The expression of C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with increased metastatic potential of cancer cells. CXCR4 increases tumor malignancy by encouraging tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis provides a good strategy to inhibit the metastatic spread of tumor cells and slow cancer progression. Various studies suggest that cannabis may have anti-proliferative as well as anti-metastatic properties, though a biochemical mechanism describing how this occurs has yet to be discovered. Our lab has confirmed that agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) can form heterodimers that play a role in decreasing cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231 and the prostate cancer cell line PC-3, with CXCL12 and AM1241, a synthetic ligand for CB2, desensitizes the intrinsic cellular response to migrate toward areas of high CXCL12 concentration. Furthermore, through co-immunoprecipitation and proximity ligation assays (PLA), we have determined that there is increased interaction between the two receptors with co-stimulation of respective agonists, providing evidence for the therapeutic notion that treating tumors that endogenously secrete CXCL12 with exogenous ligands for the cannabinoid can induce dimerization. Moreover, when CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Finally, to determine whether simultaneously–treated, dimerized receptors inhibited activity of respective receptors, calcium mobilization assays to determine G-protein coupled receptor activation were employed. Results showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treated with their individual ligands. Phosphorylation of ERK and AKT were abrogated in response to simultaneous stimulation indicating loss in downstream signaling. Therefore, we believe that the interaction of CB2 with CXCR4 may play a role in inhibiting the cells response to CXCL12, leading to a loss in metastatic potential of cells expressing these receptors.

Pericentrin is a molecular scaffold protein. It anchors protein kinases, (PKB, (Purohit, personal communication), PKC, (Chen et al., 2004), PKA Diviani et al., 2000), the γ tubulin ring complex, (γ TuRC) (Zimmerman et al., 2004), and possibly dynein (Purohit et al., 1999) to the spindle pole. The γ TuRC is a ~ 2 MDa complex which binds the minus ends of microtubules and nucleates microtubules in vitro, (Zheng et al., 1995). Prior to this work, nothing was known about the association of the γTuRC with pericentrin. Herein I report the biochemical identification of a large protein complex in Xenopus extracts containing pericentrin, the γ TuRC, and other as yet unidentified proteins. Immunodepletion of γ tubulin results in co-depletion of pericentrin, indicating that virtually all the pericentrin in a Xenopus extract is associated with γ tubulin. However, pericentrin is not a member of the, γ TuRC, since isolated γ TuRCs do not contain pericentrin. The association of pericentrin with the γ TuRC is readily disrupted, resulting in two separable complexes, a small pericentrin containing complex of approximately 740 KDa and the the γ TuRC, 1.9 MDa in Xenopus. Co overexpression/ coimmunoprecipitation and yeast two hybrid studies demonstrate that pericentrin binds the γTuRC through interactions with both GCP2 and GCP3. When added to Xenopus mitotic extracts, the GCP2/3 binding domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly and induced rapid disassembly of pre-assembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding, and were specific for mitotic centro somal asters as I observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Overexpression of the GCP2/3 binding domain of pericentrin in somatic cells perturbed mitotic astral microtubules and spindle bipolarity. Likewise pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. I conclude that pericentrin anchoring of γ tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death. Additionally, I provide functional and in vitro evidence to suggest that the larger pericentrin isoform (pericentrin B/ Kendrin) is not functionally homologous to pericentrin/pericentrin A in regard to it's interaction with the γ TuRC.

In response to the growing rates of antibiotic resistance in human bacterial pathogens, this study explores the natural products involved in the defensive symbiosis between actinobacteria and fungus-growing ants to uncover new potential antibiotics. This study also seeks to understand the function of natural antibiotics in their ecological contexts, especially those involved in defensive symbioses. Defensive symbiosis can be a beneficial platform for discovering useful antibiotics, because antibiotics in these relationships must be able to selectively inhibit enemies without harming hosts, and are therefore likely more specific and less toxic. Pseudonocardia sp. associated with Trachymyrmex septentrionalis ants demonstrated antibiotic activity against several gram-positive bacteria. Therefore, the natural products from this strain were extracted and purified through activity-guided fractionation. Using mass spectrometry, the structure of the active compound was elucidated as GE37468A, an antibiotic that has been previously identified from Streptomyces sp. ATCC 55365 from Italy. This compound had never before been characterized in a defensive symbiosis, which demonstrates the use of the molecule in a new context. Antibiotic GE37468A is a thiopeptide, which is a group of antibiotics that has previously demonstrated strong activity against many gram-positive bacteria, including bacterial human pathogens. Due to its potency against dangerous bacteria and its likely low toxicity, this antibiotic could therefore hold potential pharmacological uses.

Voltage-gated K+ channels associate with multiple regulatory proteins to form complexes with diverse gating properties and pharmacological sensitivities. Small molecules which activate or inhibit channel function are valuable tools for dissecting the assembly and function of these macromolecular complexes. My thesis focuses on the discovery and use of small molecules to probe the structure and function of the KCNQ family of voltage-gated K+ channels. One protein that obligatorily assembles with KCNQ channels to mediate proper assembly, trafficking, and gating is the calcium sensor, calmodulin. Although resolution of the crystal structures of calmodulin associated with isolated peptide fragments from other ion channels has provided some insight into how calmodulin interacts with and modulates KCNQ channels, structural information for calmodulin bound to a fully folded ion channel in the membrane is unknown. In Chapter II, I developed an intracellular tethered blocker approach to determine the location of calmodulin binding with respect to the KCNQ ion-conducting pathway. Using distance restraints from a panel of these intracellular tethered blockers we then generated models of the KCNQ-calmodulin complex. Our model places calmodulin close to the gate of KCNQ channels, providing structural insight into how CaM is able to communicate changes in intracellular calcium levels to KCNQ channel complexes. In addition to pore blockers, chemical modification of ion channels has been used to probe ion channel function. During my initial attempt to chemically activate KCNQ channels, I discovered that some boronates modulate KCNQ complexes. In Chapter III, the activating derivative, phenylboronic acid, is characterized. Characterization of activation by phenylboronic acid showed that it targeted the ion conduction pathway of KCNQ channels with some specificity over other voltage-gated K+ channels. The commercial availability of thousands of boronic acid derivatives provides a large class of compounds with which to systematically dissect the mechanisms of KCNQ gating and may lead to the discovery of a potent activator of KCNQ complexes for the treatment of channelopathies. All of the electrophysiological studies presented in this thesis were conducted in Xenopus oocytes. Unexpectedly, during the studies described above, the quality of our Xenopus oocytes declined. The afflicted oocytes developed black foci on their membranes, had negligible electric resting potentials, and poor viability. Culturing the compromised oocytes determined that they were infected with multi-drug resistant Stenotrophomonas maltophilia, Pseudomonas fluorescens and Pseudomonas putida. Antibiotic testing showed that all three species of bacteria were susceptible to amikacin and ciprofloxacin, which when included in the oocyte storage media prevented the appearance of black foci and resulted in oocytes that were usable for electrophysiological recordings. This study provides a solution to a common issue that plagues many electrophysiologists who use Xenopus oocytes. Taken together, these findings provide new insights into activation of KCNQ channel complexes and provide new tools to study the structure-function relationship of voltage-gated K+ channels.

Dynactin is a multi-subunit complex which was initially identified in 1991 as an activator of cytoplasmic dynein-driven microtubule-based organelle motility in vitro. Although genetic studies also supported the involvement of both complexes in the same functional pathways in yeast, filamentous fungi, and Drosophila, none of these findings yielded significant insights into dynactin's mechanism of action. The full range of cytoplasmic dynein functions in vertebrate cells has also remained poorly understood, due, in large part, to the lack of a specific method of inhibition. The present thesis work was designed to investigate these issues through a study of the 50 kDa subunit of dynactin. As a first step (Chapter 1), I cloned mammalian p50 and characterized its expression at the tissue and subcellular levels. Rat and human cDNA clones revealed p50 to be a novel α-helix-rich protein containing several highly-conserved structural features including one predicted coiled-coil domain. Immunofluorescence staining of p50, as well as other dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase and concentrate near spindle poles thereafter. These findings represented the first evidence for dynactin and cytoplasmic dynein co-localization within cells, and for the presence of dynactin at kinetochores. The second major phase of the thesis (Chapter 2) was focused on investigating dynactin and cytoplasmic dynein function in cultured cells in vivo using a dominant negative inhibition approach based on transient transfections of p50 constructs. Overexpression of wild type human p50 in cultured cells resulted in a dramatic fragmentation and dispersal of the Golgi apparatus. Time-lapse fluorescence microscopy analysis of p50-overexpressing cells revealed that microtubule-based vesicle transport from the endoplasmic reticulum to the Golgi was inhibited. Also, the interphase microtubule organizing center was found to be less well-focused in some but not all transfected cells. Overexpression of p50 also disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still generally bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings provided the first in vivoevidence for the role of dynactin in cytoplasmic dynein function, i.e. mediating the motor's binding to at least one "cargo" organelle, the kinetochore, and probably also to others such as vesicles destined for the Golgi complex. These data also strongly implicated both dynactin and dynein in Golgi organization during interphase, and chromosome alignment and spindle organization during mitosis. Based on the remarkable disruptive phenotypic effects associated with overexpressing of p50, the name of dynamitin was proposed for this polypeptide. In the third and last phase of the thesis (Chapter 3), two issues were addressed: first, the dynamitin-induced mitotic arrest phenotype was studied in greater detail to better understand the exact sites of dynactin and cytoplasmic dynein activity throughout mitosis. Second, a domain analysis of dynamitin was performed to gain insight into its function within the dynactin complex. A time-lapse fluorescence microscopy study of mitosis in living dynamitin-overexpressing COS-7 cells strongly suggested specific defects in interactions of astral microtubules with the cell cortex, and in both spindle pole assembly and maintenance. Analysis of the mitotic arrest phenotype in a second cell line revealed a second arrest point at metaphase, and a clear effect of dynamitin overexpression on spindle axis orientation, again consistent with defects in interactions between microtubules and the cell cortex. Refined analyses of kinetochore and spindle pole components also confirmed specific defects in kinetochore function and spindle pole organization. Taken together, these findings support three main sites of dynactin and cytoplasmic dynein activity during vertebrate mitosis: prometaphase kinetochores, spindle poles, and the cell cortex. Finally, the domain analysis revealed dynamitin to be capable of self-association through at least two separate interaction domains, consistent with models of the mechanism underlying dynamitin-induced dynactin dissociation, and therefore, yielding important new insights into dynactin assembly. This study also indicated that a third region within dynamitin, residues 105 to 154, is essential for dynamitin and dynactin function. An independent study confirmed this finding, implicating this region in binding to ZW10, an upstream kinetochore protein. Dynamitin has therefore been revealed to be the kinetochore-targeting subunit of dynactin, and indirectly, cytoplasmic dynein. Through the body of this thesis work, dynamitin has also emerged as a powerful new tool for studying vertebrate dynactin and cytoplasmic dynein function in vivo and in vitro.

Hindbrain patterning requires many factors involved in early segmentation and later segment identity of the specific domains of the hindbrain. Hox proteins and their cofactors are of great importance during segmentation of the hindbrain, because segmentation and/or segment identity are lost when any of them are lost. Previously, we have reported that Meis proteins synergize with Pbx, another Hox cofactor, and Hox proteins expressed in the hindbrain. To further investigate Meis function during hindbrain development, we utilized a Meis dominant-negative molecule, ΔCPbx4, and expressed it in zebrafish embryos. We find that ΔCPbx4 affects gene expression and neuronal differentiation especially in r3 through r5. Further, we combined ΔCPbx4 with another Meis dominant-negative molecule (ΔHDCMeis) to disrupt Meis function more extensively. Under these conditions, we find that the entire hindbrain loses gene expression as well as its complement of neuronal differentiation. This phenotype is strikingly similar to that of loss of Pbx function, suggesting that Meis proteins act in the same pathway as Pbx. Therefore, Meis family proteins are indispensable for the entire hindbrain segmentation. In addition to the milder effect on hindbrain patterning, we also found upon expressing ΔCPbx4 that the caudal hindbrain transforms to r4-like fates, supported by expression of r4-specific marker gene (hoxbla) and specification of r4-specifc Mauthner neurons in the domain. This phenotype is not reported upon loss of Pbx function, suggesting that Meis proteins may play a more modulatory role, while Pbx is absolutely required during hindbrain development. Through several in vivo assays, we find that this r4 transformation is induced by Hox PG1 proteins and that vhnf1 represses r4 fates in the caudal hindbrain to further specify caudal fates in this region. Based on these results, we propose a model by which hindbrain patterning is achieved. Initially, un-segmented hindbrain is segmented into two domains wherein the caudal domain displays an r4 fate. This caudal r4 fate is then repressed by vhnf1 function which restricts the r4 fate to the presumptive r4 domain and specifies r5 and r6 by inducing its downstream genes such as valentino and hox PG3. Taken together, we conclude that Meis family proteins are essentially involved in function of Hox complexes to specify distinct rhombomeres during segmentation of the zebrafish hindbrain.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.

Author did not provide abstract.

The large conductance Ca++-activated K+ channel (BKCa) regulates neuronal excitability through the efflux of K+, in response to membrane depolarization and increases in intracellular Ca++. The activity of the BKCa channel is increased by acute exposure to ethanol (EtOH), which is thought to underlie, in part, the influence of the drug on peptide hormone release from neurohypophysial nerve terminals (Dopico et al., 1996, 1998). Moreover, chronic EtOH exposure attenuates acute drug action on hormone release, and reduces the sensitivity of BKCa channels to acute EtOH exposure (Knott et al., 2002). The factors regulating EtOH action on BKCa channels are not well understood. Several lines of evidence suggest, however, that the lipid composition of the plasma membrane may influence channel sensitivity to the drug. The plasma membrane is highly complex in its organization (Welti and Glaser, 1994; Brown and London, 1998). There is a growing body of literature indicating that the local lipid composition of the membrane can influence the function of ion channels, including BKCa (Chang et al., 1995a, b; Moczydlowski et al., 1985; Park et al., 2003; Turnheim et al., 1999). Interestingly, chronic exposure to EtOH in animal models results in alterations in the composition of synaptic plasma membranes, including changes in the amount and distribution of membrane cholesterol (CHS) (Chin et al., 1978; Chin et al., 1979; Wood et al., 1989). The significance of these alterations is unclear. Here, we set out to determine the ability of membrane lipids to modulate BKCa channel activity and EtOH sensitivity. To address this, we implement the planar lipid bilayer technique, allowing control of both the protein and lipid components of the membrane. Native BKCa channels retain EtOH sensitivity in this reductionist preparation (Chu et al., 1998), and we extend the study here to examine cloned human brain (hslo) BKCachannels. We show here that hslo channels maintain their characteristic large conductance, voltage and Ca++-dependent gating, and sensitivity to 50 mM EtOH in bilayers cast from a 3:1 mixture of 1-pamiltoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-pamiltoyl-2-oleoyl-phosphatidylserine (POPS). The addition of CHS to the bilayer decreases both the basal activity and EtOH sensitivity of the channels, in a concentration-dependent manner. This lends support to the notion that alterations in plasma membrane CHS levels following chronic EtOH exposure may reflect adaptations to the acute actions of the drug on ion channels. Furthermore, the EtOH sensitivity and CHS modulation of these reconstituted hslo channels are greatly reduced in the absence of negatively charged POPS in the bilayer (pure POPE). Based on these findings, we look to gain mechanistic insight into the lipid headgroup and acyl chain properties that may regulate BKCa channel modulation by EtOH and CHS. When POPS is replaced with the uncharged lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the hslo response to EtOH and CHS is restored, suggesting that the loss of negative surface charge or PS headgroup structure itself cannot explain the lack of channel modulation by these agents in POPE bilayers. Moreover, increases in the proportion of unsaturated acyl chains in the bilayer cannot significantly influence the hslo response to EtOH. The loss of EtOH sensitivity in pure POPE and CHS-containing bilayers may, therefore, reflect the propensity of POPE and CHS to form nonlamellar (nonbilayer) structures. Regarding the basal activity of the channel, we demonstrate that decreases in negative surface charge, increases in the proportion of unsaturated acyl chains, and increases in the complexity of head group interactions can all influence the steady-state activity of reconstituted hslochannels, relative to control POPE/POPS (3:1) bilayers. Overall, these data further suggest the ability of the local lipid environment to regulate the basal function and EtOH sensitivity of an ion channel protein. Parts of this dissertation have appeared in separate publications: Treistman, S.N., O'Connell, R.J., and Crowley, J.J. (2002). Artificial Bilayer Techniques in Ion Channel Study. In Methods in Alcohol-Related Neuroscience Research, D. Lovinger and Y. Liu, eds. (Boca Raton, Florida: CRC Press) Crowley, J.J., Treistman, S.N., and Dopico, A.M. (2003). Cholesterol antagonizes ethanol potentiation of human BKCA channels in binary phospholipid bilayers. Mol. Pharma. 64(2):364-372.

The purpose of this study was to investigate the mechanism of action of lipolysis by growth hormone in rat adipocytes. GH-induced lipolysis, in contrast to that of isoproterenol (ISO), is slow in onset (lag time >1h), small in magnitude (~2X basal). and requires corticosteroid. Evidence for direct coupling between GH receptors and adenylyl cyclase or G-proteins is lacking, and although we could detect no measurable change in cAMP content after treatment with GH + dexamethasone (Dex), it is likely that cAMP activation of protein kinase A is a central event in GH-induced lipolysis. Rp-cAMPS, a competitive antagonist of cAMP was equally effective in decreasing lipolysis in tissues treated with GH/Dex or a comparably lipolytic dose of ISO. Incorporation of 32P from γ-32P-ATP into kemptide, a synthetic oligopeptide substrate for protein kinase A, was increased in homogenates of GH/Dex-treated tissue. This increase was correlated with increased lipolysis. Earlier estimates based upon 32P-ribosylation of Gi catalysed by pertussis toxin (PTx) suggested that the abundance of Gi in adipocyte membranes was decreased 4h after treatment of hypophysectomized rats with GH. We therefore examined the possibility that changes in amount or distribution of G-proteins in adipocyte membranes might account for the lipolytic action of GH. Homogenates of GH/Dex-treated and control adipocytes were subjected to differential centrifugation and the abundance of G-proteins in low speed, l6k x g (16k), pellets and high speed, 100k x g (100k), pellets were determined by quantitative Western analysis with densitometry. A 35% loss of Giα2 from the l6k pellet compared from tissues treated with GH/Dex was associated with a 70% increase of Giα2 in the 100k pellet. No change in Gsα was observed in the l6k pellet but a 35% loss of Gsα was seen in the 100k pellet. The G proteins in the l6k pellet were fractionated on a continuous sucrose gradient followed by quantitation with Western analysis or autoradiography after 32P-NAD ribosylation. Giα2 was consistently shifted from heavier to lighter fractions of the l6k pellet after treatment with GH/Dex. Similar shifts of Gsα were not seen. The distribution of 32P-labelled proteins was comparably altered after incubation of homogenates of control and GH/Dex treated adipocytes with PTx and 32P-NAD. These shifts were blocked by treatment of adipocytes with 100μM colchicine which also blocked the lipolytic action of GH/Dex. We propose that an action of GH/Dex on the cytoskeleton of fat cells may change the cellular distribution of G-proteins in a manner that produces a relative decrease in the tonic inhibitory influence of Gi on adenylyl cyclase.