominantly inherited movement disorder, early-onset torsion dystonia (DYT1). TorsinA, a member of the AAA+ ATPase superfamily, is located within the lumen of the nuclear envelope (NE) and endoplasmic reticulum (ER). We investigated an association between torsinA and nesprin-3, which spans the outer nuclear membrane (ONM) of the NE and links it to vimentin via plectin in fibroblasts. Mouse nesprin-3α co-immunoprecipitated with torsinA and this involved the C-terminal region of torsinA and the KASH domain of nesprin-3α. This association with human nesprin-3 appeared to be stronger for torsinAΔE than for torsinA. TorsinA also associated with the KASH domains of nesprin-1 and -2 (SYNE1 and 2), which link to actin. In the absence of torsinA, in knockout mouse embryonic fibroblasts (MEFs), nesprin-3 was localized predominantly in the ER. Enrichment of yellow fluorescent protein (YFP)-nesprin-3 in the ER was also seen in the fibroblasts of DYT1 patients, with formation of YFP-positive globular structures enriched in torsinA, vimentin and actin. TorsinA-null MEFs had normal NE structure, but nuclear polarization and cell migration were delayed in a wound-healing assay, as compared with wild-type MEFs. These studies support a role for torsinA in dynamic interactions between the KASH domains of nesprins and their protein partners in the lumen of the NE, with torsinA influencing the localization of nesprins and associated cytoskeletal elements and affecting their role in nuclear and cell movement.
PMCID: PMC3539201  PMID: 18827015
Nesprin; Dystonia; Cell migration; Nuclear polarization; DYT1; Vimentin; Actin
20.  Sorting nexin-2 is associated with tubular elements of the early endosome, but is not essential for retromer-mediated endosome-to-TGN transport 
Journal of cell science  2005;118(Pt 19):4527-4539.
Sorting nexins are a large family of phox-homology-domain-containing proteins that have been implicated in the control of endosomal sorting. Sorting nexin-1 is a component of the mammalian retromer complex that regulates retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network. In yeast, retromer is composed of Vps5p (the orthologue of sorting nexin-1), Vps17p (a related sorting nexin) and a cargo selective subcomplex composed of Vps26p, Vps29p and Vps35p. With the exception of Vps17p, mammalian orthologues of all yeast retromer components have been identified. For Vps17p, one potential mammalian orthologue is sorting nexin-2. Here we show that, like sorting nexin-1, sorting nexin-2 binds phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,5-bisphosphate, and possesses a Bin/Amphiphysin/Rvs domain that can sense membrane curvature. However, in contrast to sorting nexin-1, sorting nexin-2 could not induce membrane tubulation in vitro or in vivo. Functionally, we show that endogenous sorting nexin-1 and sorting nexin-2 co-localise on high curvature tubular elements of the 3-phosphoinositide-enriched early endosome, and that suppression of sorting nexin-2 does not perturb the degradative sorting of receptors for epidermal growth factor or transferrin, nor the steady-state distribution of the cation-independent mannose 6-phosphate receptor. However, suppression of sorting nexin-2 results in a subtle alteration in the kinetics of cation-independent mannose 6-phosphate receptor retrieval. These data suggest that although sorting nexin-2 may be a component of the retromer complex, its presence is not essential for the regulation of endosome-to-trans Golgi network retrieval of the cation-independent mannose 6-phosphate receptor.
PMCID: PMC1904489  PMID: 16179610
Sorting nexin; Retromer; CI-MPR; Phosphoinositide; PX-domain
21.  The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport 
Journal of cell science  2006;119(Pt 19):3944-3957.
The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. A shared feature of fab1Δ yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P2, in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.
PMCID: PMC1904490  PMID: 16954148
PIKfyve; Fab1p; Early endosome; Phosphatidylinositol (3,5)-bisphosphate; Endosomal sorting
22.  Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation 
Journal of cell science  2012;125(Pt 10):2436-2445.
To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell cycle regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2/M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. In contrast, when Cdt1 is overexpressed or is stabilised by the Neddylation inhibitor MLN4924, re-replication can occur throughout S phase.
PMCID: PMC3481538  PMID: 22366459
Geminin; DNA replication; replication licensing; re-replication; deep sequencing
23.  Trimerization is important for the function of clathrin at the mitotic spindle 
Journal of cell science  2006;119(Pt 19):4071-4078.
Clathrin is a triskelion consisting of three heavy chains each with an associated light chain. During mitosis, clathrin contributes to kinetochore fibre stability. As the N-terminal domain at the foot of each leg can bind to the mitotic spindle, we proposed previously a “bridge hypothesis” wherein clathrin acts as a brace between two or three microtubules within a kinetochore fibre to increase fibre stability. Here, we have tested this hypothesis by replacing endogenous clathrin heavy chain in human cells with a panel of clathrin constructs. Mutants designed to abolish trimerization were unable to rescue the mitotic defects caused by depletion of endogenous clathrin. In contrast, stunted triskelia with contracted legs could partially rescue normal mitosis. These results indicate that the key structural features of clathrin that are necessary for its function in mitosis are a trimeric molecule with a spindle interaction domain at each end, supporting the “bridge hypothesis” for clathrin function in mitosis.
PMCID: PMC3475310  PMID: 16968737
Clathrin; mitosis; endocytosis; RNAi
24.  Viral oncoprotein-induced mislocalization of select PDZ proteins disrupts tight junctions and causes polarity defects in epithelial cells 
Journal of cell science  2005;118(Pt 18):4283-4293.
The development of human cancers is frequently associated with a failure of epithelial cells to form tight junctions and to establish proper apicobasal polarity. Interestingly, the oncogenic potential of the adenovirus E4-ORF1 protein correlates with its binding to the cellular PDZ proteins MUPP1, MAGI-1, ZO-2 and SAP97, the first three of which assemble protein complexes at tight junctions. Given that E4-ORF1 sequesters these three PDZ proteins in the cytoplasm of fibroblasts, we postulated that E4-ORF1 would inhibit tight junction formation in epithelial cells. Providing further support for this idea, we identified MUPP1-related PATJ, a key component of the tight junction-associated CRB3-PALS1-PATJ polarity complex, as a new PDZ-protein target for both the E4-ORF1 and high-risk human papillomavirus type 18 E6 oncoproteins. Moreover, in epithelial cells, E4-ORF1 blocked the tight junction localization of PATJ and ZO-2, as well as their interacting partners, and disrupted both the tight junction barrier and apicobasal polarity. These significant findings expose a direct link between the tumorigenic potential of E4-ORF1 and inactivation of cellular PDZ proteins involved in tight junction assembly and polarity establishment.
PMCID: PMC3471650  PMID: 16141229
Tight junction; Polarity; PATJ; ZO-2; E4-ORF1; E6
25.  Sumoylation Dynamics During Keratinocyte Differentiation 
Journal of cell science  2006;120(Pt 1):125-136.
SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several critical keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), calcium-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding critical sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASxβ), SUMO2, and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similar to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Lastly, abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process.
PMCID: PMC3470114  PMID: 17164289
Keratinocyte; differentiation; SUMO; HaCaT; Ubc9

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