Intracellular pH distribution is a critical physiological determinant of diverse cellular functions such as gene expression, vesicular transportations, and lysosomal degradation. Inhibition of the pH regulatory mechanisms in astrocytes and osteoclasts was shown to induce serious pathological defects. As a consequence, accurate and efficient imaging techniques to follow the pH distributions in living cells are essential to biomedical research. Fluorescence-based detections have become popular techniques in determining intracellular pH. The general detection principles include wavelength shifts, intensity-ratio changes, and lifetime changes. The recent progress of time-resolved detection has made fluorescence lifetime imaging microscopy a potential pH-imaging tool. However, reports focusing on lifetime-based pH imaging in organelles are scarce. The objective of this dissertation is to use the frequency-domain FLIM technique to image the pH distributions in cytosol and in vesicular organelles lysosomes. In addition, we evaluated the performance of FLIM in correlating intracellular pH variations with specific pH regulatory processes. Based on the previous lifetime characterization results, Carboxy-SNAFL2 was used for cytosolic pH imaging. We are able to image the cytosolic pH in different adherent cells and follow the transient cytosolic pH variations in living CHO cells. DM-NERF dextrans, OG-514 carboxylic acid dextrans, and LysoSensor DND-160 were used to image the pH distributions in acidic compartments since they showed appropriate low pH working range. The resting lysosomal pH values determined from 3T3 fibroblasts are between pH 4.5 and 4.9. It was also possible to follow the significant alkalization of the lysosomal pH after the treatments of proton-pump inhibitors and ionophores with FLIM. These imaging results demonstrate that our FLIM apparatus is able to provide comparable pH resolution and sensitivity to those obtained from intensity ratio imaging measurements and from other lifetime imaging techniques. In order to resolve the cellular structures at the sub-micron level, our present FLIM apparatus needs to be upgraded with the pseudo-confocal capability. We expect the future installation of multi-photon multi-focal microscopy to the FLIM instrumentation will greatly improve the z-axis resolution of the lifetime images. The future plans are to resolve the possible pH gradients existing in cytosol and describe the pH evolution in endosomal vesicles during endocytosis processes.

Store-operated Ca2+ entry (SOCaE) is involved in a host of processes within cells, including Ca2+ store replenishment, growth, and gene transcription. Although this process has long since been identified, the molecular nature of the store-operated Ca2+ channel (SOC) as well as the activation and regulatory mechanisms underlying its control have remained elusive. Studies began, using a cellular approach integrated with fluorescent techniques to measure intracellular Ca 2+, examining the possible involvement of the actin cytoskeleton in the stimulation or regulation of SOCaE using cultured smooth muscle cells. Manipulation of the cytoskeleton, either its removal or redistribution, demonstrated that the actin cytoskeleton did not actively participate in the activation or control of SOCaE. Compelling evidence that internal membrane association with the plasma membrane is necessary for the activation of SOCaE was provided by loss of SOCaE when cells were induced to form cortical actin subjacent to the plasma membrane. In addition, these interactions were demonstrated to occur in a reversible and labile manner by disruption of this cortical actin, restoring the activation and function of SOC's. This data led to the proposal of a new model for SOCaE, due to its close parallels to secretion. Using a more molecular approach, activation of human transient receptor potential channel 3 (TRP3), a possible model for SOC, was compared to SOCaE. Using HEK-293 cells stably transfected with TRP3, cortical actin formation demonstrated that TRP3 also required internal membrane associations at the plasma membrane for activation, although TRP3 did not prove to be activated by Ca2+ store-depletion. The direct activator of TRP3, diacylglycerol, could activate TRP3 in the presence or absence of cortical actin, proving that a functional channel exists proceeding cortical actin formation. In conjunction with cortical actin formation inhibiting activation of TRP3 by InsP3 , this provides even more evidence for a "secretion-like" activation process. Use of the inositol tri-phosphate receptor (InsP 3-receptor) antagonists 2-amino-diphenylborate (2-APB) and xestaspongin C had identical properties to cortical actin formation in the inhibition of SOCaE and TRP3. From this we concluded that the InsP3-receptor was an integral member of the SOCaE activation pathway. Use of 2-APB in combination with a novel inhibitor of Ca2+ channels, MDL-12,330A, allowed for a pharmacological profile of SOC, TRP3 and Ca2+ channels activated by S-nitrosylation. Only SOC and TRP3 proved to be sensitive to 2-APB, suggesting that Ca2+ channels activated by S-nitrosylation are either not activated by the InsP3-receptor, or that the InsP 3-receptor is not required for their activation in this manner. MDL-12,330A proved to be a useful tool, not only as a reliable, rapidly reversible antagonist, but also as a direct modifier of Ca2+ channels, providing indirect evidence that a conserved region exists within all of the Ca2+ channels studied. This work taken as a whole implies an important role of internal membranes and inositol signaling in the activation and regulation of SOCaE, in addition to providing evidence that SOC's and TRP's may be closely related, but differentially activated.

Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset familial Alzheimer's disease (FAD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. We describe here the properties of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interactions, GST pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, we show that ubiquilin promotes presenilin protein accumulation, suggesting that it functions in promoting presenilin stability. While overexpression of ubiquilin by itself does not affect cell survival, when coexpressed with PS2, ubiquilin enhances cell death resulting from PS2 overexpression. Our studies suggest that ubiquilin is an important modulator of presenilin function. Moreover, immunostaining of tissue sections from AD brain, Parkinson's disease brain, and diffuse Lewy body disease brain revealed immunoreactive structures to anti-ubiquilin antibodies including senile plaques, Hirano bodies, and Lewy bodies. Since these diseases involve abnormally aggregated protein species, it raises the possibility of a link between ubiquilin and the protein folding or degradation pathways.

Celiac disease (CD) is an autoimmune disease of the small intestine that affects nearly 1% of the general population in the U.S. and in Europe. CD is unique because it is currently the only form of autoimmunity in which both the major genetic (90% HLA-DQ2+, the others HLA-DQ8) and etiologic factors (dietary glutens) for susceptibility are known. Still, the HLA association is thought to account for only 40% of the genetic requirement, and does not explain why most individuals that carry these alleles and consume dietary glutens on a regular basis never develop CD. IL-23 is a recently described cytokine thought to participate in both the innate and adaptive arms of the immune system. While the downstream effects of IL-23 on TH17 cells and their role in disease pathogenesis have been the primary focus of autoimmune research, the agents that initiate its production in the context of autoimmunity remain elusive. Thus, this dissertation investigated the IL-23 response induced by wheat gliadin, the major etiologic agent in CD. The results establish that enzymatically digested gliadin stimulates significantly greater production of IL-23, IL-1beta and TNFalpha and reduced levels of IL-1ra from CD patients compared to HLA-DQ2+ healthy controls, providing the first evidence that IL-23 is involved in the pathogenesis of CD. In addition, CD16+ monocytes were identified as the primary source of IL-23 induced by gliadin and were found to be overrepresented in CD patients, implicating this subset in the disease process. Moreover, these studies present convincing evidence that IL-1 cytokines control IL-23 production in human monocytes and in addition, offer insight on cytokine-dependent and FcR-mediated regulation of IL-1beta and IL-1ra secretion in myeloid-derived cell populations. These novel findings identify a functional role for a number of candidate genes that have been associated with CD and provide information that will guide studies of innate immune pathways in other inflammatory diseases. Importantly, the results of this study may lead to the discovery of therapeutic targets for this disease and other conditions characterized by an imbalance in the ratio of IL-1beta/IL-1ra and/or elevated levels of IL-23, as the two are likely synonymous.

Marginal zone (MZ) B cells are the major cell population in the spleen to respond to T-independent type II antigens (Ti-II) in the blood. However, the evidence regarding the potential role of MZ B cells in response to T-dependent Ag (Td-Ag) is still rudimentary and somewhat controversial, as studies with Pyk-2-/- and RBP-J conditional deficient mice showed opposite results. The current study compared in detail, the responses of MZ and follicular (FO) B cell to a Td Ag, Nitrophenyl-hapten (NP) coupled to chicken gamma globulin (NP-CGG) in an adoptive transfer experiment. The results showed that MZ B cells are the major population to generate early antibody-forming cells (AFC) and they produce about 10-fold higher IgM and slightly higher IgG throughout the entire primary response observed (3 months) compared to FO B cells. In addition, MZ B cells gave rise to germinal center (GC) formation albeit with a delayed onset. GCs from both MZ and FO B cells displayed similar levels of somatic hypermutation (SHM) frequencies, clonal selection, and IgG memory responses. Surprisingly, MZ B cells also produced an IgM memory response. The VH gene repertoire of the early antibody forming cells (AFCs) from MZ B cells is different from that of GCs and late AFCs, suggesting their origins from different precursors. Studies on gene expression profile showed that MZ B cells express some early activation markers both on cell surface, such as B7-2, CD54, and CD44, and intracellularly, such as c-myc, pim-1, and fos proteins, consistent with their rapid response to stimulation. Most importantly, MZ B cells upregulate gene expression of the Blimp-1, which confers MZ B cells the propensity to rapidly develop into plasma cells. In addition, MZ B cells differentially express higher levels of RP105, a LPS receptor on B cells, which may explain the stronger response of MZ B cells to LPS stimulation and the predominant role in Ti responses. In conclusion, MZ B cells are phenotypically and functionally heterogeneous and they have the potential to respond to both Ti and Td Ags.

The polarized expression of disparate transport proteins on two distinct membrane domains is an essential prerequisite for the vectorial transport of water, solutes and ions across epithelia. The renal cortical collecting duct, the site of potassium secretion in the kidney, provides a salient example. In these cells, the asymmetric expression of weakly inward rectifying potassium channels on the apical membrane and strongly rectifying potassium channel on the basolateral membrane increases the fidelity of the secretory process, ensuring that potassium preferentially exits the cell across the apical membrane into the lumen of the tubule in concert with the demands of potassium homeostasis (Giebisch, 1998). The identification of a plausible gene candidate, Kir 2.3, for the basolateral potassium channel (Welling, 1997) provided the impetus to elucidate the basis for polarized membrane targeting of a native channel (Le Maout et al., 1997). The basolateral membrane sorting determinant of Kir 2.3 is comprised of a unique arrangement of trafficking motifs, containing juxtaposing biosynthetic targeting and PDZ-based signals (Le Maout et al., 2001). In the present study, the mechanism by which the PDZ interactions coordinate the basolateral membrane expression of Kir 2.3 was elucidated. In contrast to apical missorting of Kir 2.3 channels lacking the basolateral sorting domain (Le Maout et al., 2001), deletion of the downstream PDZ binding domain causes channels to accumulate in an endosomal compartment. To identify PDZ proteins that functionally interact with the Kir 2.3 sorting signal, the yeast two-hybrid interaction system was employed. Two PDZ proteins that differentially regulate sorting of Kir 2.3 were identified. Consistent with a retention mechanism, one of these PDZ proteins, hLin-7b, couples Kir 2.3 to a multimeric scaffolding complex at the basolateral membrane in epithelial cells. The second PDZ protein, MOPP, has unique structural properties, allowing it to function as a natural dominant-negative PDZ protein. MOPP competes with hLin-7b for interaction with Kir 2.3, thereby regulating basolateral membrane expression of the channel. In conclusion, we propose that basolateral membrane expression of Kir 2.3 is coordinated by the sequential use of different sorting machinery in a multi-step basolateral sorting program.

Despite the precision with which spatial and temporal details of Ca 2+ signals have been resolved, a fundamental aspect of the generation of Ca2+ signals, the activation of "store-operated channels" (SOCs), remains a molecular and mechanistic mystery. The TRP family of receptor-operated channels share several operational parameters with SOCs and the question of whether activation of SOCs and TRP channels is mediated by the inositol-1,4,5-trisphosphate (InsP3) receptor (InsP3R) is examined. The permeant InsP3R-antagonist, 2-aminoethoxydiphenyl borate (2-APB) was previously reported to block SOCs and TRPs in an InsP3R-dependent fashion. Electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated light response is inhibited by 2-APB. This action of 2-APB is likely to be InsP3R-independent since InsP3Rs are dispensable for the light response. We used a triple InsP3R knockout variant line of DT40 chicken B cells to further assess the role of InsP3Rs in SOC and TRP activation. 45Ca2+ flux analysis revealed that only the wild-type DT40 cells retain normal InsP3R-mediated, 2-APB-sensitive, Ca 2+ release. In intact cells, all parameters of Ca2+ store-function and coupling to activate SOCs were identical in DT40 wt and DT40InsP3R-k/o cells. Moreover, in both cell lines SOC-activation is completely blocked by 2-APB with identical kinetics of inhibition. We transiently transfected TRPC3 channels into the DT40 cells, and found that (a) endogenous B-cell receptors (BCR) coupled to phospholipase C-gamma (PLC-gamma); (b) exogenously expressed muscarinic receptors coupled to phospholipase c-gamma (PLC-gamma), and (c) the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) activate the expressed TRPC3 channels in both DT40w/t and DT40InsP3R-k/o cells. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor U73122, and by wortmannin-induced PLC substrate depletion. However OAG-induced TRPC3 activation and store-operated channel activation remained unaffected under these conditions. We found that in the DT40 cells, 2-APB was a powerful InsP3R independent activator of store-operated Ca2+ entry between 1--10 muM. 2-APB activated authentic SOCs since the entry was selective for Ca2+ and highly sensitive to inhibition by La3+. With both w/t and InsP3R-k/o DT40 cells, the actions of 2-APB were restricted to SOCs in a store-coupled state. The results indicate that SOC and TRPC3 activation can occur independent of functional InsP 3Rs, and that in the DT40 cells TRPC3 channels are activated almost entirely by DAG following the stimulation of PLC-beta or -gamma. Furthermore, the biphasic effects of 2-APB reflect activation of authentic SOCs and 2-APB appears to modify SOCs by targeting the elusive coupling mechanism.

Germline mutations of the breast cancer associated gene 1 (BRCA1 ) predispose women to breast and ovarian cancers. In mice, loss of function mutations of Brca1 results in recessive lethality during gestation. Analysis of these mutant embryos revealed that Brca1 plays essential roles in maintaining genomic integrity. However the mechanisms underlying these defects remain to be illustrated. To further define the processes through which BRCA1 maintains genomic integrity, meiosis in mutant mice that are homozygous for a targeted deletion of Brca1 exon 11 (Brca1Delta11/Delta11) was studied. Brca1Delta11/Delta11 embryos died at early gestation, but survived to adulthood if either one or both wild type p53 alleles were also mutated. This study showed that all Brca1Delta11/Delta11 p53+/- and Brca1 Delta11/Delta11p53-/- males were infertile and females exhibited decreased fertility. Mutant males had significantly reduced testicular size and contain no spermatozoa even though mutant testes contained spermatogonia, stem cells, and early stage primary spermatocytes; in their seminiferous tubes. Chromosomal analyses on the spermatocytes indicated that mutant homologous chromosomes paired and advanced to the pachytene stage, however mutant mice were unable to form chiasma, and no spermatocytes developed to the diplotene stage. The block of spermatogenesis occurred right after homologous chromosome pairing at meiosis I. Brca1-Delta11 mutation decreased the frequency of Mlh1 foci formation at crossover sites, affected Rad51 foci formation, and interfered with the relocalization of gamma-H2AX from sites of DNA double strand breaks to the XY body during spermatogenesis. The expression levels of a number of genes that are involved in DNA damage repair were reduced including helicase and silent information regulatory proteins. Termination of spermatogenesis at pachytene stage was accompanied by increased rates of apoptosis. cDNA microarray analyses demonstrated changes in expression of genes involved in both p53-dependent and -independent apoptotic pathways. In conclusion, this study revealed an essential role for Brca1 in meiotic recombination repair and regulation of genetic exchange of homologous chromosomes during spermatogenesis. Loss of Brca1 function led to the accumulation of DNA damage, genomic instability, activation of both p53-dependent and -independent apoptotic pathways and, eventually, failure of spermatogenesis.

Microfluorimetric, electrophysiological, pharmacological, and intracellular photorelease techniques were used to study Ca2+ physiology in rabbit vagal sensory neurons (nodose ganglion neurons, NGNs). NGNs exhibit robust Ca2+-induced Ca2+ release (CICR) that can be triggered by caffeine, the classic CICR agonist. A caffeine-induced increase in cytosolic free Ca2+ concentration ([Ca2+ ]i) was traditionally taken as diagnostic of the existence of CICR. However, when CICR was disabled through depletion of intracellular Ca2+ stores or pharmacological blockade of intracellular Ca 2+ release channels (ryanodine receptors, RyRs), caffeine still elicited a significant rise in [Ca2+]i in ∼50% of NGNs. We demonstrated that this rise in [Ca2+]i results from Ca2+ influx, and that Ca2+ influx is one component of a non-selective cation current that is activated by caffeine. Therefore, in approximately half of all NGNs, caffeine elicits both CICR and Ca2+ influx. We determined that d-myo-inositol 1,4,5-trisphosphate receptors (IP3Rs), another type of intracellular Ca2+ release channel, coexist with RyRs in NGNs. ATP, an extracellular physiological signaling molecule, consistently evoked robust transient increases in [Ca 2+]i (Ca2+ transients) that have a dual origin: (1) Ca2+ influx via P2X receptors and voltage-gated Ca2+ channels; and (2) intracellular Ca2+ release, via IP3Rs and RyRs, that requires activation of P2Y receptors and phospholipase C. We determined that Ca2+ transients evoked by IP3 photorelease can gate a Ca2+-activated K+ current (I IP3) in NGNs. IIP3 is unaffected by three common antagonists of Ca2+-activated K+ currents: iberiotoxin, apamin, and 8-Br-CAMP. Using caffeine to selectively deplete intracellular Ca2+ stores, we show that while CICR does not contribute significantly to global IP3-evoked Ca2+ transients, surprisingly, ∼20% of IIP3 is activated by CICR. We propose a model of Ca2+ signaling microdomains that rationalize these observations. We showed that ATP can evoke a Ca2+-activated K + current in 57% of NGNs. ATP-evoked Ca2+ influx activates ∼75% of this current, while ATP-evoked intracellular Ca2+ release activates the other ∼25%. We also determined that NGNs express P2X receptors that mediate robust influx, therefore our data suggest that ATP can exert both excitatory and inhibitory effects in NGNs.

Neurotransmission at the neuromuscular junction involves the release of acetylcholine from the presynaptic nerve terminal, and following its action at the postsynaptic receptor, termination by the serine hydrolase, acetylcholinesterase (AChE). AChE is tightly regulated by muscle and nerve activity, in part through elicitation of muscle contractions. Denervation increases AChE activity in chicken muscle but decreases it in rat muscle. The aim of this dissertation was to delineate the role of transcriptional activation in the regulation of AChE by identifying genomic elements involved in the transcriptional regulation of AChE by muscle contraction in vitro and in vivo . The in vitro paradigm used rat primary muscle cultures that were transfected with genomic fragments containing flanking sequences of the AChE promoter, driving the expression of a luciferase reporter gene, and either electrically stimulated to enhance contractile activity or treated with the Na+ channel blocker, tetrodotoxin, to inhibit contractions. One cis-element within intron I, an N-box, was necessary for the response to contraction and its mutagenesis or co-transfection with a dominant negative form of GABP, a transcription factor that binds to the N-box, eliminated the response to contraction. Another cis-element involved in regulation of AChE was identified in the region upstream of the basal promoter as a binding site for NFAT, a transcription factor implicated in the regulation of gene expression by contractile activity. Overexpression of NFAT in muscle cultures transfected with AChE reporter constructs containing this element increased reporter activity and probes containing this element interacted with NFAT in an in vitro binding assay. Studies in vivo employed transient transfection by intramuscular plasmid injection in innervated and denervated muscle of mice and chicks. These studies demonstrated that intron I is necessary for decreased reporter activity in response to denervation and that the differential response between mice and chicks is due to different cis-elements. Together, these findings provide clear support that AChE transcription is regulated by muscle contraction and that elements within intron I as well as the upstream regulatory region play a role in the regulation.