Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels.
journal of cell biology
2015; 209 (5): 653-670
Calcium signaling in neocortical development.
Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca(2+) stores and activate Orai channels in the plasma membrane. Although the amplitude and dynamics of SOCE are considered important determinants of Ca(2+)-dependent responses, the underlying modulatory mechanisms are unclear. In this paper, we identify STIM2?, a highly conserved alternatively spliced isoform of STIM2, which, in contrast to all known STIM isoforms, is a potent inhibitor of SOCE. Although STIM2? does not by itself strongly bind Orai1, it is recruited to Orai1 channels by forming heterodimers with other STIM isoforms. Analysis of STIM2? mutants and Orai1-STIM2? chimeras suggested that it actively inhibits SOCE through a sequence-specific allosteric interaction with Orai1. Our results reveal a previously unrecognized functional flexibility in the STIM protein family by which alternative splicing creates negative and positive regulators of SOCE to shape the amplitude and dynamics of Ca(2+) signals.
View details for DOI 10.1083/jcb.201412060
View details for PubMedID 26033257
A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration
NATURE CELL BIOLOGY
2014; 16 (2): 133-144
The calcium ion (Ca(2+) ) is an essential second messenger that plays a pivotal role in neurogenesis. In the ventricular zone (VZ) of the neocortex, neural stem cells linger to produce progenitor cells and subsequently neurons and glial cells, which together build up the entire adult brain. The radial glial cells, with their characteristic radial fibers that stretch from the inner ventricular wall to the outer cortex, are known to be the neural stem cells of the neocortex. Migrating neurons use these radial fibers to climb from the proliferative VZ in the inner part of the brain to the outer layers of the cortex, where differentiation processes continue. To establish the complex structures that constitute the adult cerebral cortex, proliferation, migration, and differentiation must be tightly controlled by various signaling events, including cytosolic Ca(2+) signaling. During development, cells regularly exhibit spontaneous Ca(2+) activity that stimulates downstream effectors, which can elicit these fundamental cell processes. Spontaneous Ca(2+) activity during early neocortical development depends heavily on gap junctions and voltage dependent Ca(2+) channels, whereas later in development neurotransmitters and synapses exert an influence. Here, we provide an overview of the literature on Ca(2+) signaling and its impact on cell proliferation, migration, and differentiation in the neocortex. We point out important historical studies and review recent progress in determining the role of Ca(2+) signaling in neocortical development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol, 2015.
View details for DOI 10.1002/dneu.22273
View details for PubMedID 25652687
Network analysis of time-lapse microscopy recordings.
Frontiers in neural circuits
2014; 8: 111
Ca(2+) signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca(2+)-regulatory proteins and second messengers are spatially organized in migrating cells. Here we show that receptor tyrosine kinase and phospholipase C signalling are restricted to the front of migrating endothelial leader cells, triggering local Ca(2+) pulses, local depletion of Ca(2+) in the endoplasmic reticulum and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca(2+) influx, STIM1, is transported by microtubule plus ends to the front. Furthermore, higher Ca(2+) pump rates in the front relative to the back of the plasma membrane enable effective local Ca(2+) signalling by locally decreasing basal Ca(2+). Finally, polarized phospholipase C signalling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca(2+) control system with polarized Ca(2+) signalling proteins and second messengers to synergistically promote directed cell migration.
View details for DOI 10.1038/ncb2906
View details for Web of Science ID 000331161400003
Small-world networks of spontaneous Ca(2+) activity.
Communicative & integrative biology
2013; 6 (4)
Multicellular organisms rely on intercellular communication to regulate important cellular processes critical to life. To further our understanding of those processes there is a need to scrutinize dynamical signaling events and their functions in both cells and organisms. Here, we report a method and provide MATLAB code that analyzes time-lapse microscopy recordings to identify and characterize network structures within large cell populations, such as interconnected neurons. The approach is demonstrated using intracellular calcium (Ca(2+)) recordings in neural progenitors and cardiac myocytes, but could be applied to a wide variety of biosensors employed in diverse cell types and organisms. In this method, network structures are analyzed by applying cross-correlation signal processing and graph theory to single-cell recordings. The goal of the analysis is to determine if the single cell activity constitutes a network of interconnected cells and to decipher the properties of this network. The method can be applied in many fields of biology in which biosensors are used to monitor signaling events in living cells. Analyzing intercellular communication in cell ensembles can reveal essential network structures that provide important biological insights.
View details for DOI 10.3389/fncir.2014.00111
View details for PubMedID 25278844
Inside-Out Connections: The ER Meets the Plasma Membrane.
2013; 153 (7): 1423-1424
Synchronized network activity among groups of interconnected cells is essential for diverse functions in the brain. However, most studies have been made on cellular networks in the mature brain when chemical synapses have been formed. Much less is known about the situation earlier in development. When studying neural progenitors derived from embryonic stem cells and neural progenitors from mice embryos, we found networks of gap junction coupled cells with vivid spontaneous non-random calcium (Ca(2+)) activity driven by electrical depolarization that stimulated cell growth. Network activity was revealed by single-cell live Ca(2+) imaging and further analyzed for correlations and network topology. The analysis revealed the networks to have small-world characteristics with scale-free properties. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.
View details for DOI 10.4161/cib.24788
View details for PubMedID 23986813
Neural progenitors organize in small-world networks to promote cell proliferation.
Proceedings of the National Academy of Sciences of the United States of America
2013; 110 (16): E1524-32
Junctions that connect the endoplasmic reticulum (ER) and the plasma membrane (PM) are unique yet ubiquitous subcellular compartments. Giordano et al. now report that extended synaptotagmins (E-Syts) promote their formation, providing fundamental insight into the molecular machinery controlling ER and plasma membrane crosstalk.
View details for DOI 10.1016/j.cell.2013.05.054
View details for PubMedID 23791170
Intracellular calcium release modulates polycystin-2 trafficking
Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca(2+)) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca(2+) channels that triggered Ca(2+) oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca(2+) activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.
View details for DOI 10.1073/pnas.1220179110
View details for PubMedID 23576737
Regulators of Calcium Homeostasis Identified by Inference of Kinetic Model Parameters from Live Single Cells Perturbed by siRNA.
2013; 6 (283): ra56
Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca(2+)) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells.We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca(2+) signaling. Plasma membrane (PM) Ca(2+) permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy.We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca(2+) signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca(2+) oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca(2+) permeability. Intracellular Ca(2+) buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca(2+)/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM.These novel findings demonstrate intracellular Ca(2+)-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.
View details for DOI 10.1186/1471-2369-14-34
View details for Web of Science ID 000315121300003
View details for PubMedID 23398808
Ca2+ and cAMP Signaling in Human Embryonic Stem Cell-Derived Dopamine Neurons
STEM CELLS AND DEVELOPMENT
2010; 19 (9): 1355-1364
Assigning molecular functions and revealing dynamic connections between large numbers of partially characterized proteins in regulatory networks are challenges in systems biology. We showed that functions of signaling proteins can be discovered with a differential equations model of the underlying signaling process to extract specific molecular parameter values from single-cell, time-course measurements. By analyzing the effects of 250 small interfering RNAs on Ca(2+) signals in single cells over time, we identified parameters that were specifically altered in the Ca(2+) regulatory system. Analysis of the screen confirmed known functions of the Ca(2+) sensors STIM1 (stromal interaction molecule 1) and calmodulin and of Ca(2+) channels and pumps localized in the endoplasmic reticulum (ER) or plasma membrane. Furthermore, we showed that the Alzheimer's disease-linked protein presenilin-2 and the channel protein ORAI2 prevented overload of ER Ca(2+) and that feedback from Ca(2+) to phosphatidylinositol 4-kinase and PLC? (phospholipase C?) may regulate the abundance of the plasma membrane lipid PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) to control Ca(2+) extrusion. Thus, functions of signaling proteins and dynamic regulatory connections can be identified by extracting molecular parameter values from single-cell, time-course data.
View details for PubMedID 23838183
Na,K-ATPase signal transduction triggers CREB activation and dendritic growth
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (7): 2212-2217
Human embryonic stem (hES) cell differentiation into dopamine neurons is considered a promising strategy for cell replacement therapy in Parkinson's disease, yet the functional properties of hES cell-derived dopamine neurons remain poorly defined. The objective of this study was to characterize intracellular calcium (Ca(2+)) and sub-plasma membrane cyclic AMP-signaling properties in hES cell-derived dopamine neurons. We found that hES cell-derived dopamine neurons and neural progenitors raised Ca(2+) from intra- and extracellular compartments in response to depolarization, glutamate, ATP, and dopamine D(2) receptor activation, while undifferentiated hES cells only mobilized Ca(2+) from intracellular stores in response to ATP and D(2) receptor-induced activation. Interestingly, we also found that hES cell-derived dopamine neurons in addition to primary ventral midbrain dopamine neurons were more prone to release Ca(2+) from intracellular stores than non-dopamine neurons following treatment with the neuropeptide neurotensin. Furthermore, hES cell-derived dopamine neurons showed cAMP elevations in response to forskolin and 3-isobutyl-methylxanthine, similar to primary dopamine neurons. Taken together, these results unravel the temporal sequence by which hES cells acquire Ca(2+) and cAMP signaling competence during dopamine differentiation.
View details for DOI 10.1089/scd.2009.0436
View details for Web of Science ID 000281517700008
View details for PubMedID 20043754
alpha-chemokines regulate proliferation, neurogenesis, and dopaminergic differentiation of ventral midbrain precursors and neurospheres
2008; 26 (7): 1891-1900
Dendritic growth is pivotal in the neurogenesis of cortical neurons. The sodium pump, or Na,K-ATPase, is an evolutionarily conserved protein that, in addition to its central role in establishing the electrochemical gradient, has recently been reported to function as a receptor and signaling mediator. Although a large body of evidence points toward a dual function for the Na,K-ATPase, few biological implications of this signaling pathway have been described. Here we report that Na,K-ATPase signal transduction triggers dendritic growth as well as a transcriptional program dependent on cAMP response element binding protein (CREB) and cAMP response element (CRE)-mediated gene expression, primarily regulated via Ca(2+)/calmodulin-dependent protein (CaM) kinases. The signaling cascade mediating dendritic arbor growth also involves intracellular Ca(2+) oscillations and sustained phosphorylation of mitogen-activated protein (MAP) kinases. Thus, our results suggest a novel role for the Na,K-ATPase as a modulator of dendritic growth in developing neurons.
View details for DOI 10.1073/pnas.0809253106
View details for Web of Science ID 000263516100026
View details for PubMedID 19164762
Distinct role of the N-terminal tail of the Na,K-ATPase catalytic subunit as a signal transducer
JOURNAL OF BIOLOGICAL CHEMISTRY
2006; 281 (31): 21954-21962
Increasing evidence suggests that alpha-chemokines serve several important functions in the nervous system, including regulation of neuroimmune responses, neurotransmission, neuronal survival, and central nervous system development. In this study, we first examined the function of two alpha-chemokines, chemokine ligand (CXCL) 6 and CXCL8, and their receptors, CXCR1 and CXCR2, in the developing rat ventral midbrain (VM). We found that CXCR2 and CXCL6 are regulated during VM development and that CXCL6 promotes the differentiation of nurr77-related receptor (Nurr1)+ precursors into dopaminergic (DA) neurons in vitro. Intriguingly, CXCL8, a ligand expressed only in Homo sapiens, enhanced progenitor cell division, neurogenesis, and tyrosine hydroxylase-positive (TH+) cell number in rodent precursor and neurosphere cultures. CXCL1, the murine ortholog of CXCL8, was developmentally regulated in the VM and exhibited activities similar but not identical to those of CXCL8. TH+ cells derived from chemokine-treated VM neurospheres coexpressed Nurr1 and VMAT and were functionally active, as shown by calcium (Ca(2+)) fluxes in response to AMPA. In conclusion, our data demonstrate that CXCL1, CXCL6, and CXCL8 increase the number of DA neurons in VM precursor and neurosphere cultures by diverse mechanisms. Thus, alpha-chemokines may find an application in the preparation of cells for drug development or Parkinson's disease cell replacement therapy.
View details for DOI 10.1634/stemcells.2007-0753
View details for Web of Science ID 000258004400025
View details for PubMedID 18436867
Allosteric changes of the NMDA receptor trap diffusible dopamine 1 receptors in spines
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (3): 762-767
Mounting evidence suggests that the ion pump, Na,K-ATPase, can, in the presence of ouabain, act as a signal transducer. A prominent binding motif linking the Na,K-ATPase to intracellular signaling effectors has, however, not yet been identified. Here we report that the N-terminal tail of the Na,K-ATPase catalytic alpha-subunit (alphaNT-t) binds directly to the N terminus of the inositol 1,4,5-trisphosphate receptor. Three amino acid residues, LKK, conserved in most species and most alpha-isoforms, are essential for the binding to occur. In wild-type cells, low concentrations of ouabain trigger low frequency calcium oscillations that activate NF-kappaB and protect from apoptosis. All of these effects are suppressed in cells overexpressing a peptide corresponding to alphaNT-t but not in cells overexpressing a peptide corresponding to alphaNT-t deltaLKK. Thus we have identified a well conserved Na,K-ATPase motif that binds to the inositol 1,4,5-trisphosphate receptor and can trigger an anti-apoptotic calcium signal.
View details for DOI 10.1074/jbc.M601578200
View details for Web of Science ID 000239387100042
View details for PubMedID 16723354
The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors. Exposure to NMDA reduces the diffusion coefficient for D1R and causes an increase in the number of D1R-positive spines. Unexpectedly, the action of NMDA in potentiating D1R recruitment was independent of calcium flow via the NMDA receptor channel. Thus, a highly energy-efficient, diffusion-trap mechanism can account for intraneuronal interaction between the glutamatergic and dopaminergic systems and for regulation of the number of D1R-positive spines. This diffusion trap system represents a molecular mechanism for brain plasticity and offers a promising target for development of antipsychotic therapy.
View details for Web of Science ID 000234727800047
View details for PubMedID 16407151