The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant natural killer T (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. Here, we report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 (B6) mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFNγ, which we demonstrate controls the severity of murine Lyme carditis by at least two mechanisms. First, IFNγ greatly enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Secondly, IFNγ activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate acute murine Lyme carditis through the action of IFNγ, which appears to self-renew through a positive feedback loop during infection. Inflammation during infection with B. burgdorferi is dependent on the ability of the spirochete to evade local mechanisms of clearance. Even though macrophages are the main infiltrating cell during Lyme carditis, the identification of a receptor capable of mediating phagocytosis of B. burgdorferi has been elusive. Here, we demonstrate that the integrin CR3 is able to mediate binding to the spirochete and facilitate phagocytosis in a complement-dependent and independent manner. Expression of CR3, but not CR4, in CHO cells markedly enhanced their capacity to interact with B. burgdorferi, in the absence and presence of complement opsonization. Furthermore, the interaction between CR3 and B. burgdorferi is dependent on the metal-ion-dependent adhesion site (MIDAS) and could be blocked with EDTA. Inhibition of CR3 with blocking antibody was able to completely abrogate phagocytosis of B. burgdorferi by the macrophage-like RAW264.7 cells and partially block uptake by bone marrow-derived macrophages (BMMs), a finding that was recapitulated with CD11b-deficient BMMs. We further show that activation with recombinant IFNγ increases the transcription of CD11b and CD18, which correlates with increased surface expression of CR3, and that the effect of IFNγ on the phagocytosis of B. burgdorferi is circumscribed to CR3 activity, because inhibition of CR3 is able to completely diminish the effect of IFNγ on the phagocytosis of the B. burgdorferi. Lastly, our results demonstrate that CR3 is a negative regulator of proinflammatory cytokine induction in macrophages responding to B. burgdorferi. Overall, our data demonstrate roles for CR3 in the binding, phagocytosis and proinflammatory cytokine elicited by B. burgdorferi and shed light on the role of IFNγ in mediating the clearance of the spirochete during Lyme disease.

Mouse metaphase II (MII)-stage eggs exhibit oscillatory Ca2+ responses ([Ca2+]i oscillations) following fertilization. The wealth of information regarding Ca2+ signaling pathways in eggs has allowed these cells to become an ideal model system for the study of general Ca2+ signaling pathways. This dissertation provides data that contribute to the elucidation of the mechanism that culminates in Ca2+ release at fertilization, and to our understanding of the functional regulation of the inositol 1,4,5-trisphosphate receptor (IP 3R) using the mouse egg as a model system. We first present data indicating that injection of mouse eggs with porcine sperm factor (SF) induces [Ca2+]i oscillations through activation of a phospholipase C (PLC). U73122, a PLC inhibitor, prevented SF-induced [Ca2+]i oscillations whether SF or eggs were treated with the inhibitor. We also show that SF injection elicits inositol 1,4,5-trisphosphate (IP3) production and Ca2+ release in single Xenopus oocytes. Thus, SF induces [Ca2+]i release by stimulating the phosphoinositide pathway. We next show that KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, antagonizes IP3R function independently of effects on CaMKII in mouse eggs and permeabilized A7r5 cells. This inhibition is not due to a block of IP3 production, Ca2+ store filling, or IP3 binding to the IP3R. KN-93 interferes with Ca2+-induced Ca2+ release by the IP 3R, indicating that KN-93 may prevent the ability of IP3 and/or Ca2+ to induce activatory conformational changes to the IP3R. KN-93 directly interacts with and alters the conformation of the IP3R, based on in vitro and in vivo proteolysis experiments. Finally, KN-93 does not interact with the IP3R via a calmodulin binding site, as hypothesized based on its mechanism of CaMKII inhibition. Lastly, we present preliminary data toward the development of an IP 3R overexpression system in mouse eggs. We successfully generated enhanced yellow fluorescent protein (eYFP)-tagged murine IP3R-1 mRNA in vitro. Injection of eYFP-IP3R-1 mRNA into mouse eggs resulted in expression of exogenous IP3R-1 protein. This system will provide new opportunities for the use of the mouse egg as a model system for the study of IP3R signaling, and may help facilitate the elucidation of the mechanism by which KN-93 inhibits the IP3R.

Cell adhesion to an extracellular matrix plays a critical role in many aspects of normal cell function. Cells display various modes of interaction with the extracellular matrix; they may attach and spread, become immobilized, or become motile. These cellular responses are regulated by intracellular signals, which modify the organization of the cytoskeleton. One common characteristic of malignantly transformed cells is alteration in one or more aspects of adhesion. Most notably, cancer cells often display enhanced motility and there is a positive correlation between cell mobility and metastatic potential in situ. HeLa cells, a cell line derived from a cervical carcinoma, were used as a model system for this investigation. It has been shown, in HeLa cells, that cell attachment to a gelatin-coated substrate results in the release of arachidonic acid, which is metabolized by lipoxygenase. A subsequent cascade of lipid second messengers activates protein kinase C, which triggers actin polymerization leading to cell spreading. This work employed inhibitor studies, and biochemical analysis to elucidate a parallel branch of arachidonic acid signaling that reorganizes the actin cytoskeleton into small bundles. This branch of the pathway is initiated by cyclooxygenase, which generates prostaglandins and causes the downstream activation of cyclic AMP-dependent protein kinase. The results suggest that arachidonic acid functions at a branch point in signaling to the cytoskeleton. The lipoxygenase branch provides polymerized actin; the actin filaments then act as a substrate for the cylooxygenase branch to generate actin bundles. These actin bundles were shown to associate with myosin and small adhesion complexes. Activation of cyclooxygenase signaling and the subsequent cytoskeletal organization were found to increase cell motility. Overexpression of the small GTPases rho and cdc42, also induces cell crawling, and these signaling molecules seem to interact with cyclooxygenase in directing organization of the cytoskeleton. In sum these results suggest that faulty regulation of arachidonic acid signaling can result in the pathological cell motility that characterizes the most aggressive cancers.

The zygotic centrosome, contributed by the sperm, forms an array of microtubules that spread throughout the ooplasm. These microtubules are used by the female pronucleus to come into juxtaposition with the male pronucleus in order to form the zygotic nucleus. Zygotic centrosome regulation has been shown to involve an MPM-2 phosphoepitope that is dephosphorylated prior to aster formation. The MPM-2 reactive candidate appears as a triplet of proteins with MWapp of 85, 81, and 77 kDa on SDS-PAGE. The objectives of this work were to investigate the relationship among the triplet proteins, determine physical characteristics of the MPM-2 reactive protein, and investigate potential kinases that may act on the MPM-2 target epitope. Amino acid analysis and peptide mapping show that the triplet proteins are nearly identical with all of the peptide fragments of the smaller proteins contained in the largest of the triplet. Physical properties, immunoreactivity, and isolation characteristics demonstrate that the reactive protein is part of the outer dense fiber/segmented column complex. The protein is highly insoluble in aqueous solutions and requires a reducing agent and a chaotropic agent, such as urea, to remain in solution. Kinase studies reveal that the MPM-2 reactive protein is maintained in a phosphorylated state by a kinase that is Maturation Promoting Factor (MPF) or dependent on MPF. Further, the epitope is dephosphorylated coincident with a drop in MPF in activated oocyte extract while MAP kinase levels remain high. This information indicates that the MPM-2 reactive sperm protein is part of the segmented columns or dense fibers and likely exerts its influence on the zygotic centrosome indirectly, perhaps through blocking. The epitope on the protein appears to be regulated through the action of kinases that maintain the phosphorylation of the protein. Dephosphorylation is thought to occur by ubiquitous phosphatases that are favored upon deactivation of MPF and downstream kinases.

The chloroplast envelope plays critical roles in the synthesis and regulated transport of key metabolites, including intermediates in photosynthesis and lipid metabolism. Despite this importance, the biogenesis of the envelope membranes has not been investigated in detail. To identify the determinants of protein targeting to the inner envelope membrane (IM), I investigated the targeting of the nucleus-encoded integral IM protein, atTic40. I found that pre-atTic40 is imported into chloroplasts and processed to an intermediate size (int-atTic40) before insertion into the IM. Int-atTic40 is soluble and inserts into the IM from the internal stromal compartment. I also show that atTic40 and a second IM protein, atTic110, can target and insert into isolated IM vesicles in vitro. These in vitro studies are further supported by in vivo evidence showing pre-atTic40 directly engineered into the chloroplast genome and expressed within chloroplasts can efficiently target to the inner envelope membrane. Taken together, my experiments are consistent with a "post import" mechanism in which the IM proteins are first imported from the cytoplasm and subsequently inserted into the IM from the stroma.

Cytokinesis in plant cells involves the formation and insertion of a new cell wall that separates daughter nuclei after mitosis. The deposition of the new wall is regulated by a structure called the phragmoplast which contains microtubules, actin filaments and membranes. The experiments described in this thesis, emphasize the participation of the actin filaments in the cytokinetic process. Caffeine inhibition of cytokinesis shows that two distinct phases cell plate development can be distinguished; an early initiation phase and a late lateral expansion phase. Caffeine completely inhibits the latter stage. I have looked at the dynamics of actin filaments, as well the microtubules, during the inhibition of cytokinesis by caffeine. I found that formation of the cytoskeletal torus, associated with the later stages of cell plate formation, is completely inhibited in the presence of caffeine. The functional role of actin filaments was probed by microinjection of the actin monomer binding protein profilin. The results show a dose-dependent effect on cytokinesis, in which the effects range from delayed cell plate formation to complete inhibition. To asses the localization of profilin, I microinjected a fluorescent profilin probe into living cells and analyzed the localization throughout mitosis. Profilin is accumulated in the nucleus during interphase and prophase and is released in the accessible volume after nuclear envelope break down at the onset of metaphase. It remains in the accessible volume during the subsequent mitotic stages until it re-accumulates in the telophase nuclei. No specific localization of profilin in the phragmoplast was observed. Also, the role of actomyosin in plant cytokinesis was studied by using the myosin ATPase inhibitor, 2,3-butanedione monoxime (BDM). Cytokinetic cells, treated with BDM, displayed buckled and thin cell plates that were often tilted at a significant angle. In addition, late lateral cell plate expansion was inhibited and actin filaments in the phragmoplast lost their proper orientation. These results show that actomyosin plays a crucial role in cell plate alignment and late lateral expansion of the cell plate. Finally, a new cell plate formation model is presented that combines some of the results of this thesis with existing models and previous findings.

During fertilization the sperm activates the mammalian egg by eliciting [Ca2+]i oscillations. However, how the sperm triggers [Ca2+]i oscillations in mammalian eggs remains unknown. To test the possibility that a factors) from the sperm is able to elicit [Ca 2+]i oscillations, sperm fractions (factors) were prepared from different species and injected into mammalian oocytes or eggs. The results show that injection of sperm factor from either porcine or human sperm triggered long-lasting [Ca2+]i oscillations in mouse oocytes and bovine eggs in a pattern similar to the physiological responses observed in each of these species, implying that the sperm factor is functionally conserved among mammalian species. In addition, sperm factor-induced [Ca2+] i oscillations appeared to be mediated by the IP3 receptor, while the ryanodine receptor may be involved in the modulation of these oscillations. Furthermore, [Ca2+]i oscillations induced by sperm factor are capable of initiating normal egg activation and parthenogenetic development. In order to isolate and characterize the unknown active molecule(s) in sperm factor, different tissues or cell extracts were screened for the presence of sperm factor like activity. It appears that the [Ca2+] i oscillation-inducing activity is sperm/testis specific. In addition, the results showed that the active component contains a protein moeity and that the same single active component of porcine sperm factor is present in both soluble and in less soluble sperm compartments. We also demonstrated that gpd/oscillin, a proposed active component of mammalian sperm factor, is not responsible for the [Ca2+]i oscillation-inducing activity as shown by the lack of effects on Ca2+ responses or absence in active fractions following immunodepletion or a combination of fractionation techniques. Similarly, our results also showed that neither PLCγ1, PLCγ2 nor tr-c-Kit is likely to be the active component of sperm factor. Although we were unable to identify a specific candidate molecule, these experiments have led to the identification of three polypeptides in the final active fraction after sequential chromatographic steps. Our results also show that the active component has an isoelectric point of 6.5–7.0 and a relative molecular weight ranging from 29–68 kDa. These polypeptides will be sequenced and used to raise monoclonal or polyclonal antibodies, which will be used to screen a testis cDNA library. This last step is expected to lead to the isolation of the gene encoding sperm factor.

Microtubules are required for several cellular processes including vesicle transport, cytoplasmic organization, cell motility, maintenance of cellular polarity, and mitosis. The mechanisms by which microtubule arrays are established, maintained and remodeled are important for understanding these vital processes. I have used fluorescence analog chemistry and live cell imaging approaches in mammalian cells to address these questions. I have documented the de novo formation of microtubules in peripheral regions of epithelial-like cells, at sites distant from the centrosome, challenging the traditional doctrine that microtubules are nucleated solely at microtubule organizing centers (MTOCs). Historically, the behavior of minus ends has been difficult to study in vivo, due to the high density of microtubules in central cellular regions; however, the peripheral position of the non-centrosomal microtubules allowed me to quantify the dynamic behavior of both microtubule ends. The results demonstrate that individual minus ends are remarkably stable, suggesting that their stability is either intrinsic to their structure or is the result of a molecular cap. During cell motility, microtubules must populate the advancing lamella and be removed from retracting regions; however, the mechanisms that cells utilize to remodel microtubule arrays are complex and poorly defined. My experiments, using photoactivated fluorescent tubulin to mark the microtubule lattice, demonstrate that microtubules are transported in motile cells, and that transport is a two component process. The first component is the unidirectional transport of microtubules in the direction of the dominant actomyosin-generated contractile force; the second is the bidirectional movement of individual microtubules. In cells with numerous noncentrosomal microtubules, transport is likely to play a significant, and previously unrecognized, role in microtubule reorganization. The mechanism of the bidirectional component was analyzed; inhibition of myosin II abolished microtubule movement, while inhibition of cytoplasmic dynein increased it. In addition, the absence of myosin function resulted in slower turnover of the microtubule array, which is the first direct evidence for myosin-generated forces modulating the kinetic behavior of microtubules. The results support a role for cytoplasmic dynein in tethering microtubules and resisting actomyosin-generated forces, suggesting that the antagonistic forces of these motors contribute to the organization of the microtubule array.

Immature or incompletely assembled proteins that do not fold correctly are retained in the endoplasmic reticulum (ER) by quality control factors. Terminally misfolded glycoproteins are eventually sorted for dislocation out of the ER and ubiquitinated, leading to degradation by the 26S proteasome in a process termed ER-associated degradation (ERAD). Mannose-trimming of glycans has been proposed to act as a sorting mechanism for the degradation of misfolded glycoproteins. EDEM1 (ER degradation-enhancing α-mannosidase-like 1) is hypothesized to extract misfolded proteins out of the calnexin cycle and sort them for degradation by recognizing a mannose-trimmed glycan. This extraction is proposed to be assisted by a direct interaction between the transmembrane domain of calnexin and a putative transmembrane domain of EDEM1. To investigate the role of EDEM1 in the quality control and degradation of misfolded glycoproteins, initially, the fundamental properties of EDEM1 were characterized. We observed that endogenous EDEM1 matures to a soluble protein that is heterogeneously glycosylated. This predominantly soluble phenotype was in disagreement with the proposed mechanism for EDEM1 extraction of misfolded glycoproteins from the calnexin binding cycle. EDEM1 binding to misfolded proteins has been proposed to be mediated through the presence of a mannose-trimmed glycan on the misfolded substrate. After establishing an EDEM1 binding assay, we found that EDEM1 bound transiently associated with misfolded glycoproteins in a glycan-independent manner. EDEM1 was also found to bind SEL1L, an ERAD dislocation and ubiquitination complex adapter glycoprotein. Inhibition of mannose trimming with kifunensine or disruption of the EDEM1 mannosidase-like domain by mutation had no effect on EDEM1 substrate binding, but diminished its association with the SEL1L. Therefore, we propose a model where EDEM1 binds to misfolded glycoproteins in a glycan-independent manner and delivers the non-native cargo to the ERAD dislocation and ubiquitination complex using its mannosidase-like domain, which associates with SEL1L. This investigation presents an alternative hypothesis of the function of EDEM1 in the degradation of misfolded glycoproteins while also emphasizing the importance of glycans in the degradation of misfolded glycoproteins.

Hic-5 is a focal adhesion protein of paxillin superfamily that was initially cloned from mouse osteoblasts as a TGF-β or H2O2 inducible cDNA. As well, Hic-5 was independently identified as an Androgen receptor activator (ARA55). Conflicting data have implicated Hic-5 in opposing processes. With two Hic-5 isoforms documented, I hypothesized that multiple Hic-5 isoforms may exist that have both overlapping and isoform-specific functions, which may explain those discrepancies. To test this hypothesis, I have utilized C2C12 myoblasts and analyzed the roles of Hic-5 isoforms in development and homeostasis. 1. I have confirmed the presence of the two previous reported Hic-5 isoforms (α and β) and uncovered 10 additional novel Hic-5 transcripts. Conceptually translated proteins from these transcripts significantly differ at the N-terminal region and likely have distinct binding properties and functions. Hic-5 isoforms have distinct tissue distribution and are developmentally regulated in the mouse mammary gland in vivo (Chapter two). 2. I found that: (a) myoblasts express multiple Hic-5 isoforms; (b) the two predominant isoforms, Hic-5α and Hic-5β, are differentially expressed during myogenesis; (c) any experimentally-induced change in Hic-5 expression results in a substantial increase in apoptosis during differentiation; (d) ectopic expression of Hic-5α is permissive to differentiation while expression of either Hic-5β or antisense Hic-5 reduces myoblast chemo-differentiation and blocks fusion; (e) Hic-5 localizes to focal adhesion in C2C12 myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (f) perturbations of Hic-5 expression interfere with the normal expression dynamics of laminin; and (g) the rescue of myoblast survival and differentiation by laminin but not fibronectin suggests that Hic-5 isoforms differentially regulate myogenesis due to their different impacts on cell-ECM interaction, focal adhesion dynamics and integrin signaling (Chapter Three). In summary, the roles Hic-5 may assume in development and homeostasis are complex and the different Hic-5 isoforms may mediate distinct physiological and/or pathological responses in cells. Therefore, a more precise analysis of Hic-5 isoforms is required to more fully understand the roles of not only Hic-5, but also integrin signaling in normal and diseased cells ( Chapter four and five).