No description available.

No description available.

Ca2+ signaling is essential for a wide variety of cellular activities, ranging from short term activities, such as synaptic and muscle contraction, to long term processes, such as proliferation and differentiation. Store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in non-excitable cells, well coordinates Ca2+ release from ER and Ca2+ influx through plasma membrane. STIM1 and Orai1, serving as ER Ca2+ sensor and pore forming subunit, respectively, are the two essential components of SOCE machinery. In addition to activate Orai1 channel, studies have shown that STIM1 regulates other plasma membrane Ca2+ channels and senses a variety of cellular stresses to regulate SOCE. Therefore, it is of great interests to investigate the mechanisms and physiological functions of STIM1 and Orai1 mediated SOCE. Here, we performed tandem affinity purification to identify STIM1 associated proteins in Hela cells stably expressing STIM1-His6-3×Flag. Four candidate proteins, including GRP78, HSP70, IQGAP1, and Actin, were identified by mass spectrometry analyses. Surprisingly, IQGAP1 failed to affect the activity of SOCE. Interestingly, GRP78 knockdown only affected the inactivation phase while exerted no effect on the activation phase of SOCE. In addition, GRP78 knockdown markedly induced cell apoptosis and dramatically increased the ER Ca2+ concentration. Moreover, GRP78 was involved in the regulation of SOCE by the ER stress. These data indicate that GRP78 is an important regulator of SOCE to prevent Ca2+ overload in cells. HSP70, however, significantly reduced the activity of SOCE by inhibiting STIM1 translocation to ER-PM junctions. Future studies will explore the mechanism of GRP78 and HSP70 in regulating SOCE by confocal and TIRF imaging. Embryonic stem (ES) cells proliferate unlimitedly and can differentiate into all fetal and adult cell types. This property endows ES cells to be the promising candidates in the therapy of neurodegenerative diseases. Thus, it is important to identify novel signaling molecules or events that could play a role in the neural commitment of ES cells. Accumulated evidences have documented the role of STIM1 and Orai1 mediated SOCE in neuronal activities. Yet, the role of SOCE in early neural development remains to be determined. Here we examined the role of STIM1 and Orai1 during neural differentiation of mouse ES cells. Both of STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is required for neural differentiation of mouse ES cells independent of Orai1-mediated SOCE. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

No description available.

Abstract Stretch of the cardiac muscle activates several physiological processes leading to changes in the function of the muscle. These changes include increase of the contraction force accompanied by changes in the intracellular calcium concentration. This phenomenon is known as Frank-Starling relation of the heart. In addition to this, stretch also influences the membrane voltage of individual myocytes predisposing the cardiac muscle to arrhythmias. In atrial muscle stretch augments the secretion of the atrial natriuretic peptide (ANP). Although several cellular components are known to be sensitive to mechanical stimulus the precise mechanisms participating to these stretch-induced changes are not known in detail. Further it is not known if these changes are causally related or if they share a common causal factor. This research was aimed to study the stretch-induced changes in the rat atrium. The possible interactive mechanisms were studied by recording intracellular action potentials, changes in the intracellular calcium concentration, contraction force and ANP secretion during stretch. The plausible mechanosensitive cellular components were incorporated into a mathematical model that was used to further study the mechanisms. The role of intracellular acidosis as a possible modulator of the mechanotransduction was of special interest. In isolated rat left atrium moderate stretch produced by increasing the intra-atrial pressure increased the contraction force in a biphasic manner. The immediate increase of the force was caused by altered properties of the contractile element, but the following slow increase was accompanied by an increase of the Ca2+ transient. These changes were followed by lengthening of the late phase of action potentials and augmented secretion of the ANP. Intensive sustained stretch was also found to induce delayed afterdepolarizations (DADs). Gadolinium (Gd3+), blocker of stretch-activated ion channels reduced the stretch-dependent activation of the contraction and inhibited the stretch-induced DADs. The mathematical model simulated the experimental findings at best when stretch-activated channel (SA-channel) activation and increased troponin-C affinity were used to mimic the stretch. The modelling data suggested that the SA-channel current increases the sarcoplasmic reticulum calcium content in a time dependent manner leading to Ca2+ transient augmentation during systole. Bigger Ca2+ transients induce a depolarizing current during the late phase of the action potential (AP) repolarization via the Na+/Ca2+ exchanger causing the lengthening of the action potentials. A small reduction of the intracellular pH (0.18 units) with 20 mM propionate was found to modulate the stretch-induced changes in the rat atrium. Acidosis leads to an increase in the diastolic [Ca2+]i during stretch, inhibits the stretch-induced changes in action potentials and slows down the contraction development during stretch by inhibiting the fast component of the force increase. These changes in E-C-coupling (excitation-contraction-coupling) were accompanied by a simultaneous augmentation of the ANP secretion. Furthermore, it was shown that contraction force and diastolic [Ca2+]i of the stretched tissue are more sensitive to acidosis than in non-stretched tissue. In conclusion, the stretch-induced changes in rat atrial myocytes are mediated by at least two mechanisms; stretch-activated Ca2+ influx and change in the properties of the contractile element. The action potential changes can be largely explained by modulation of the membrane voltage by intracellular calcium via Na+/Ca2+-exchanger. The co-occurrence of the changes in the [Ca2+]i and ANP secretion suggests that the stretch-induced ANP secretion can be mediated by [Ca2+]i.

Eosinophils are end-point effectors of inflammation that contribute to the clinical severity of asthma. Eosinophil homing to the asthmatic lung is primarily guided by eotaxin-1, which is an eosinophil-selective chemokine. The mechanism by which eotaxin-1 augments intracellular calcium during cell migration is incompletely understood but is integral to the extravasation of eosinophils at sites of inflammation. We consequently report here that fluid shear stress, like eotaxin-1, unexpectedly activates human eosinophils in a calcium-dependent manner. We used confocal fluorescence microscopy to study calcium-handling in purified human eosinophils. Application of eotaxin-1 augmented the [Ca2+]i in a concentration-dependent manner. Pre-treatment of cells with ryanodine (10 μM) completely abolished the eotaxin-mediated calcium response, indicating that this phenomenon is dependent on Ca2+-release from the ER. Several SOCC blockers (2-APB, 100 μM; Gd3+, 10 μM; SKF-96365, 100 μM) attenuated SOCE, suggesting that these channels may directly contribute towards the eotaxin-1 calcium response in human eosinophils. In the presence of fluid-perfusion, eosinophils displayed a robust perfusion-induced calcium response (PICR) demonstrating that eosinophils are mechanically sensitive. The PICR rapidly induced adhesion and non-directional migration in eosinophils, suggesting that some hitherto unknown molecular mechanosensor permits these cells to detect and respond to changes in shear-stress. Pre-treatment of eosinophils with the non-selective tripeptide integrin receptor blocker, Arg-Gly-Asp (RGD), abrogated the PICR. The highly selective, dual α4β7/α4β1 integrin receptor blocker, CDP-323, was used to ascertain whether these highly expressed integrin subtypes mediate the PICR in eosinophils. Pre-treatment of cells with CDP-323 completely abolished the PICR, in addition to the eotaxin-mediated calcium response in a shear-dependent manner. Taken together, our results support a novel role for the α4β7/α4β1 integrin receptors as mechanosensors that directly modulate [Ca2+]i, adhesion and migration in human eosinophils. On-going experiments will seek to quantify the shear-response thresholds at which eosinophils activate and the time-course of the associated calcium response. This study suggests that the recruitment and activation of eosinophils are regulated by chemical and mechanical stimuli via overlapping, calcium-dependent signal transduction cascades. Given that the PICR is mediated by the eosinophil-specific α4β7/α4β1 integrin receptors, we conclude that integrin receptors are molecular mechanosensors that may facilitate eosinophil activation, adhesion and non-directional migration independently of, or in conjunction with, chemokine signaling. / Thesis / Master of Science in Medical Sciences (MSMS)

Presenilin (PS) is the catalytic subunit of the gamma-secretase which is responsible for the cleavage of amyloid precursor protein to form beta amyloid (Aβ). Mutations in PS cause familial Alzheimer’s disease (FAD) by increasing the Aβ plaques formation in the brain and thereby induce neurodegeneration. Apart from this, FAD-linked PS mutations have been demonstrated to disrupt cellular calcium (Ca2+) homeostasis. Ca2+is a vital secondary messenger that involved in various neurophysiological functions, including memory, learning, and neuroplasticity and mounting evidence suggesting that Ca2+dysregulation associated with PS mutations may play a proximal role in the AD pathogenesis. Yet, the molecular mechanism for Ca2+dysregulation in AD remains debatable. It has been reported that cellular Ca2+homeostatsis can be disrupted in various ways. On the one hand, mutant PS has been demonstrated to exaggerate Ca2+release from the endoplasmic reticulum (ER) through different pathways. On the other hand, attenuatedCa2+influx from the extracellular medium through the capacitative Ca2+entry (CCE) pathway has also been reported to bring about cellular Ca2+disruption. However, the molecular mechanism for the PS mutation-mediated CCE deficits is largely unknown. For this reason, the objective of the current study is to elucidate the underlying molecular mechanism for attenuated CCE in AD. In this study, human neuronal cell line SH-SY5Y is employed as a cellular model to investigate the effect of wild-type or FAD-linked PS1 mutation on CCE pathway. Using single cell Ca2+imaging technique, significant CCE deficits was observed in SH-SY5Y stably expressing FAD-linked PS1mutation, PS1M146L. Interestingly, this CCE attenuation in PS1 mutant expressing cells was not mediated by the down-regulation of STIM1 and Orai1 expression, the known essential molecular players in the CCE pathway. Instead, co-immunoprecipitation and proximity ligation assay have suggested a physical interaction between PS1 and STIM1 proteins. Moreover, a putative gamma-secretase mediated STIM1 cleavage was discovered by western blotting. In addition, confocal imaging showed that PS1M146L significantlyreduceSTIM1 puncta formation and ER translocation followed by the activation of CCE pathway by ER Ca2+store depletion with thapsigargin. This indicated that mutant PS1 attenuates CCE by affecting STIM1 oligomerization or its recruitment with Orai1. Taken together, our results suggested the negative regulatory role of PS on CCE pathway and hypothesized the molecular mechanism of CCE where FAD-linked PS mutation is perceived as a gain-of-function mutation and enhanced the negative impact on STIM1 to inhibit Ca2+entry.This hypothetic model provides new insights into the molecular regulation for CCE pathway and the identification of the interacting domains between PS1 and STIM1 may suggest novel targets for the development of therapeutic agents that help to treat the disease. / published_or_final_version / Physiology / Master / Master of Philosophy

Ca2+ is an important mediator of stimulus-secretion coupling in beta cells of the pancreatic islets, which secrete insulin in response to elevation in plasma glucose concentration. I studied the actions of two lipids, arachidonic acid (AA) and cholesterol, on enzymatically-dissociated single beta cells of rat and mouse, using cytosolic Ca2+ ([Ca2+]i) measurement in conjunction with whole-cell patch-clamp techniques. AA, which is produced in the beta cell upon stimulation with either glucose or acetylcholine, was found to induce a large increase in [Ca2+]i that was dependent on both extracellular Ca2+ entry and intracellular Ca2+ release. Part of the AA-mediated extracellular Ca2+ entry was due to Ca2+ influx through the arachidonate-regulated Ca2+ (ARC) channels, which have not previously been reported in beta cells. The AA-mediated intracellular Ca2+ release was a result of Ca2+ mobilization from multiple inositol trisphosphate (IP3)-sensitive intracellular stores, including the endoplasmic reticulum (ER) and an acidic Ca2+ store that is probably the secretory granules. Therefore, in beta cells, the AA-mediated Ca2+ signal may amplify the [Ca2+]i rise induced by insulin secretagogues. Cholesterol is an integral component of cellular membranes and an important regulator of cellular functions. However, elevation of cholesterol level in the pancreatic islets reduces glucose-stimulated insulin secretion. I found that cholesterol overload impairs the glucose-stimulated [Ca2+]i increase in beta cells by two major mechanisms: the first is a decrease in glucose-stimulated ATP production, which is partly mediated by a decrease in glucose uptake, and the second is the reduction of voltage-gated Ca2+ current density. These effects of cholesterol may partly account for the decreased insulin secretion that develops in patients with type II diabetes, who typically exhibit hypercholesterolemia. In summary, different lipids may mediate beneficial or detrimental effects on Ca2+ regulation in rodent pancreatic beta cells.

No description available.

Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated to such a transport has been relatively slow, even though significant progress has been made in the characterization of the intracellular trafficking of cationic lipid/DNA complexes. Surprisingly, little is known about the effects of these delivery vectors on cell functioning. In the present thesis, we show that cationic lipids and cationic lipid/DNA complexes strongly increase the intracellular Ca2+ concentration. The end point of the Ca2+ increase was ~400 nM from a basal level of ~100 nM. The [Ca2+]i increase was studied using K562 and Jurkat cells cultured in vitro. This effect is weakened following addition of DNA to cationic liposomes, although remaining very large at cationic lipid/DNA ratios commonly used for cell transfection experiments. Removal of extracellular Ca2+ did not abolish this effect significantly and preincubating K562 cells with the Ca2+-ATPase inhibitor thapsigargin strongly abolished intracellular Ca2+ concentration increase, indicating that Ca2+ was released mainly from internal Ca2+ stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122 blocked the intracellular Ca2+ concentration rise, suggesting an inositol pathway-dependent mechanism. LDH release assay indicates that in the conditions used for fluorescence measurement and in those used to transfer DNA into cells, cationic liposomes diC14-amidine and DOTAP had no massive cytotoxic effects. Cationic liposomes showed more toxicity than their corresponding complexes; this toxicity decreases in the presence of serum. The effect of cationic lipids on phosphatidylinositol-specific phospholipase C (PI-PLC) was quantitatively assessed using phosphatidylinositol (PI) and radiolabeled phosphatidylinositol ([3H]-PI). Incorporation of diC14-amidine into PC/PI vesicle activated PI-PLC and was shown to activate the hydrolysis of PI and [3H]-PI. Our data may suggest that mobilization of intracellular Ca2+ by complex could have an effect on the transfection process itself. These results indicate for the first time that cationic lipids and cationic lipid/DNA complexes are not inert and can affect the functioning of the cells by increasing their intracellular Ca2+.