The mechanisms underlying the gestational and circadian timing of parturition in humans are not fully understood. Studies of the timing of initiation of spontaneous labor show a peak between 2400 and 0500. This peak in labor onset coincides with peak serum melatonin levels in humans. Melatonin, N-acetyl-5-methoxytryptamine, is the molecular messenger of circadian night. A monoamine hormone produced by the pineal gland, melatonin, is released into the blood directly in a circadian manner controlled by input from suprachiasmatic nuclei (SCN). Peak levels occur several hours after darkness and its release is inhibited by light via photic input transmitted from the eye via the retino-hypothalamic tract to the SCN. Once in the circulation melatonin can act on numerous tissues via its receptors or via antioxidant mechanisms inferred by its indole ring. Our laboratory recently characterized the expression of the melatonin receptors in the human myometrium and showed that the expression of these receptors is suppressed until late pregnancy. In an effort to understand better the significance of melatonin in the human myometrium, we explored the mechanisms through which this hormone influences the expression of the oxytocin receptor in vitro. The stable melatonin analog iodo-melatonin (I-MEL) was presented to cultured telomerase-immortalized smooth muscle myometrial cells of the human telomerase reverse transcriptase line under physiological doses and durations. Pharmacological inhibitors of melatonin binding (4P-PDOT), gene transcription (actinomycin), phospholipase C (U73122), and protein kinase C (C1) signaling were used to define the mechanism of melatonin action. Our results reveal that melatonin significantly reduces oxytocin receptor mRNA levels primarily via the melatonin type 2 receptor, MT2R. We assayed OTR mRNA levels over 24 hours after treatment with the transcriptional inhibitor, Actinomycin, with and without cotreatment melatonin. Our data suggest the melatonin-dependent decrease in oxytocin receptor transcripts involves reduction of the OTR mRNA accumulation rate rather than enhanced rates of transcript degradation. Melatonin effects were abolished by pre-treating the cells with the phospholipase C inhibitor U73122 or the protein kinase C inhibitor C1. Melatonin, like oxytocin, can negatively regulate oxytocin receptor transcription in human myometrial cells via modulation of protein kinase C signaling. Due to the similarities between the melatonin and oxytocin signaling pathways including reduction of OTR mRNA levels, we next sought to determine the effects of melatonin on contractility and the contractile machinery in telomerase-immortalized human myometrial cells. To ascertain the effect of melatonin on myometrial contractility in cell cultures, we performed gel retraction assays with cells exposed to I-MEL, oxytocin and the pharmacological inhibitors 4P-PDOT, U73122, C1 and combinations of ligands and inhibitors. I-MEL was found to synergistically enhance oxytocin-induced contractility via the MT2R, which is coupled to a protein kinase C-dependent increase in phosphorylation of the myosin light chain protein. The effects of I-MEL on gap junctions were also investigated as gap junction proteins have been shown to be upregulated by melatonin in other tissues and have also been shown to be important for coordination of contractions in the laboring uterus. I-MEL increased expression of the gap junction protein, connexin 43. In vitro dye spread assays showed that I-MEL-treated cells displayed substantially increased intercellular coupling. Increases in connexin 43 mRNA and cell to cell coupling were also found to be mediated via the MT2R in a protein kinase C-dependent manner. Additionally, expression levels of the type 2 melatonin receptor (MT2R) were assessed in myometrial biopsies from term pregnant women with or without labor. MT2R expression was markedly elevated in samples from pregnant women who had entered labor, as compared to matched non-laboring pregnant women. To ascertain the signaling pathway of melatonin and leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to I-MEL with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on IP3/DAG/ Protein Kinase C (PKC) signaling were also investigated as these signaling molecules were implicated by our previous pharmacology experiments. I-MEL was found to activate PKCα via the phospholipase C/IP3/DAG signaling pathway which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity and its effects on contractility were insensitive to Rho kinase inhibition. In order to examine another possible method of contractile sensitization, we assayed for caldesmon phosphorylation and upstream Erk1/2 activation. I-MEL did increase phosphorylation of Erk1/2 and caldesmon, which was inhibited by the MEK inhibitor, PD98059 or the PKC inhibitor, C1. These findings lead us to surmise that melatonin sensitizes myometrial cells to subsequent pro-contractile signals in vitro through activation of the phospholipase C/IP3/DAG signaling pathway resulting in specific activation of PKCα and Erk1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and crossbridging. This research revealed a new role for melatonin in reproductive physiology, sensitizing myometrial cells to a subsequent pro-contractile oxytocin signal. This function would help explain the increased nocturnal uterine contractility and increased incidence of parturition observed in late term human pregnancy. Synergistic actions of melatonin on oxytocin-induced contractility may be of clinical relevance in that it could provide a means to lower the oxytocin dose used in the induction of labor and thus reducing the contraindications associated with oxytocin induction of labor. / A Dissertation Submitted to the Department of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Fall Semester, 2009. / November 4, 2009. / Myometrium, Oxytocin, Parturition, Contractility, Melatonin / Includes bibliographical references. / James M. Olcese, Professor Directing Dissertation; Hank W. Bass, University Representative; Akash Gunjan, Committee Member; Choogon Lee, Committee Member; Branko Stefanovic, Committee Member.

The hepatitis C virus (HCV) is a serious health threat globally. Current therapies are not tolerated well, have a low response rate, and there is no available vaccine. New viral targets for drugs are urgently needed. The aim of this study was to characterize resistance to chemical and non-chemical treatments of the HCV replicon and determine a viral target for new treatment options. Replicon cells were treated with both Cyclosporine A (CsA) and temperature shift treatment (39°C). Resistant replicon cells were attained by double treatment with selection antibiotics and anti-viral treatments, in concert with live cell sorting techniques. Resistant cell lines were analyzed and RNA was extracted. This RNA was electroporated into naïve or cured cells, creating new cell lines. These new cell lines were then tested for resistance. Resistant replicon RNA was also sequenced and compared to non-resistant strains. After cell lines had been attained with high levels of resistance, and RNA was electroporated into naïve or cured replicon cells, these new cell lines also showed resistance. This indicated that the viral RNA was the source of the treatment resistance. There were unique mutations at the amino acid level in both CsA and temperature shift resistant cell lines. These unique mutations in both the CsA and temperature shift resistant replicon genomes indicate changes in the NS5B and NS3 proteins, respectively. Further work on the protein structures with the amino acid substitutions and their interactions could lead to new targets for therapies in patients. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. / Fall Semester, 2009. / July 17, 2009. / Temperature Shift, Hepatitis C Virus. Drug Resistance / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Thomas Keller, Committee Member; Kenneth Roux, Committee Member; Fanxiu Zhu, Committee Member.

Activation of the type I interferon (IFN) pathway by small interfering RNA (siRNA) is a major contributor to the off-target effects of RNA interference in mammalian cells. While IFN induction complicates gene function studies, immunostimulation by siRNAs may be beneficial in certain therapeutic settings. Various forms of siRNA, meeting different compositional and structural requirements, have been reported to trigger IFN activation. The consensus is that intracellularly expressed short- hairpin RNAs (shRNAs) are less prone to IFN activation because they are not detected by the cell-surface receptors. In particular, lentiviral vector-mediated transduction of shRNAs has been reported to avoid IFN response. Here we identify an shRNA that potently activates the IFN pathway in human cells in a sequence- and 50-triphosphate- dependent manner. In addition to suppressing its intended mRNA target, expression of the shRNA results in dimerization of interferon regulatory factor-3, activation of IFN promoters and secretion of biologically active IFNs into the extracellular medium. Delivery by lentiviral vector transduction did not avoid IFN activation by this and another, unrelated shRNA. We also demonstrated that retinoic-acid-inducible gene I, and not melanoma differentiation associated gene 5 or toll-like receptor 3, is the cytoplasmic sensor for intracellularly expressed shRNAs that trigger IFN activation. Hepatitis C virus (HCV) is a leading cause of liver disease and much about its life cycle is still poorly understood. We have isolated a mutant virus, named SAV III, though serially passaging JFH-1 through a cell line expressing siRNA against SRBI, a known HCV receptor. We have shown that this virus spreads faster in cell culture and produces more infectious viral particles as compared to wild type HCV. In addition, we have also shown that the HCV core protein is present in higher concentrations despite other viral proteins having similar levels of expression between WT and SAV III. SAV III also has 10 fold more intracellular and extracellular infectious virus as compared to WT, suggesting enhanced viral assembly. We propose that more SAV III core is assembled into viral particles, resulting in the core protein being stabilized. Several mutations have been identified and we are working to determine what mutation, or groups of mutations are responsible for the mutant phenotype. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2011. / April 4, 2011. / Adaptive Mutations, Innate Immunity, Hepatitis C Virus / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Brian Miller, University Representative; Wu-Min Deng, Committee Member; Kenneth Roux, Committee Member; Fanxiu Zhu, Committee Member.

Conditioned taste aversion (CTA) learning occurs after the pairing of a novel taste with a toxin (e.g. sucrose taste with LiCl toxin). The immediate early gene c-Fos is necessary for CTA learning, but c-Fos alone cannot be sufficient for CTA consolidation. The expression of other activator protein 1 (AP-1) proteins from the Fos (c-Fos, FosB, Fra-1 and Fra-2)- and Jun (c-Jun, JunB and JunD)-families may also be required shortly after conditioning for CTA consolidation. To screen for the expression of AP-1 transcription factors within small subregions, RT-PCR analysis was used after laser capture microdissection (LCM) of the amygdala. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry showed that changes in Fra-2 and c-Fos expression in the BLA and CeA at the time of conditioning, together with constitutive expression of c-Jun and JunD, may contribute to CTA learning. Using double immunolabeling, I confirmed that c-Fos co-localized with Fra-2 in a majority of LiCl-induced c-Fos-positive cells in the CeA. This co-localization of c-Fos and Fra-2 following LiCl suggests that the transcriptional regulation of AP-1 dimeric complexes of c-Fos and Fra-2 may occur in a subset of cells in the CeA. AP-1 family members bind each other to make homo- or hetero-dimers that regulate expression of target genes. For example, c-Fos needs to dimerize with a complementary member of other AP-1 proteins, specially Jun members. Therefore, it can be postulated that there may be transcriptional modulation of a set of AP-1 genes to form a functional AP-1 complex in a neuronal unit that is expressing c-Fos during CTA learning. I examined changes in mRNA expression of immediate early genes including AP-1 family members in c-Fos-specific neurons of the amygdala during CTA learning. Using X-Gal staining, single-cell LCM and RT-PCR, I detected mRNA expression of c-fos and β-actin genes in LiCl-induced lacZ-positive cells in the CeA, cortex and hippocampus of c-fos-lacZ transgenic mice. This result confirmed that endogenous c-fos gene and the c-fos-lacZ transgene were expressed in same cells of the brain. LiCl administration increases c-Fos expression in some brain regions including the CeA. Recent studies show that c-Fos expression in the brain after certain stimuli may be affected by histone modification such as acetylation and phosphorylation. Continuing our studies on gene expression in the amygdala in CTA learning, I investigated if LiCl-induced c-Fos expression in the amygdala is correlated with histone acetylation and phospho-acetylation. LiCl significantly increased the level of acetylated histone H3 (AcH3) in the CeA at 0.5 h and the number of phospho-acetyl-histone H3 (pAcH3)-positive cells in the CeA at 0.5 and 1 h, and the timecourse of LiCl-induced AcH3 and pAcH3 corresponded to LiCl-induced c-Fos timecourse in the CeA. Double immunolabeling results showed that c-Fos co-localized with pAcH3 in a majority of LiCl-induced c-Fos-positive cells in the CeA. These results suggest a possible correlation between LiCl-induced c-Fos expression and modifications of histone H3 in the CeA. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2008. / August 6, 2008. / Includes bibliographical references. / Thomas A. Houpt, Professor Directing Dissertation; Samuel C. Grant, Outside Committee Member; Charles C. Ouimet, Committee Member; Frank Johnson, Committee Member; Hank W. Bass, Committee Member.

The redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding network of amicyanin from the aforementioned study were attempted to be used as a model to increase the stability of another beta barrel protein, the immunoglobulin light chain variable domain (VL). In addition, amicyanin was used as an alternative redox partner with MauG. MauG is a diheme protein from the mau gene cluster that catalyzes the biogenesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase (MADH). The amicyanin-MauG complex was used to study the free energy dependence and impact of reorganization energy in biological electron transfer reactions. The sole tryptophan of amicyanin was converted to a tyrosine via site-directed mutagenesis. This mutation had no effect on the electron transfer parameters with its redox partners, methylamine dehydrogenase and cytochrome c-551i. However, the pKa of the pH-dependence of the redox potential of the copper site was shifted +0.5 pH units. This was a result of an additional hydrogen bond between Met51 and the copper coordinating residue His95 in the reduced form of amicyanin. This additional hydrogen bond stabilizes the reduced form. Also, the stability of the copper site and the protein overall was significantly decreased, as seen by the temperature dependence of the visible spectrum of the copper site and the circular dichroism spectrum of the protein. This destabilization is attributed to the loss of an interior, cross-barrel hydrogen bond. The VL is structurally similar to amicyanin, but it does not contain any cross-barrel hydrogen bonds. The importance of the cross-barrel hydrogen bond in stabilizing amicyanin is evident. A homologous bond in VL was attempted to be engineered by using site-directed mutagenesis to insert neutral residues with protonatable groups into the core of the protein. Wild-type (WT) VL was purified from the periplasm and found to be properly folded as determined by circular dichroism and size exclusion chromatography. Mutants were expressed in E. coli using the amicyanin signal sequence for periplasmic expression. Folded mutant protein could not be purified from the periplasm. When amicyanin is used in complex with MauG, it retains the pH-dependence of the redox potential of its copper site due to the looseness of the interprotein interface. The free energy of the reaction was manipulated by variation in pH from pH 5.8 to 8.0. The ET parameters are reorganization energy of 2.34 eV and an electronic coupling constant of 0.6 cm-1. P94A amicyanin has a potential that is 120 mV higher than WT amicyanin and was used to extend the range of the free energy dependence studied. The ET parameters of the reaction of WT and P94A amicyanin with MauG were within error of each other. This is significant because the ET reaction of P94A amicyanin with its natural electron acceptor was not able to be studied due to a kinetic coupling of the ET step with a non-ET step. This kinetic coupling obscured the parameters of the ET step because it is not kinetically distinguishable from the ET step. A Y294H MauG mutant was also studied. This mutation replaced the axial tyrosine ligand of the six-coordinate heme of MauG with a histidine. No reaction is observed with Y294H MauG in its native reaction. However, the high valent oxidation state of the five-coordinate heme of Y294H MauG reacts with reduced amicyanin. The ET rate was analyzed by ET theory to study the high valent heme in Y294H MauG. The reorganization energy of Y294H MauG was calculated to be nearly 20% lower as compared to the same reaction with WT MauG. These results provide insight into the obscured nature of reorganization energy of large redox cofactors in proteins, particularly heme cofactors, as well as to how the active sites of enzymes are optimized to perform long range ET vs catalysis with regard to balancing redox potential and reorganization energy.

Krüppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. In this study we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Furthermore, CXCR4-CXCL12 engagement downstream of KLF8 leads to the feed-forward activation of FAK. Interestingly, KLF8 expression, through CXCR4 engagement, triggered the formation of filopodium-like protrusions (FLP) and thereby enhanced the proliferation rate of breast cancer cells in 3D Matrigel-on-Top culture, under prolonged treatment with CXCL12. This indicates that KLF8 plays a major role in promoting aggressive colonization of tumor cells in a CXCL12-enriched foreign tissue microenvironment, thereby aiding in secondary macrometastasis formation. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. Apart from lung, KLF8 overexpression also induced spontaneous secondary metastasis to other CXCL12-rich organs through CXCR4 signaling. These results collectively suggest a critical role for KLF8 and the CXCR4-CXCL12 pathway in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies.

Due to cancer recurrence and the development of drug resistance, metastatic breast cancer is a leading cause of death in women. In the search for a new therapeutic to treat metastatic disease, we discovered CT20p, an amphipathic peptide based on the C-terminus of Bax. Due to inherent properties of its sequence and similarity to antimicrobial peptides, CT20p is a promising cytotoxic agent whose activity is distinct from the parent protein (e.g. does not cause apoptosis). CT20p is not membrane permeable but can be introduced to cells using polymeric nanoparticles, a method that promotes efficient delivery of the peptide into the intracellular environment. We demonstrated that CT20p was cytotoxic using triple negative breast cancer (TNBC) cell lines, primary breast tumor tissue, and breast tumor murine xenografts. Importantly, normal breast epithelial cells and normal primary breast cells were resistant to the lethal effects of the peptide. Examination of multiple cellular processes showed that CT20p causes cell death by promoting cytoskeletal disruption, cell detachment, and loss of substrate-mediated survival signals. In order to identify the intracellular target of CT20p, we performed pull-down experiments using a biotinylated peptide and found that CT20p binds directly to a type II chaperonin called chaperonin containing T-complex (CCT), which is essential for the folding of actin and tubulin into their native forms. The resulting effect of CT20p upon the cytoskeleton of cancer cells is disruption of vital cellular processes such as migration and adhesion. CCT gene expression and protein levels were examined across several breast cancer cell lines, and we found that susceptibility to CT20p correlated with higher CCT levels. Using human cancer tissue microarrays, we determined that CCT was present in significantly higher amounts in tumor tissues compared to normal tissues and that expression often increased with advanced cancer stage. These results indicate that CCT is a promising therapeutic target for the treatment of metastatic breast cancer and suggest that the use of cancer-targeted nanoparticles loaded with CT20p is a novel and effective therapeutic strategy for cancers, such as TNBC, that recur and are refractory to current treatments.

Protein-derived cofactors are redox and catalytic centers that are generally formed by the posttranslational modifications of one or more amino acids. An important class of these cofactors are the quinones derived from tyrosine and tryptophan. Amongst redox proteins, it has been known till now that oxidases either contain a flavin or a tyrosylquinone cofactor, whereas tryptophylquinone ones are present within the dehydrogenases. In recent times, oxidases from a marine bacterium, Marinomonas mediterranea, have been shown to possess the latter. This study involves the characterization of two such proteins, lysine-?-oxidase (LodA) and glycine oxidase (GoxA). They have been reported to contain the same cysteine tryptophylquinone (CTQ) cofactor. Both require the co-expression of a second protein, LodB and GoxB respectively to generate matured CTQ containing active protein. Kinetic analysis of the reaction catalyzed by LodA showed that it followed the usual Michaelis-menten mode of interaction with its substrates. GoxA on the other hand exhibited allosteric cooperativity for its substrate glycine. This was attributed to the dimeric conformation of the wildtype GoxA based on size exclusion chromatographic studies. Mutagenesis study of amino acid residues based on the crystal structure of LodA and a homology model of GoxA, have given a detailed idea about their structure-function relationship. Kinetic studies on mutants of Tyr211 of LodA along with Lys530 present at the substrate channel, showed effects on both Km for the substrates and kcat for the reaction. As a result these residues have shown their involvement in forming a gate-like structure to control the to and fro movement of the substrate and products. Corresponding to this residue, the Phe237 of GoxA has proved to be important in maintaining the allostericity, by mediating the stable dimer formation. From the kinetic parameters, Cys448 of LodA was found to be responsible for substrate specificity and affinity. Whereas, mutants of His466 of GoxA that correspond to the Cys448 residue, were unable to yield CTQ containing active GoxA. On the other hand, Asp512 of LodA and Asp547 of GoxA that correspond to each other, have been implicated for their involvement in CTQ biogenesis. This study therefore highlights how even though this new pool of enzymes have great degrees of similarity in terms of the cofactors and conserved active site residues, there are major differences in the mechanism of the reaction that they catalyze which on a broader sense could influence the overall physiological importance of the enzyme in the biological system.

Ovarian cancer is a leading cause of gynecological malignancy. Cytoreductive surgery and frontline platinum/taxane-based chemotherapy provides good initial efficacy in the treatment, but poor long-term patient survival. This is mainly caused by tumor relapse due to intraperitoneal spreading and ineffective alternate therapies to treat these resistant tumors. The challenge in the field is to develop strategies that would prove effective in these patients and extend overall survival. Over the years, various treatments have been developed for the treatment of cancer amongst which, adoptive cell immunotherapy has shown promising results. But despite the efficacy seen in the clinic, there are concerns with the complexity of treatment and associated side effects. Therefore, there is still a need for better understanding of how different components of the immune system react to the presence of tumor. In this study, healthy human peripheral blood mononuclear cells (PBMCs) were used to examine the immune response in a mouse model with residual human ovarian tumor, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion and cytotoxicity in mice treated intraperitoneally (IP) with PBMCs+Interleukin-2 (IL- 2). Intravenous (IV) adoptive transfer of isolated NK cells has been attempted in ovarian cancer patients before, but showed lack of persistence in patients resulting in lack of anti-tumor efficacy. Experiments in this study highlight the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population (when treated IP), as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients (when treated IV). NK cell immunotherapy is mainly limited by insufficient numbers generated for adoptive transfer, limited in vivo life span after adoptive transfer, lack of cytotoxicity and some logistical concerns that impede its widespread implementation. Therefore there is a need to develop methods of NK cell expansion that provide stimulation similar to other immune cell types in the PBMC population. The second part of this study utilizes a method of in vivo NK cell expansion using a particle-based approach in which plasma membranes of K562-MB21-41BBL cells (K562 cells expressing membrane-bound IL-21 and 41BB ligand) are used for specific NK cell expansion from PBMCs. NK cells expanded with this method were cytotoxic, showed in vivo persistence and biodistribution in different organs. Collectively, these studies show that NK cells are a major innate immune component that can recognize and kill the tumor. Their cytotoxic ability, using particle-based stimulation, can be enhanced for a second-line treatment of relapsed tumors such as in ovarian cancer as well as other cancer types.

Myelination is a complex process requiring coordination of directional motility and an increase in Schwann cell (SC) size to generate a multi-lamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin1-type III (NRG) expressed on the axon surface. NRG binding to ErbB2/3 receptors on the Schwann cell surface initiates signaling cascades necessary for myelination. We identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG-ErbB2/3 signaling in rat SC. A five minute exposure of SCs to NRG triggers phosphorylation of ErbB2 with concomitant dephosphorylation, and activation, of cofilin, and its upstream regulators, LIM domain kinase (LIMK) and Slingshot-1 phosphatase (SSH). This leads to cofilin activation and recruitment to the leading edge of the SC plasma membrane. These changes are associated with rapid plasma membrane expansion yielding a 35–50% increase in SC size within 30 minutes of NRG1 exposure. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs co-cultured with sensory neurons fail to elaborate myelin. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 hours of co-culturing with neurons, cofilin-deficient SCs fail to align and elongate on axons and often adhere to the underlying substrate rather than to axons. We show that the Neurofibromatosis Type II (NF2) tumor suppressor, merlin, is an upstream regulator of cofilin1, and that merlin knockdown in Schwann cells inhibits their elaboration of normal myelin sheaths in vitro. Merlin-deficient SCs form shorter myelin segments in DRG neuron/SC co-cultures. Merlin-deficient Schwann cells have increased levels of both active Rac (Rac-GTP) and F-actin indicative of a stable actin cytoskeleton. Surprisingly merlin-deficient Schwann cells fail to dephosphorylate and activate cofilin1 in response to NRG stimulation. Inhibition of LIMK restores the ability of merlin-deficient SCs to activate cofilin in response to NRG. In developing rat sciatic nerve, merlin becomes hyper-phosphorylated at S518 during the time of peak myelin formation. During this time, cofilin is localized to the inner mesaxon, and subsequently to Schmidt-Lanterman incisures in mature myelin. This study: 1) identifies cofilin and its upstream regulators, LIMK and SSH, as end targets of a NRG-ErbB2/3 signaling pathway in Schwann cells, 2) demonstrates that cofilin modulates actin dynamics in Schwann cells allowing for motility needed to effectively engage and myelinate axons, 3) shows that merlin regulates NRG-ErbB2/3-cofilin-actin signaling during SC myelination to determine the myelin segment length.