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1.  MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division 
EMBO Reports  2014;15(4):383-391.
The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.
doi:10.1002/embr.201337424
PMCID: PMC3989669  PMID: 24599748
Asymmetric division; differentiation; MYC; neural progenitor; Notch
2.  Interleukin-6 Secretion by Astrocytes Is Dynamically Regulated by PI3K-mTOR-Calcium Signaling 
PLoS ONE  2014;9(3):e92649.
After contusion spinal cord injury (SCI), astrocytes become reactive and form a glial scar. While this reduces spreading of the damage by containing the area of injury, it inhibits regeneration. One strategy to improve the recovery after SCI is therefore to reduce the inhibitory effect of the scar, once the acute phase of the injury has passed. The pleiotropic cytokine interleukin-6 (IL-6) is secreted immediately after injury and regulates scar formation; however, little is known about the role of IL-6 in the sub-acute phases of SCI. Interestingly, IL-6 also promotes axon regeneration, and therefore its induction in reactive astrocytes may improve regeneration after SCI. We found that IL-6 is expressed by astrocytes and neurons one week post-injury and then declines. Using primary cultures of rat astrocytes we delineated the molecular mechanisms that regulate IL-6 expression and secretion. IL-6 expression requires activation of p38 and depends on NF-κB transcriptional activity. Activation of these pathways in astrocytes occurs when the PI3K-mTOR-AKT pathway is inhibited. Furthermore, we found that an increase in cytosolic calcium concentration was necessary for IL-6 secretion. To induce IL-6 secretion in astrocytes, we used torin2 and rapamycin to block the PI3K-mTOR pathway and increase cytosolic calcium, respectively. Treating injured animals with torin2 and rapamycin for two weeks, starting two weeks after injury when the scar has been formed, lead to a modest effect on mechanical hypersensitivity, limited to the period of treatment. These data, taken together, suggest that treatment with torin2 and rapamycin induces IL-6 secretion by astrocytes and may contribute to the reduction of mechanical hypersensitivity after SCI.
doi:10.1371/journal.pone.0092649
PMCID: PMC3965459  PMID: 24667246
3.  Network analysis of time-lapse microscopy recordings 
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 (Ca2+) 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.
doi:10.3389/fncir.2014.00111
PMCID: PMC4166320  PMID: 25278844
networks; graph theory; neural networks; cross correlation; small-world; scale-free; calcium signaling
4.  Small-world networks of spontaneous Ca2+ activity 
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 (Ca2+) activity driven by electrical depolarization that stimulated cell growth. Network activity was revealed by single-cell live Ca2+ 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.
doi:10.4161/cib.24788
PMCID: PMC3742060  PMID: 23986813
neural progenitors; networks; calcium signaling; stem cells; gap junctions
5.  Intracellular calcium release modulates polycystin-2 trafficking 
BMC Nephrology  2013;14:34.
Background
Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells.
Methods
We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM) Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy.
Results
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 Ca2+ 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 Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM.
Conclusions
These novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.
doi:10.1186/1471-2369-14-34
PMCID: PMC3577431  PMID: 23398808
Polycystin-2; Protein trafficking; Calcium signaling; Kidney cells; Autosomal dominant polycystic kidney disease
6.  Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart 
BMC Physiology  2012;12:3.
Background
Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.
Results
Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.
Conclusion
The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.
doi:10.1186/1472-6793-12-3
PMCID: PMC3334682  PMID: 22449203
Heart; Screening; Zebrafish; Small molecule; Ex vivo; Ca2+ signaling
7.  RET PLCγ Phosphotyrosine Binding Domain Regulates Ca2+ Signaling and Neocortical Neuronal Migration 
PLoS ONE  2012;7(2):e31258.
The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca2+) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca2+ from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca2+ and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.
doi:10.1371/journal.pone.0031258
PMCID: PMC3280273  PMID: 22355350
8.  PfMDR1: Mechanisms of Transport Modulation by Functional Polymorphisms 
PLoS ONE  2011;6(9):e23875.
ATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.
doi:10.1371/journal.pone.0023875
PMCID: PMC3164660  PMID: 21912647
9.  Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission 
The EMBO Journal  2011;30(14):2762-2778.
Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission
Mitochondrial morphology depends on the balance between fission and fusion events. This study identifies a receptor for the fission factor Drp1 within the mitochondrial outer membrane, which inhibits Drp1-mediated fission and activates fusion.
Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion–fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.
doi:10.1038/emboj.2011.198
PMCID: PMC3160255  PMID: 21701560
Drp1; hFis1; mitochondrial fusion and fission; SMCR7L; MIEF1/MiD51
10.  Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations 
Neurochemical Research  2011;36(7):1175-1185.
Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca2+) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) are intracellular Ca2+ release channels that mediate Ca2+ release from endoplasmic reticulum (ER) Ca2+ stores. The three IP3R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP3R by the endogenous modulators IP3, Ca2+, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP3R subtype in shaping cytosolic Ca2+ oscillations.
doi:10.1007/s11064-011-0457-7
PMCID: PMC3111726  PMID: 21479917
Inositol 1,4,5-trisphosphate receptor; Inositol 1,4,5-trisphosphate receptor-associated protein; Calcium signaling; Calcium oscillations
11.  Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis 
The EMBO Journal  2010;29(22):3869-3878.
Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis
This study establishes HCN2 channels as physiological relevant ‘calcium gates' that mediate apoptosis-inducing factor-dependent cell death in cancer and primary neuronal cells.
Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca2+ influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca2+ import through hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis-inducing factor (AIF)-mediated apoptosis. Downregulation of HCN2 prevented the drug-induced Ca2+ increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca2+ entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412-induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C-terminal domain of HCN2 revealed that dephosphorylation of Thr549 was critical for the prolonged Ca2+ entry required for AIF-mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.
doi:10.1038/emboj.2010.253
PMCID: PMC2989107  PMID: 21037554
apoptosis; apoptosis-inducing factor; calcium signalling; calpain; HCN channel
12.  MYC proteins promote neuronal differentiation by controlling the mode of progenitor cell division 
EMBO Reports  2014;15(4):383-391.
The role of MYC proteins in somatic stem and progenitor cells during development is poorly understood. We have taken advantage of a chick in vivo model to examine their role in progenitor cells of the developing neural tube. Our results show that depletion of endogenous MYC in radial glial precursors (RGPs) is incompatible with differentiation and conversely, that overexpression of MYC induces neurogenesis independently of premature or upregulated expression of proneural gene programs. Unexpectedly, the neurogenic function of MYC depends on the integrity of the polarized neural tissue, in contrast to the situation in dissociated RGPs where MYC is mitogenic. Within the polarized RGPs of the neural tube, MYC drives differentiation by inhibiting Notch signaling and by increasing neurogenic cell division, eventually resulting in a depletion of progenitor cells. These results reveal an unexpected role of MYC in the control of stemness versus differentiation of neural stem cells in vivo.
doi:10.1002/embr.201337424
PMCID: PMC3989669  PMID: 24599748
Asymmetric division; differentiation; MYC; neural progenitor; Notch

Results 1-12 (12)