Laura B. Grabel - US grants

The long term goals of this study are to identify and characterize the molecules involved in the intercellular adhesion of teratocarcinoma stem cells, to describe their mode of action in adhesion, and their possible role during stem cell differentiation. The value of undertaking this study using teratocarcinoma stem cells is underscored by the implication that aggregation is a prelude to differentiation in this system. Studying the basis of adhesion and differentiation of teratocarcinoma stem cells is of particular interest because the stem cells lose their tumorigenecity when they differentiate, thereby providing a model for evaluating the regulation of the neoplastic state. In preliminary studies based upon the lectin hypothesis of cell-cell recognition, we have described a fucan-mannan specific lectin activity, present on the surface of the stem cells, which appears to mediate a divalent cation-independent component of cell adhesion. We have recently identified the protein responsible for this activity. The specific aims of this proposal include employing antibody specific to the lectin in studies designed to 1) determine the cell type specificity of expression of the lectin, 2) verify the role of the protein in stem cell adhesion, and 3) determine if this protein is involved in stem cell differentiation. In addition we propose to identify an endogenous carbohydrate-containing receptor for the lectin, and to decipher the mode of action of these interacting components during cell adhesion. The lectin receptor may be found among the previously described stem cell glycoconjugates reported to contain terminal fucose. The tissue specificity of the divalent cation-dependent and the divalent cation-independent systems of adhesion will be determined and the role of extracellular multicomponent particles as aggregation promoters will be evaluated. These experiments will provide detailed information on the moelcular mechanism of the intercelluar adhesion of teratcarcinoma stem cells.

centrifugation; biomedical equipment resource;

Grabel 9604367 The long term goals of this investigation are to identity the genes involved in making, early lineage decisions during mammalian embryogenesis and to define how the products of these genes interact. Dr. Grabel propose that the Indian hedgehog (Ihh) gene is involved in initiating both extraembryonic endoderm and mesoderm differentiation. Evidence accumulated by us using the P9 teratocarcinoma system, as well as ES cells and mouse blastocyst cultures, suggests a role for Ihh in establishing extraernbryonic endoderm. Proposed studies will substantiate this role, begin to define the molecular mechanism of action of Ihh in early mouse embryogenesis, and determine if mesoderm induction involves Ihh action. Specific Aims are: 1. To substantiate the role of Ihh in extraembryonic endoderm differentiation. These studies will include situ hybridization analyses and immunocytochemistry, generating an Ihh-deficient P9 cell line by targeted mutagenesis, and testing the effect of hh peptides. 2. To identify gene products involved in the hh signaling pathway active during early mouse embryogenesis. These studies will test the role of the patched (ptc) gene and of protein kinase A as downstream mediators of Ihh. 3. To determine if Ihh provides a signal that acts via BMP-2 or BMP-4 to induce mesoderrn. Hedgehog genes clearly play a critical role in patterning the embryo. These studies promise to define the role of Ihh in signaling the earliest lineage decision in the mammalian embryo, distinguishing embryonic from extraembryonic and establishing primary germ layers.

DESCRIPTION (provided by applicant) Integrins are cell surface receptors that play a key role in morphogenetic events during development, and in a variety of pathogenic conditions, such as metastasis. The long-term goal of this project is to understand how integrin function is modulated during differentiation. Our work to date has focused predominantly on the B1 integrin subunit and the functional role of phosphorylation on a serine residue in its cytoplasmic domain. Specific aims will continue to study the functional significance of Bl phosphorylation and to examine how this phosphorylation event is regulated. In addition, we will begin to study the role of tetraspanin proteins in modulating integrin function. Specific Aim 1: To determine the role of serine phosphorylation of the cytoplasmic domain in B1 function. Preliminary studies suggest that cells expressing B1 with amino acid substitutions that mimic a phosphorylated serine in the cytoplasmic domain behave differently from cells expressing the wild type serine or neutral amino acids substitutions at this site. We will extend these studies using the 131-deficient parental GD25 fibroblast cell line and transfected cell lines expressing site-specific mutations. We will also investigate the ability of wild type and the site-specific mutant forms of BI to associate with a variety of focal adhesion proteins using a number of experimental approaches including co-immunoprecipitation and GST-pull down experiments. Specific Aim 2: To investigate how serine phosphorylation of the B1 integrin subunit is regulated in F9 cells. Our preliminary studies suggest roles for the phosphatase PP2A and the kinase ILK. Approaches will include determining if phosphorylated B1 can be de-phosphorylated in vitro by PP2A and investigating the behavior of F9 cells either overexpressing ILK or expressing a dominant negative form of the kinase. Specific Aim 3: To examine the role of the tetraspanins CD81 and CD9 in a6B1-mediated cell migration. These tetraspanins associate with a6B1, and CD9 appears to assist integrin-mediated sperm-egg fusion. We will first determine if these tetraspanins associate with the integrin in F9 cells using co-immunoprecipitation analysis. We will then use function-blocking antibodies to test the relative roles of these proteins in cell migration.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to define the mechanisms regulating motility of the first migratory cell type in the mammalian embryo, parietal endoderm (PE). The F9 teratocarcinoma system provides an in vitro model in which F9-derived embryoid bodies generate PE outgrowth when plated on an extracellular matrix (ECM)-coated substrate. Cells leave the embryoid body and migrate away as a sheet of PE. At the edge of the outgrowth, the outermost ring cells extend filopodia and lamellipodia and actively migrate away from the embryoid body, while remaining attached to the cells behind them. We have shown that focal adhesion turnover and lamellipodia formation by the outermost ring cells contribute to PE outgrowth. We also established a role for the small GTPase Rho and its downstream effector ROCK in maintaining robust focal adhesions and applying the brakes to PE migration. Time-lapse analysis led us to hypothesize a "push/pull" model for PE outgrowth in which the cells leaving the embryoid body spread and intercalate, pushing the outgrowth away from the embryoid body, while the outermost ring cells pull the outgrowth away from the embryoid body. Specific Aim 1. To define the role of Rho family GTPases in PE outgrowth. These studies will continue to examine the role of Rho and initiate analysis of the roles of Rac and Cdc42 using gain and loss of function approaches. We will identify major effectors and determine the subcellular localization of the active GTPases in migrating cells. Specific Aim 2. To determine if directed migration of PE is regulated by the PCP pathway. This aim tests the hypothesis that PE outgrowth is an example of directed migration using intercalation movements mediated by the PCP pathway. We will use gain and loss of function approaches. Specific Aim 3. To examine PE migration on a complex ECM and evaluate the role of integrins in modulating the mode of migration. The in vivo substrate for PE migration consists initially of fibronectin, and then Reichert's membrane, a complex matrix similar to the components of Matrigel. We will therefore examine PE outgrowth on Matrigel, plus and minus fibronectin, with a particular focus on examining the role of integrins in promoting directed versus random migration. [unreadable] [unreadable] [unreadable]

An award is made to Wesleyan University to support the acquisition of a confocal microscope for use by faculty and students across multiple departments and disciplines. The new instrument will profoundly enhance the training environment and be used during many highly interactive laboratory courses taught by Wesleyan faculty to promote STEM career advancement. The proposed microscope will be housed in an advanced imaging facility that includes scanning and transmission electron microscopes. To support dissemination of science to the general public, as exemplified by the large number of articles authored by Wesleyan faculty that are printed in the popular media, data generated by the new system will be used in outreach presentations, science camps, local high-schools, an annual scientific imaging student prize, and similar forums.

Wesleyan boasts an unusually broad portfolio of research topics that will benefit from the new system, including evolution and development of morphology, optogenetic-based control of behavior, cell biology, physiology, developmental neuroscience, and chromosomal dynamics. Each of these areas is experiencing an expansion of research into spatial relationships, driven by advances in microscopy, and by the realization that researchers must study three-dimensional shapes and how those change over time to understand the structure and function of biological systems. Several of the research projects will only be possible because the local availability of the system. Data and analyses from these studies will be published in peer-reviewed scientific journals, presented at scientific meetings, and used in both educational and public outreach activities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.