The cellular machinery responsible for Cu(+)-stimulated delivery of the Wilson-disease-associated protein ATP7B to the apical domain of hepatocytes is poorly understood. We demonstrate that myosin Vb regulates the Cu(+)-stimulated delivery of ATP7B to the apical domain of polarized hepatic cells, and that disruption of the ATP7B-myosin Vb interaction reduces the apical surface expression of ATP7B. Overexpression of the myosin Vb tail, which competes for binding of subapical cargos to myosin Vb bound to subapical actin, disrupted the surface expression of ATP7B, leading to reduced cellular Cu(+) export. The myosin-Vb-dependent targeting step occurred in parallel with hepatocyte-like polarity. If the myosin Vb tail was expressed acutely in cells just prior to the establishment of polarity, it appeared as part of an intracellular apical compartment, centered on γ-tubulin. ATP7B became selectively arrested in this compartment at high [Cu(+)] in the presence of myosin Vb tail, suggesting that these compartments are precursors of donor-acceptor transfer stations for apically targeted cargos of myosin Vb. Our data suggest that reduced hepatic Cu(+) clearance in idiopathic non-Wilsonian types of disease might be associated with the loss of function of myosin Vb.

Brain injury is a major cause of long-term disability. The possibility exists for exogenously derived neural progenitor cells to repair damage resulting from brain injury, although more information is needed to successfully implement this promising therapy. To test the ability of neural progenitor cells (NPCs) obtained from rats to repair damaged neo-cortex, we transplanted neural progenitor cell suspensions into normal and injured slice cultures of the neocortex acquired from rats on postnatal day 0-3. Donor cells from E16 embryos were obtained from either the neocortex, including the ventricular zone (VZ) for excitatory cells, ganglionic eminence (GE) for inhibitory cells or a mixed population of the two. Cells were injected into the ventricular/subventricular zone (VZ/SVZ) or directly into the wounded region. Transplanted cells migrated throughout the cortical plate with GE and mixed population donor cells predominately targeting the upper cortical layers, while neocortically derived NPCs from the VZ/SVZ migrated less extensively. In the injured neocortex, transplanted cells moved predominantly into the wounded area. NPCs derived from the GE tended to be immunoreactive for GABAergic markers while those derived from the neocortex were more strongly immunoreactive for other neuronal markers such as MAP2, TUJ1, or Milli-Mark. Cells transplanted in vitro acquired the electrophysiological characteristics of neurons, including action potential generation and reception of spontaneous synaptic activity. This suggests that transplanted cells differentiate into neurons capable of functionally integrating with the host tissue. Together, our data suggest that transplantation of neural progenitor cells holds great potential as an emerging therapeutic intervention for restoring function lost to brain damage.

Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu‐directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF‐B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans‐Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton‐pump inhibitor bafilomycin‐A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu‐stimulated) case. Overall, loss of acidification‐impaired Cu‐regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane.

Ca(2+) and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca(2+) and cAMP signals, including some that are Ca(2+)-responsive, some that are cAMP-responsive and some that detect coincident Ca(2+) and cAMP signals. Because Ca(2+) and cAMP can influence each other's amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca(2+) are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca(2+) buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP(3) levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements - the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP(3) metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca(2+) stores. Our data indicate that Ca(2+) release from IP(3)-sensitive pools is required for cAMP-induced transcription in hippocampal neurons.

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4 + T cells whose etiology is thought to be associated with the viral trans -activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27 Kip1 and p21 CIP1/WAF1 through protein stabilization and mRNA trans -activation and stabilization, respectively. The Tax-induced surge in p21 CIP1/WAF1 and p27 Kip1 begins in S phase and results in cellular senescence. Importantly, HeLa and SupT1 T cells infected by HTLV-1 also arrest in senescence, thus challenging the notion that HTLV-1 infection causes cell proliferation. Here we use time-lapse microscopy to investigate the effect of Tax on cell cycle progression in two reporter cell lines, HeLa/18x21-EGFP and HeLa-FUCCI, that express enhanced green fluorescent protein (EGFP) under the control of 18 copies of the Tax-responsive 21-bp repeat element and f luorescent u biquitin c ell c ycle i ndicators, respectively. Tax-expressing HeLa cells exhibit elongated or stalled cell cycle phases. Many of them bypass mitosis and become single senescent cells as evidenced by the expression of senescence-associated β-galactosidase. Such cells have twice the normal equivalent of cellular contents and hence are enlarged, with exaggerated nuclei. Interestingly, nocodazole treatment revealed a small variant population of HeLa/18x21-EGFP cells that could progress into mitosis normally with high levels of Tax expression, suggesting that genetic or epigenetic changes that prevent Tax-induced senescence can occur spontaneously at a detectable frequency.

The localized control of second messenger levels sculpts dynamic and persistent changes in cell physiology and structure. Inositol trisphosphate [Ins(1,4,5)P(3)] 3-kinases (ITPKs) phosphorylate the intracellular second messenger Ins(1,4,5)P(3). These enzymes terminate the signal to release Ca(2+) from the endoplasmic reticulum and produce the messenger inositol tetrakisphosphate [Ins(1,3,4,5)P(4)]. Independent of their enzymatic activity, ITPKs regulate the microstructure of the actin cytoskeleton. The immune phenotypes of ITPK knockout mice raise new questions about how ITPKs control inositol phosphate lifetimes within spatial and temporal domains during lymphocyte maturation. The intense concentration of ITPK on actin inside the dendritic spines of pyramidal neurons suggests a role in signal integration and structural plasticity in the dendrite, and mice lacking neuronal ITPK exhibit memory deficits. Thus, the molecular and anatomical features of ITPKs allow them to regulate the spatiotemporal properties of intracellular signals, leading to the formation of persistent molecular memories.

Inositol hexakisphosphate (InsP(6)) is an important component of cells, and its mass levels are usually assayed by either (a) equilibrium labelling of cell cultures with radiolabelled inositol or (b) by a variety of mass assays of differing sensitivities and ambiguities. Here, we describe a mass assay for InsP(6) that is based on phosphorylating InsP(6) with [(32)P]-ATP to 5-(PP)InsP(5) using a recombinant Giardia InsP(6) kinase and quantification of the radiolabelled 5-[(32)P](PP)InsP(5) product by anion exchange HPLC with an internal [(3)H]-(PP)InsP(5) standard. Interference with the enzyme reaction by other factors in the tissue extract is corrected for by assay of identical aliquots of tissue spiked with known amounts of InsP(6). This assay only measures InsP(6) (and not other inositol phosphates), and although it is simple in principle and requires no dedicated or specialised equipment, it is quite time-consuming. But the assay is unambiguous and is capable of quantifying accurately as little as 10 fmol of InsP(6) in a cell extract.

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate‐limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDHα). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorlyated PDHα. Dephosphorylation of astrocytic PDHα restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. © 2010 Wiley‐Liss, Inc.

The actin microstructure in dendritic spines is involved in synaptic plasticity. Inositol trisphosphate 3-kinase A (ITPKA) terminates Ins(1,4,5)P(3) signals emanating from spines and also binds filamentous actin (F-actin) through its amino terminal region (amino acids 1-66, N66). Here we investigated how ITPKA, independent of its kinase activity, regulates dendritic spine F-actin microstructure. We show that the N66 region of the protein mediates F-actin bundling. An N66 fusion protein bundled F-actin in vitro, and the bundling involved N66 dimerization. By mutagenesis we identified a point mutation in a predicted helical region that eliminated both F-actin binding and bundling, rendering the enzyme cytosolic. A fusion protein containing a minimal helical region (amino acids 9-52, N9-52) bound F-actin in vitro and in cells, but had lower affinity. In hippocampal neurons, GFP-tagged N66 expression was highly polarized, with targeting of the enzyme predominantly to spines. By contrast, N9-52-GFP expression occurred in actin-rich structures in dendrites and growth cones. Expression of N66-GFP tripled the length of dendritic protrusions, induced longer dendritic spine necks, and induced polarized actin motility in time-lapse assays. These results suggest that, in addition to its ability to regulate intracellular Ca(2+) via Ins(1,4,5)P(3) metabolism, ITPKA regulates structural plasticity.

A highly specific and sensitive mass assay for inositol hexakisphosphate (InsP6) was characterized. This centres around phosphorylating InsP6 with [32P]ATP using a recombinant InsP6 kinase from Giardia lambia, followed by HPLC of the 32P-labelled products with an internal [3H]InsP7 standard. This assay was used to quantify InsP6 levels in a variety of biological samples. Concentrations of InsP6 in rat tissues varied from 10–20 μM (assuming 64% of wet weight of tissue is cytosol water), whereas using the same assumption axenic Dictyostelium discoideum cells contained 352±11 μM InsP6. HeLa cells were seeded at low density and grown to confluence, at which point they contained InsP6 levels per mg of protein similar to rat tissues. This amounted to 1.952±0.117 nmol InsP6 per culture dish, despite the cells being grown in serum shown to contain no detectable (less than 20 pmol per dish) InsP6. These results demonstrate that mammalian cells synthesize all their own InsP6. Human blood was analysed, and although the white cell fraction contained InsP6 at a concentration comparable with other tissues, in serum and platelet-free plasma no InsP6 was detected (<1 nM InsP6). Human urine was also examined, and also contained no detectable (<5 nM) InsP6. These results suggest that dietary studies purporting to measure InsP6 at micromolar concentrations in human plasma or urine may not have been quantifying this inositol phosphate. Therefore claims that administrating InsP6 in the diet or applying it topically can produce health benefits by increasing extracellular InsP6 levels may require reassessment.

High lactate generation and low glucose oxidation, despite normal oxygen conditions, are commonly seen in cancer cells and tumors. Historically known as the Warburg effect, this altered metabolic phenotype has long been correlated with malignant progression and poor clinical outcome. However, the mechanistic relationship between altered glucose metabolism and malignancy remains poorly understood. Here we show that inhibition of pyruvate dehydrogenase complex (PDC) activity contributes to the Warburg metabolic and malignant phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which results in inhibitory phosphorylation of the pyruvate dehydrogenase alpha (PDHalpha) subunit. We also demonstrate that PDC inhibition in cancer cells is associated with normoxic stabilization of the malignancy-promoting transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha) by glycolytic metabolites. Knockdown of PDK-1 via short hairpin RNA lowers PDHalpha phosphorylation, restores PDC activity, reverts the Warburg metabolic phenotype, decreases normoxic HIF-1alpha expression, lowers hypoxic cell survival, decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated gene, and these data suggest that the buildup of glycolytic metabolites, resulting from high PDK-1 expression, may in turn promote HIF-1 activation, thus sustaining a feed-forward loop for malignant progression. In addition to providing anabolic support for cancer cells, altered fuel metabolism thus supports a malignant phenotype. Correction of metabolic abnormalities offers unique opportunities for cancer treatment and may potentially synergize with other cancer therapies.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinases (IP3Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P3 into inositol 1,3,4,5-tetrakisphosphate. However, what they contribute to the complexities of Ca2+ signaling, and how, is still not fully understood. In this study, we have used a simple Ca2+ imaging assay to compare the abilities of various Ins (1,4,5)P3-metabolizing enzymes to regulate a maximal histamine-stimulated Ca2+ signal in HeLa cells. Using transient transfection, we overexpressed green fluorescent protein-tagged versions of all three mammalian IP3K isoforms, including mutants with disrupted cellular localization or calmodulin regulation, and then imaged the Ca2+ release stimulated by 100 μm histamine. Both localization to the F-actin cytoskeleton and calmodulin regulation enhance the efficiency of mammalian IP3Ks to dampen the Ins (1,4,5)P3-mediated Ca2+ signals. We also compared the effects of the these IP3Ks with other enzymes that metabolize Ins(1,4,5)P3, including the Type I Ins(1,4,5)P3 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP3K found in Drosophila. All reduce the Ca2+ signal but to varying degrees. We demonstrate that the activity of only one of two IP3K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP3Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P3 signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.

It is 20 yr since the Ins(1,4,5)P3 3-kinase reaction was discovered, and this review summarizes and discusses some of the advances we have made in our understanding of the physiological significance of this reaction. The three major potential functions of Ins(1,4,5)P3 3-kinases are discussed in the context of their localization and regulation, the possible functions of their product, Ins(1,3,4,5)P4 and their potential contribution to the synthesis of higher inositol polyphosphates.

The structure of the actin cytoskeleton in dendritic spines is thought to underlie some forms of synaptic plasticity. We have used fixed and live-cell imaging in rat primary hippocampal cultures to characterize the synaptic dynamics of the F-actin binding protein inositol trisphosphate 3-kinase A (IP3K), which is localized in the spines of pyramidal neurons derived from the CA1 region. IP3K was intensely concentrated as puncta in spine heads when Ca2+ influx was low, but rapidly and reversibly redistributed to a striated morphology in the main dendrite when Ca2+ influx was high. Glutamate stimulated the exit of IP3K from spines within 10 s, and re-entry following blockage of Ca2+ influx commenced within a minute; IP3K appeared to remain associated with F-actin throughout this process. Ca2+-triggered F-actin relocalization occurred in about 90% of the cells expressing IP3K endogenously, and was modulated by the synaptic activity of the cultures, suggesting that it is a physiological process. F-actin relocalization was blocked by cytochalasins, jasplakinolide and by the over-expression of actin fused to green fluorescent protein. We also used deconvolution microscopy to visualize the relationship between F-actin and endoplasmic reticulum inside dendritic spines, revealing a delicate microorganization of IP3K near the Ca2+ stores. We conclude that Ca2+ influx into the spines of CA1 pyramidal neurons triggers the rapid and reversible retraction of F-actin from the dendritic spine head. This process contributes to changes in spine F-actin shape and content during synaptic activity, and might also regulate spine IP3 signals.

The size and number of IP3R (inositol 1,4,5-trisphosphate receptor) clusters located on the surface of the ER (endoplasmic reticulum) is hypothesized to regulate the propagation of Ca2+ waves in cells, but the mechanisms by which the receptors cluster are not understood. Using immunocytochemistry, live-cell imaging and heterologous expression of ER membrane proteins we have investigated IP3R clustering in the basophilic cell line RBL-2H3 following the activation of native cell-surface antigen receptors. IP3R clusters are present in resting cells, and upon receptor stimulation, form larger aggregates. Cluster formation and maintenance required the presence of extracellular Ca2+ in both resting and stimulated cells. Using transfection with a marker of the ER, we found that the ER itself also showed structural changes, leading to an increased number of 'hotspots', following antigen stimulation. Surprisingly, however, when we compared the ER hotspots and IP3R clusters, we found them to be distinct. Imaging of YFP (yellow fluorescent protein)-IP3R transfected in to living cells confirmed that IP3R clustering increased upon stimulation. Photobleaching experiments showed that the IP3R occupied a single contiguous ER compartment both before and after stimulation, suggesting a dynamic exchange of IP3R molecules between the clusters and the surrounding ER membrane. It also showed a decrease in the mobile fraction after cell activation, consistent with receptor anchoring within clusters. We conclude that IP3R clustering in RBL-2H3 cells is not simply a reflection of bulk-changes in ER structure, but rather is due to the receptor undergoing homotypic or heterotypic protein-protein interactions in response to agonist stimulation.

Using single cell Ca(2+) imaging and whole cell current clamp recordings, this study aimed to identify the signal transduction mechanisms involved in mACh receptor-mediated, enhanced synaptic signaling in primary cultures of hippocampal neurons. Activation of M(1) mACh receptors produced a 2.48 +/- 0.26-fold enhancement of Ca(2+) transients arising from spontaneous synaptic activity in hippocampal neurons. Combined imaging of spontaneous Ca(2+) signals with inositol 1,4,5-trisphosphate (IP(3)) production in single neurons demonstrated that the methacholine (MCh)-mediated enhancement required activated G(q/11)alpha subunits and phospholipase C activity but did not require measurable increases in IP(3). Electrophysiological studies demonstrated that MCh treatment depolarized neurons from -64 +/- 3 to -45 +/- 3 mV and increased action potential generation. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) enhanced neuronal excitability and prolonged the action of MCh. These studies suggest that, in addition to producing the second messengers IP(3) and diacylglycerol, mACh receptor activation may directly utilize PIP(2) hydrolysis to regulate neuronal excitability.

IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the localization and activity of IP3KB and its modulation by proteolysis. We found that the N- and C-termini (either side of residue 262) of IP3KB localized predominantly to the actin cytoskeleton and ER (endoplasmic reticulum) respectively, both in COS-7 cells and in primary astrocytes. The functional relevance of this was demonstrated by showing that full-length (actin-localized) IP3KB abolished the histamine-induced Ca2+ response in HeLa cells more effectively than truncated constructs localized to the ER or cytosol. The superior efficacy of full-length IP3KB was also attenuated by disruption of the actin cytoskeleton. By transfecting COS-7 cells with double-tagged IP3KB, we show that the translocation from actin to ER may be a physiologically regulated process caused by Ca2+-modulated constitutive proteolysis in intact cells.

Using fluorescently tagged markers for organelles in conjunction with confocal microscopy, we have studied the effects of agonist-induced Ca2+ signals on the motility of mitochondria and the ER (endoplasmic reticulum). We observed that the muscarinic agonist carbachol produced a rapid, simultaneous and reversible cessation of the movements of both organelles, which was dependent on a rise in cytosolic Ca2+. This rise in Ca2+ was shown to cause a fall in cellular ATP levels, and the effect of carbachol on organelle movement could be mimicked by depleting ATP with metabolic inhibitors in the absence of any such rise in Ca2+. However, a Ca2+-sensing process independent of ATP appears also to be involved, because we identified conditions where the ATP depletion was blocked (by inhibitors of the Ca2+ pumps), but the organelle movements still ceased following a rise in cytosolic Ca2+. We conclude that the co-ordinated cessation of mitochondria and ER motility is a process regulated by the cytosolic concentration of both Ca2+ and ATP, and that these two parameters are likely to synergize to regulate the localization of the two organelles, and to facilitate the transfer of Ca2+ between them.