Master of Science (MS), Bowling Green State University, 2019, Biological Sciences

Filopodia are finger-like projections on cells that allow the cells to sense and explore their external microenvironment. They are required for cell migration, neurogenesis and several other physiological processes. Filopodia dynamics have been widely studied in nerve cells. Here, I investigated the role of increased cytoplasmic calcium levels in the regulation of filopodia through activation of store-operated calcium entry in epithelial cells. The depletion of the endoplasmic reticulum store caused a transient increase in filopodia. The increase in fraction of cells with filopodia and percentage coverage of filopodia was seen at 10 min followed by a decrease at 30 min of treatment with cyclopiazonic acid (CPA). Filopodia dynamics are more sensitive to and regulated by Transient receptor potential cation (TRPC) channels. The downstream effectors of calcium such as calpain and calmodulin are negative regulators of filopodia. The possible mechanisms by which calcium regulates filopodia through these downstream effectors were reported here. The effect of calcium-like protein 2 (CALP2) in filopodia dynamics was first described in this study.

Committee: Carol Heckman Ph.D (Advisor); Michael Geusz Ph.D (Committee Member); David Giovannucci Ph.D (Committee Member) Subjects: Biology

Master of Science (MS), Bowling Green State University, 2016, Biological Sciences

Filopodia are the sensory appendages of the cell and have an important role in directing motility, wound healing, and axon pathfinding. Previous work showed that oncogenic transformation of epithelial cells was accompanied by the loss of filopodia. The role of filopodia as sites for adhesion and signaling is well known but the dynamics is poorly understood. The core of the filopodium is a parallel array of actin filaments.  Mallavarapu and Mitchison have proposed that filopodia are retracted by halting actin assembly at the tip of the filaments, whereupon the filaments disassemble at the opposite end. Based on previous work, it was postulated that the filopodia are affected by PKC activation in such a way that actin arrays are disassembled. To study this, synthetic peptides representing the hydrophobic segment of PKC and the effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) were designed to block PKC's effects. After treatment with peptides, cells were collected with and without a brief exposure to phorbol 12-myristate 13-acetate (PMA). PMA activates the classical and novel PKCs. The cells were evaluated by phase contrast microscopy. The mean percentage periphery of the cells covered with filopodia and the percentage of cells in a population showing filopodia were counted. When cells were treated with peptides alone, no effect was observed but when treated with PMA, PKC ε blocking peptide partially rescued the filopodia from destruction. This was significant in the t-test (P-value <0.025).  Because rescue was only pertinent to PMA-treated cells, and the same treatment had no effect on filopodia of untreated cells, I postulate that PKC ε works by a different mechanism than that governing regular dynamics. I also studied the effect of protein tyrosine phosphatase (PTP) inhibitors on filopodia. The PTP1B inhibitor enhanced filopodia in both PMA-treated and -untreated conditions. The TC-PTP inhibitor did not affect cells treated with it alone (open full item for complete abstract)

Committee: Carol Heckman PhD (Advisor); Neocles Leontis PhD (Committee Member); Zhaohui Xu PhD (Committee Member) Subjects: Biology

Master of Science (MS), Bowling Green State University, 2015, Biological Sciences

Filopodia play sensory roles by acting like `antennae' to sense the cell's surroundings. In nerve growth cone, they promote motility towards an attractive cue or away from a repulsive one. Filopodia have been reported to be involved in wound healing, adhesion to the extracellular matrix and embryonic development. A number of cytoskeletal regulatory proteins have been implicated in regulating initiation, formation, maintenance and extension/retraction cycle of such protrusions. Some of these proteins either bind to the Rho family GTPases, Cdc42 and Rac, as effectors or function downstream of such effectors to regulate signaling pathways involved in cytoskeletal reorganization. The purpose of this study was to determine whether certain proteins, which had well-defined binding interfaces with proteins downstream of GTPase-regulated proteins, were implicated in filopodial dynamics. Synthetic binding peptides (BPs) were used to impede the interaction of Cdc42 and Rac with the effectors and to interfere at protein-protein binding interfaces downstream of the GTPases. Single cell peripheries were analyzed to determine the levels of positive outgrowths of filopodia. This assay allowed for rapid determination of whether the BPs had an effect on filopodia formation. BPs, namely IQGAP (132-140), ACK and Par6 showed a negative effect on filopodia, whereas IQGAP (84-93), PAK and WASP elevated filopodia formation or had no effect compared to control. When interactions of PAK2 with Abl and PAK4 with integrin-ß5 were inhibited, filopodia prevalence increased. Based on several previous findings, the tyrosine kinase ACK is thought to mediate internalization, processing and trafficking pathways of cell surface receptors, especially epidermal growth factor receptors (EGFRs). ACK is an oncogene and its overexpression or mutation is implicated in several human cancers. During this study, when the E3 ubiquitin ligase, neural precursor cell expressed developmentally downregul (open full item for complete abstract)

Committee: Carol Heckman PhD (Advisor); Paul Morris PhD (Committee Member); Daniel Wiegmann PhD (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Molecular Biology; Neurobiology; Oncology

Master of Science (MS), Bowling Green State University, 2014, Biological Sciences

Endocytosis involves selective packaging of substances in endosomes for traffic into cells. The cell traffic pathway can be grouped into two functional compartments: Compartment I which represents the early stages of sorting and Compartment II representing the final stage of trafficking and degradation. The sorting events and fate of molecules following uptake is dependent on a variety of cell signaling pathways. The tumor promoter, phorbol 12-myristate 13-acetate (PMA) is known to cause transient loss of filopodia and increased pinocytosis in cells. Filopodia are finger-like plasma membrane protrusions of a number of animal cells containing receptors involved in cell sensing. In neurons where filopodia have been extensively studied, there is some evidence of increased endocytosis during filopodia retraction following encounters with repulsive cues. We hypothesized that, mammalian cells increase endocytic uptake during tumor promotion (with loss of filopodia), and polarity may be connected to vesicle trafficking. PMA effects its action through activation of the enzyme protein kinase C (PKC). To classify the traffic events in mammalian cells in tumor promotion, we selected conditions that enabled us to visualize the contents of Compartment II and compare them to the contents of Compartment I. To investigate the effects of PMA on trafficking three fluorescent markers were applied to rat liver cells of the IAR20 line. Using pixel-by-pixel analysis and image processing, we found an increased accumulation of fluorescent marker in compartments I and II in PMA-treated over the untreated cells. We also found that functional compartments I and II are trafficked in different directions inside cells. In another study on epithelial cells from the rat trachea, 1000W, we found an insignificant difference in fluorescent marker accumulated by endocytosis on the two halves of cells plated on a haptotactic gradient.

Committee: Carol Heckman PhD (Advisor); John Wade PhD (Committee Member); Hans Wildschutte PhD (Committee Member) Subjects: Biology; Biophysics; Cellular Biology; Molecular Biology

Bachelor of Science (BS), Ohio University, 2012, Biological Sciences

Cell migration is an essential biological process. In addition to being required for development and maintenance, cell motility also contributes to human disease. For example cancer metastasis is dependent on tumor cells gaining motility and traveling to secondary sites within the body. Cell motility requires the formation of protrusive structures including filopodia and lamellipodia. The formation of these structures requires rearrangement and coordinated organization of the actin cytoskeleton. Here I show that Drosophila Clic, a member of the CLIC family, contributes to the formation of filopodia by regulating the nucleation and elongation of these structures. This is done by examining the localization of Clic and genetic interactions with known cytoskeleton regulators. I show that there are three phases of filopodia formation,supported by the observed genetic interactions. I provide evidence that Clic is involved in all three phases of filopodia formation and interacts with Cell division cycle 42 (Cdc42), Diaphanous (Dia), Wiskott - Aldrich syndrome protein (WASp), the Actin related protein (Arp) 2/3 complex, Moesin (Moe), and Enabled (Ena). I also show that Clic may contribute to in vivo cell migration through the study of hemocyte migration in Drosophila embryos.

Committee: Dr. Soichi Tanda PhD (Advisor); Dr. Mark Berryman PhD (Advisor) Subjects: Cellular Biology; Genetics

Doctor of Philosophy, Case Western Reserve University, 2009, Pathology

Inducible Co-stimulator (ICOS) regulates effector/memory T cell activation by enhancing proliferation and cytokine production. However, the mechanisms by which ICOS ligation induces these responses have yet to be defined. We have identified both antigen dependent and antigen independent properties associated with ICOS ligation, each of which functions to promote T cell activation. In the absence of antigen, ICOS functions as an adhesion molecule, promoting effector T cell/APC conjugate formation. Subsequent ICOS-mediated cytoskeletal modifications inhibit cell motility, induce T cell spreading, and stimulate filopodia/microspike elaborations, thereby enhancing antigen recognition by promoting T cell scanning of the APC surface. While ICOS-mediated adhesion occurs independently of its ability to induce intracellular signals, T cell elongation and the anti-migratory phenotype are dependent upon PI-3 K activation, which decreases RhoA activity. Following antigen recognition, ICOS functions as a classical co-stimulatory molecule, augmenting TCR-mediated signal transduction pathways leading to T cell proliferation and cytokine production. In contrast to CD28, in which its proximity to the TCR is not important to enhance TCR-mediated T cell proliferation, ICOS co-stimulation requires both TCR and ICOS signals to be provided on the surface. While this may suggest that ICOS ligation augments functional responses by increasing the number of TCRs engaged, ICOS co-stimulation does not alter the intensity or kinetics of TCR-associated membrane proximal signaling events. Instead ICOS co-stimulation enhances and prolongs second messenger signals emanating from the TCR, as demonstrated by a delayed synergy in the elevated phosphorylation of the downstream effector molecules Akt and Erk 1/2 following co-stimulation.

Committee: Alan Levine Ph.D. (Advisor); George Dubyak Ph.D. (Committee Chair); James Finke Ph.D. (Committee Member); Gary Landreth Ph.D. (Committee Member); Clive Hamlin Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2012, Biological Sciences

Filopodia play a sensory role in directing motility during embryonic development and axon pathfinding. They also show a low prevalence in cancer cells. Here, I determined whether cultured cells from a rat tracheal epithelial line used filopodia to sense adhesive gradients. Cells exhibited haptotaxis (movement toward the more adhesive surface) when plated on tantalum (Ta) and platinum (Pt) metallic gradients. The gradients were created on glass, and high (H), middle (M), and low (L) positions defined along the gradient. Cell counts in randomly selected fields confirmed that the cells recognized the gradient. To determine whether the prevalence of protrusions differed at the H, M, and L locations, the values of latent factors 4 (filopodia), 5, and 7 were determined. Factor 4 values were high at H and significantly lower at M and L (p<0.05). Cells' ability to form larger protrusions, represented by factors 5 (lamellar distribution) and 7 (nascent neurites), was unaltered across the Ta gradient. The directional cues also appeared to be interpreted within a cell, as shown by analyzing the cells' top (T) side, i.e. the side oriented toward H location, from the bottom (B) side. Factor 4 values at H-T significantly exceed those at M-B and L-T. Trend analysis confirmed a decrease in factor 4 over the gradient with significance of P<0.001 (Ta) and P<0.023 (Pt). On Pt only, factor 5 increased (P<0.0001) as the metal content of the substrate declined. Cdc42 (cell division cycle 42) plays a crucial role in establishing polarity, and is also involved in filopodia formation, cell motility, and directional migration. Since there is some loss of polarity in preneoplastic lesions, the role of Cdc42 in filopodia-mediated sensing was of interest. To determine whether Cdc42 was implicated in reconstruction of cellular architecture and rearrangement of cytoskeletal structures, I tested the role of Cdc42 effectors in gradient sensing. While most effectors bind to Cdc42 at multiple regio (open full item for complete abstract)

Committee: Carol Heckman PhD (Advisor); Howard Cromwell PhD (Committee Member); Carmen Fioravanti PhD (Committee Member); Michael Geusz PhD (Committee Member); Wendell Griffith PhD (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2010, Biological Sciences

Committee: Carol Heckman Dr. (Advisor); Mikhail Zamkov Dr. (Committee Member); Michael Geusz Dr. (Committee Member) Subjects: Cellular Biology

Master of Science (MS), Bowling Green State University, 2008, Biological Sciences

After activation by guanine triphosphatases (GTPases) like Rac and cell division control protein (Cdc42), the p21-activated kinase (PAK) unfolds and binds tightly with SH3 domain of partner PAK-interacting exchange factor (PIX). PIX binds to G protein coupled receptor kinase interacting protein (GIT1) which binds to paxillin. Paxillin binds to focal adhesion kinase (FAK). The N-terminal region of PAK binds with Nck-alpha through its SH3 domain and Nck-alpha binds to FAK. This chain of five proteins is located at the focal contact site and believed to be important for production of filopodia and membrane protrusion. At the regulatory level, the mechanism is not known but could be clarified by further work. I studied the role of PAK in the formation of these two protrusions, by supplying PAK kinase inhibitor, PAK83-149, in the presence or absence of Nck-alpha. These two constructs were found to have an effect statistically distinguishable from control in previous experiments. Filopodia and larger membrane protrusions were affected differently by Nck-alpha alone and PAK83-149 alone. The large protrusions were more prevalent with Nck-alpha alone and unaffected by PAK83-149. Expression of both constructs together was required to affect the filopodia. When the cells were treated with tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), the results of Nck-alpha and PAK83-149 expression were different. PAK83-149 now showed a significant effect on massive protrusion formation which was statistically distinguishable from all other groups. None of the treatments any longer promoted filopodia formation. Surprisingly the effect of Nck-alpha in all treated samples was removed by PAO. Maybe the Nck-alpha alone affects the protrusions by binding PAK molecules to sites on receptors such as phosphorylated focal adhesion kinase (FAK), or transmembrane growth factor receptors. The reversal of this effect by PAO is a novel effect.

Committee: Carol Heckman PhD (Advisor); Donald Deters PhD (Committee Member); Zhaohui Xu PhD (Committee Member) Subjects: Biology