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1.  The novel endonuclease Ankle1 requires its LEM and GIY-YIG motifs for DNA cleavage in vivo 
Journal of cell science  2012;125(0 4):1048-1057.
The Lamina-associated polypeptide, Emerin, MAN1 - (LEM) domain defines a group of nuclear proteins, which bind chromatin through interaction of the LEM motif with the conserved DNA cross-linking protein, Barrier-to-Auto-Integration factor (BAF). Here, we describe a novel LEM protein, annotated in databases as “Ankyrin and LEM domain containing protein 1” (ANKLE1). We show that Ankle1 is conserved in metazoans and contains a unique C-terminal GIY-YIG motif that confers endonuclease activity in vitro and in vivo. In mammals, Ankle1 is predominantly expressed in hematopoietic tissues. While most characterized LEM proteins are components of the inner nuclear membrane, ectopic Ankle1 shuttles between cytoplasm and nucleus, and Ankle1 enriched in the nucleoplasm induces DNA cleavage and DNA damage response. This activity requires both the catalytic C-terminal GIY-YIG domain and the LEM motif, which binds chromatin via BAF. Hence, Ankle1 represents a novel LEM-protein with a GIY-YIG type endonuclease activity in higher eukaryotes.
PMCID: PMC4335191  PMID: 22399800
chromatin; DNA damage; GIY-YIG endonuclease; LEM-domain; nuclear envelope
2.  Muscle dystrophy-causing ΔK32 lamin A/C mutant does not impair functions of nucleoplasmic LAP2α - lamin A/C complexes in mice 
Journal of cell science  2013;126(0 8):1753-1762.
A-type lamins are components of the nuclear lamina, a filamentous network of the nuclear envelope in metazoans that supports nuclear architecture. In addition, lamin A/C can also be found in the nuclear interior. This nucleoplasmic lamin pool is soluble in physiological buffer, depends on the presence of the lamin-binding protein, Lamina-associated polypeptide 2α (LAP2α) and regulates cell cycle progression in tissue progenitor cells. ΔK32 mutations in A-type lamins cause severe congenital muscle disease in humans and a muscle maturation defect in LmnaΔK32/ΔK32 knock-in mice. At molecular level, mutant ΔK32 lamin A/C protein levels were reduced and all mutant lamin A/C was soluble and mislocalized to the nucleoplasm. To test the role of LAP2α in nucleoplasmic ΔK32 lamin A/C regulation and functions, we deleted LAP2α in LmnaΔK32/ΔK32 knock-in mice. In double mutant mice the LmnaΔK32/ΔK32- linked muscle defect was unaffected. LAP2α interacted with mutant lamin A/C, but unlike wild-type lamin A/C, the intranuclear localization of ΔK32 lamin A/C was not affected by loss of LAP2α. In contrast, loss of LAP2α in LmnaΔK32/ΔK32 mice impaired the regulation of tissue progenitor cells like in lamin A/C wild type animals. These data indicate that a LAP2α-independent assembly defect of ΔK32 lamin A/C is predominant for the mouse pathology, while the LAP2α-linked functions of nucleoplasmic lamin A/C in the regulation of tissue progenitor cells are not affected in LmnaΔK32/ΔK32 mice.
PMCID: PMC4333763  PMID: 23444379
congenital muscular dystrophy; nuclear envelope; lamin A/C; lamina associated polypeptide 2α; nucleoplasmic lamins
3.  A nuclear ubiquitin-proteasomal pathway targets inner nuclear membrane protein Asi2 for degradation 
Journal of cell science  2014;127(0 16):3603-3613.
The nuclear envelope consists of inner and outer nuclear membranes. While the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane represents a unique membranous environment containing specific proteins. The mechanisms of integral inner nuclear membrane protein degradation are unknown. Here we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome and independent of the vacuole exhibiting a half-life of ≈ 45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistently, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.
PMCID: PMC4333764  PMID: 24928896
ERAD; nuclear membrane; nuclear proteasome; protein degradation; ubiquitylation
4.  Nuclear location of a chromatin insulator in Drosophila melanogaster 
Journal of cell science  2004;117(0 7):1025-1032.
Chromatin-related functions are associated with spatial organization in the nucleus. We have investigated the relationship between the enhancer-blocking activity and subnuclear localization of the Drosophila melanogaster suHw insulator. Using fluorescent in situ hybridization, we observed that genomic loci containing the gypsy retrotransposon were distributed closer to the nuclear periphery than regions without the gypsy retrotransposon. However, transgenes containing a functional 340 bp suHw insulator did not exhibit such biased distribution towards the nuclear periphery, which suggests that the suHw insulator sequence is not responsible for the peripheral localization of the gypsy retrotransposon. Antibody stains showed that the two proteins essential for the suHw insulator activity, SUHW and MOD(MDG4), are not restricted to the nuclear periphery. The enhancer-blocking activity of suHw remained intact under the heat shock conditions, which was shown to disrupt the association of gypsy, SUHW and MOD(MDG4) with the nuclear periphery. Our results indicate that the suHw insulator can function in the nuclear interior, possibly through local interactions with chromatin components or other nuclear structures.
PMCID: PMC4334384  PMID: 14996934
Chromatin boundary; Insulator; suHw; SuHw; Gypsy; Drosophila
5.  Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors 
Journal of cell science  2008;121(0 8):1243-1251.
Insulin recruits two transmembrane proteins, GLUT4 and IRAP, to the plasma membrane of muscle cells and adipocytes. The subcellular trafficking and localization of GLUT4, and to a lesser extent IRAP, have been intensely studied, yet the molecular mechanisms responsible for their insulin-responsive compartmentalization remain unknown. Herein we have investigated the endocytosis and recycling of IRAP from the cell surface back to the insulin-responsive compartment (IRC). Our results show that a key dileucine motif at position 76,77 (LL76,77), although required for the initial biosynthetic entry of IRAP into the IRC, is dispensable for entry into the IRC via the endosomal system. Indeed, we found that an AA76,77 mutant of IRAP is fully capable of undergoing endocytosis and is correctly routed back to the IRC. To verify that the AA76,77 mutant enters the bona fide IRC, we show that the internalized IRAP-AA76,77 construct is sequestered in an IRC that is insensitive to brefeldin A yet sensitive to a dominant-interfering mutant of AS160 (AS160-4P). In addition, we show that the GGA clathrin adaptors are not required for the re-entry of IRAP from the cell surface back into the IRC, whereas the Q-SNARE syntaxin 6 is required for this process.
PMCID: PMC4327992  PMID: 18388312
IRAP; Insulin; AS160; Syntaxin 6; GGA; GLUT4
6.  AQP2 exocytosis in the renal collecting duct – involvement of SNARE isoforms and the regulatory role of Munc18b 
Journal of cell science  2008;121(0 12):2097-2106.
Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation.
Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.
PMCID: PMC4327994  PMID: 18505797
Aquaporin2; Vasopressin; VAMP; SNARE; Syntaxin; Munc18; Exocytosis
7.  The Wnt antagonist Dickkopf-1 and its receptors are coordinately regulated during early human adipogenesis 
Journal of cell science  2006;119(0 12):2613-2620.
Secretion of Wnts by adipose cells has an important role in the control of murine adipogenesis. We present the first evidence that a Wnt antagonist, Dickkopf 1 (Dkk1), is secreted by human preadipocytes and promotes adipogenesis. DKK1 mRNA increases six hours after onset of human adipogenesis and this is followed by an increase in Dkk1 protein. With further differentiation, the mRNA and protein levels progressively decline such that they are undetectable in mature adipocytes. The transient induction in DKK1 correlates with downregulation of cytoplasmic and nuclear β-catenin levels, this being a surrogate marker of canonical Wnt signalling, and Wnt/β-catenin transcriptional activity. In addition, constitutive expression of Dkk1 in 3T3-L1 preadipocytes promotes their differentiation, further supporting the functional significance of increased Dkk1 levels during human adipogenesis. Concomitant downregulation of the Dkk1 receptors LRP5 and LRP6 is likely to potentiate the ability of Dkk1 to inhibit Wnt signalling and promote differentiation. Notably, Dkk1 is not expressed in primary murine preadipocytes or cell lines. The involvement of Dkk1 in human but not murine adipogenesis indicates that inter-species differences exist in the molecular control of this process. Given the public health importance of disorders of adipose mass, further knowledge of the pathways involved specifically in human adipocyte differentiation might ultimately be of clinical relevance.
PMCID: PMC4304001  PMID: 16763196
Adipocyte; Adipogenesis; Wnt; Dickkopf 1; LRP5; Human
8.  Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase 
Journal of cell science  1996;109(0 2):289-300.
Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.
PMCID: PMC4303253  PMID: 8838652
Neurite outgrowth; Nerve growth factor; PC12 cell; PI3-kinase; Wortmannin
9.  The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration 
Journal of cell science  2007;120(0 17):3138-3146.
Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P3 production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P3 production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P3 production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.
PMCID: PMC4267689  PMID: 17698922
Ras; Rac; Cdc42; Rho; Lamellipodia
10.  Direct mobilisation of lysosomal Ca2+ triggers complex Ca2+ signals 
Journal of cell science  2012;126(0 1):60-66.
Accumulating evidence implicates acidic organelles of the endolysosomal system as mobilisable stores of Ca2+ but their relationship to the better-characterised endoplasmic reticulum (ER) Ca2+ store remains unclear. Here we show that rapid osmotic permeabilisation of lysosomes evokes prolonged, spatiotemporally complex Ca2+ signals in primary cultured human fibroblasts. These Ca2+ signals comprised an initial response that correlated with lysosomal disruption and secondary long-lasting spatially heterogeneous Ca2+ oscillations that required ER-localised inositol trisphosphate receptors. Electron microscopy identified extensive membrane contact sites between lysosomes and the ER. Mobilisation of lysosomal Ca2+ stores is thus sufficient to evoke ER-dependent Ca2+ release probably through lysosome-ER membrane contact sites, and akin to the proposed mechanism of action of the Ca2+ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Our data identify functional and physical association of discrete Ca2+ stores important for the genesis of Ca2+ signal complexity.
PMCID: PMC4208295  PMID: 23108667
Ca2+; Lysosomes; Endoplasmic reticulum; Membrane contact sites; NAADP
11.  Prolonged seizure activity impairs mitochondrial bioenergetics and induces cell death 
Journal of cell science  2012;125(0 7):1796-1806.
The mechanisms underlying neuronal death following excessive activity such as occurs during prolonged seizures are unclear, but mitochondrial dysfunction has been hypothesised to play a role. Here, we tested this with fluorescence imaging techniques in rat glioneuronal neocortical co-cultures using low Mg2+ levels to induce seizure-like activity. Glutamate activation of NMDA receptors resulted in Ca2+ oscillations in neurons and a sustained depolarisation of the mitochondrial membrane potential, which was cyclosporine A sensitive, indicating mitochondrial permeability and transition pore opening. It was also dependent on glutamate release and NMDA receptor activation, because depolarisation was not observed after depleting vesicular glutamate with vacuolar-type H+-ATPase concanamycin A or blocking NMDA receptors with APV. Neuronal ATP levels in soma and dendrites decreased significantly during prolonged seizures and correlated with the frequency of the oscillatory Ca2+ signal, indicative of activity-dependent ATP consumption. Blocking mitochondrial complex I, complex V or uncoupling mitochondrial oxidative phosphorylation under low-Mg2+ conditions accelerated activity-dependent neuronal ATP consumption. Neuronal death increased after two and 24 hours of low Mg2+ levels compared with control treatment, and was reduced by supplementation with the mitochondrial complex I substrate pyruvate. These findings demonstrate a crucial role for mitochondrial dysfunction in seizure-activity-induced neuronal death, and that strategies aimed at redressing this are neuroprotective.
PMCID: PMC4195235  PMID: 22328526
Cell death; Seizure; Status epilepticus; Mitochondria; ATP; Neurons
12.  Stable incorporation versus dynamic exchange of β subunits in a native Ca2+ channel complex 
Journal of cell science  2013;126(0 9):2092-2101.
Voltage-gated Ca2+ channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation–contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary β subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether β subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and β subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca2+ channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I–II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I–II loop, known to change the orientation of β relative to the α1 subunit, altered the specific dynamic properties of the β subunits. In contrast, a single residue substitution in the α interaction pocket of β1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous β1a subunit forms a stable complex, whereas the heterologous β2a and β4b subunits form dynamic complexes with the Ca2+ channel. The distinct binding properties are not determined by differences in the I–II loop sequences of the α1 subunits, but are intrinsic properties of the β subunit isoforms.
PMCID: PMC4148589  PMID: 23447673
β subunit; Ca2+ channels; CaV1.1; FRAP; Skeletal muscle
13.  Inositol-1,4,5-trisphosphate (IP3)-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake 
Journal of cell science  2014;127(0 13):2944-2955.
Mitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon the stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma membrane junctions, and opens plasma membrane Ca2+ channels. Mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study we used a shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knock-down of two proteins that are essential for mitochondrial Ca2+ uptake, either the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+-buffering or ER Ca2+ depletion by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery likely by shaping cytosolic Ca2+ micro-domains.
PMCID: PMC4077590  PMID: 24806964
Mitochondrial Ca2+ uptake; UCP2; MCU; SOCE; STIM1 oligomerization
14.  IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake 
Journal of Cell Science  2014;127(13):2944-2955.
Mitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER–plasma-membrane junctions and opens plasma membrane Ca2+ channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca2+ uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+ buffering or ER Ca2+ depletion by sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca2+ micro-domains.
PMCID: PMC4077590  PMID: 24806964
Mitochondrial Ca2+ uptake; UCP2; MCU; SOCE; STIM1 oligomerization
15.  Integrin clustering enables anandamide-induced Ca2+ signaling in endothelial cells via GPR55 by protection against CB1-receptor-triggered repression 
Journal of cell science  2008;121(0 10):1704-1717.
Although the endocannabinoid anandamide is frequently described to act predominantly in the cardiovascular system, the molecular mechanisms of its signaling remained unclear. In human endothelial cells, two receptors for anandamide were found, which were characterized as cannabinoid 1 receptor (CB1R; CNR1) and G-protein-coupled receptor 55 (GPR55). Both receptors trigger distinct signaling pathways. It crucially depends on the activation status of integrins which signaling cascade becomes promoted upon anandamide stimulation. Under conditions of inactive integrins, anandamide initiates CB1R-derived signaling, including Gi-protein-mediated activation of spleen tyrosine kinase (Syk), resulting in NFκB translocation. Furthermore, Syk inhibits phosphoinositide 3-kinase (PI3K) that represents a key protein in the transduction of GPR55-originated signaling. However, once integrins are clustered, CB1R splits from integrins and, thus, Syk cannot further inhibit GPR55-triggered signaling resulting in intracellular Ca2+ mobilization from the endoplasmic reticulum (ER) via a PI3K-Bmx-phospholipase C (PLC) pathway and activation of nuclear factor of activated T-cells. Altogether, these data demonstrate that the physiological effects of anandamide on endothelial cells depend on the status of integrin clustering.
PMCID: PMC4067516  PMID: 18445684
Anandamide; Bmx/Etk; Cannabinoid signaling; CB1 receptor; GPR55; Ca2+ signaling; Integrins; Syk
16.  Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells 
Journal of cell science  2007;120(0 19):3465-3474.
Cdc42 plays a central role in regulating the actin cytoskeleton and maintaining cell polarity. Here, we show that Cdc42 is crucial for epidermal growth factor (EGF)-stimulated protrusion in MTLn3 carcinoma cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the protruding edge of the cells. siRNA-mediated knockdown of Cdc42 expression caused a decrease in EGF-stimulated protrusion and reduced cell motility in time-lapse studies. These changes were correlated with a decrease in barbed-end formation and Arp2/3 localization at the cell edge, and a marked defect in actin filament branching, as revealed by rotary-shadowing scanning electron microscopy. Upstream of Arp2/3, Cdc42 knockdown inhibited EGF-stimulated activation of PI 3-kinase at early (within 1 minute) but not late (within 3 minutes) time points. Membrane targeting of N-WASP, WAVE2 and IRSp53 were also inhibited. Effects on WAVE2 were not owing to Rac1 inhibition, because WAVE2 recruitment is unaffected by Rac1 knockdown. Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac.
PMCID: PMC4066376  PMID: 17855387
Cdc42; Arp2/3; WAVE2; EGF; Metastasis
17.  The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation 
Journal of cell science  2008;121(0 23):3912-3921.
Nek6 and Nercc1/Nek9 belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase/metaphase arrest, and depletion of XNercc from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5, a kinesin necessary for spindle bipolarity. Nek6 phosphorylates Eg5 at several sites in vitro, and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. While Cdk1 phosphorylates nearly all Eg5 during mitosis at Thr926, Nek6 phosphorylates ~3% of Eg5, primarily at the spindle poles. Eg5 depletion arrests cells with a monopolar spindle; this can be rescued by Eg5 wildtype but not by Eg5(Thr926Ala). Eg5(Ser1033Ala) rescues half as well as wildtype whereas an Eg5(Ser1033Asp) mutant is nearly as effective. Thus Nek6 phosphorylates a subset of Eg5 polypeptides during mitosis at a conserved site, whose phosphorylation is critical for the mitotic function of Eg5.
PMCID: PMC4066659  PMID: 19001501
18.  Cytosolic Ca2+ prevents the subplasmalemmal clustering of STIM1: an intrinsic mechanism to avoid Ca2+ overload 
Journal of cell science  2008;121(0 19):3133-3139.
The stromal interacting molecule (STIM1) is pivotal for store-operated Ca2+ entry (SOC). STIM1 proteins sense the Ca2+ concentration within the lumen of the endoplasmic reticulum (ER) via an EF-hand domain. Dissociation of Ca2+ from this domain allows fast oligomerization of STIM1 and the formation of spatially discrete clusters close to the plasma membrane. By lifetime-imaging of STIM1 interaction, the rearrangement of STIM1, ER Ca2+ concentration ([Ca2+]ER) and cytosolic Ca2+ signals ([Ca2+]cyto) we show that [Ca2+]cyto affects the subcellular distribution of STIM1 oligomers and prevents subplasmalemmal STIM clustering, even if the ER is depleted. These data indicate that [Ca2+]cyto, independently of the ER Ca2+ filling state, crucially tunes the formation and disassembly of subplasmalemmal STIM1 clusters, and, thus, protects cells against Ca2+ overload resulting from excessive SOC activity.
PMCID: PMC4064434  PMID: 18765567
ER Ca2+ dynamics; FRET; STIM1 oligomerization; Store-operated Ca2+ entry
19.  Podosomes of dendritic cells facilitate antigen sampling 
Journal of cell science  2014;127(0 5):1052-1064.
Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for podosomes of dendritic cells.
PMCID: PMC4054684  PMID: 24424029
Antigen presentation; podosomes; dendritic cells; receptor mediated endocytosis
20.  Sphingolipid organization in biomembranes: what physical studies of model membranes reveal 
Journal of cell science  1998;111(0 1):1-9.
Recent cell biological studies suggest that sphingolipids and cholesterol may cluster in biomembranes to form raft-like microdomains. Such lipid domains are postulated to function as platforms involved in the lateral sorting of certain proteins during their trafficking within cells as well as during signal transduction events. Here, the physical interactions that occur between cholesterol and sphingolipids in model membrane systems are discussed within the context of microdomain formation. A model is presented in which the role of cholesterol is refined compared to earlier models.
PMCID: PMC4043137  PMID: 9394007
Sphingolipid-cholesterol raft; Caveolar lipid; Lipid solubilization via detergent
21.  The formation of epithelial tubes 
Journal of cell science  2008;121(0 21):3501-3504.
PMCID: PMC3992995  PMID: 18946020
22.  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
23.  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
24.  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
25.  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

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