Integrins are a large family of transmembrane cell adhesion receptors that are found on the surface of eukaryotic cells. Integrins act predominantly as cell surface receptors for extracellular matrix (ECM) proteins, but also have bidirectional signaling properties that allow them to play fundamental roles in development and cancer metastasis. It has become clear that during gastrulation – a period during which cells participate in morphogenetic movements that lead to the generation of a tripoblastic embryo – the integrin repertoire of each cell is in constant flux. This change in cell surface receptors is mediated through intracellular pathways, which in turn, are regulated by associations with cytoplasmic proteins. One such molecule, GIPC (GAIP-interacting protein, C-terminus), is thought to have a role in regulating α5β1 integrin surface expression, as well as integrin-mediated inside-out and outside-in signaling pathways by mediating the integrin’s ability to interact with the ECM protein, fibronectin (FN). I use Xenopus laevis as my experimental model system to study GIPC-regulated integrin function. Xenopus provides a useful model system for regulation of integrin function as α5β1-FN interactions are spatially and temporally regulated. Additionally Xenopus embryos are amenable to molecular manipulations in vivo, and the tissue can be excised from embryos and cultured in vitro. I have investigated the function of GIPC using site-directed mutagenesis to alter the PDZ domain site in Xenopus GIPC (XGIPC). Expression of dominant negative XGIPC results in the interruption of gastrulation movements in the early embryo. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that XGIPC physically interacts with the cytoplasmic domain of the α5 and α6 integrin subunit. Furthermore, I have determined that the interaction of XGIPC with α5β1 is required for assembly of a FN matrix. Cell migration and convergent extension assays demonstrate that XGIPC likely plays other undefined roles in modulating α5β1 function. XGIPC was found to be required for efficient trafficking of α5β1, as determined by α5β1 internalization assays in A6 cells. Together, my data indicate a critical role for XGIPC in modulating α5β1 integrin function during early embryonic morphogenesis.

Successful synaptic transmission at the neuromuscular junction depends on the precise alignment of the nerve terminals with the postsynaptic specialization of the muscle fiber. It is increasingly apparent that this precision is achieved during development and maintained in the adult through signals exchanged between motoneurons and their target muscle fibers that serve to coordinate their spatial and temporal differentiation. Several aspects of neuronal differentiation appear to be dependent on retrograde signals from the target and studies about synaptic modulation have now focused attention on the characterization of proteins that mediate retrograde signals regulating the organization and function of nerve terminals. According to the published evidences, we find Insulin-like growth factor-I (IGF-I ) might be one of these potential factors. The acute application of IGF-I, a factor which has been addressed to widely express in developing myocyte, dose-dependently enhances the spontaneous acetylcholine secretion at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch clamp recording. The IGF-I-induced potentiating effect is not abolished when calcium is eliminated from culture medium or bath application of pharmacological calcium channel blocker cadmium, indicating calcium influx through voltage-activated calcium channels are not required. We further define the roles of intracellular Ca2+ stores in IGF-I-induced synaptic potentiation. To approach this problem, Ca2+-ATPase inhibitor thapsigargin were initially used to deplete internal Ca2+ stores. IGF-I no longer elicited any changes in SSC frequency in thapsigargin-treated synapses suggesting that an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores may contribute to the facilitation of transmitter release induced by IGF-I. Application of membrane-permeable inhibitors of IP3-induced Ca2+ release 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) effectively occluded the increase of SSC frequency elicited by IGF-I. Furthermore, pretreatment of the cultures with ryanodine receptor antagonist 8-(dethylamino) octyl 3, 4, 5-trimethoxybenzoate (TMB-8) also blocked the IGF-I effects indicating that IGF-I activates IP3 and/or ryanodine pathway to initiate calcium release from intracellular stores which subsequently potentiate transmitter release. Treating cells with inhibitors of phosphoinositide-3 kinase (wortmannin and LY294002) and Phospholipase C-g (U73122), but not inhibitor of MAP kinase (PD98059) abolishes IGF-1-induced potentiation of synaptic transmission. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by KN-62 effectively blocks the effect of IGF-I. Taken collectively, our results obtained suggest that IGF-I potentiates neurotransmitter secretion by stimulating Ca2+ release from IP3 and ryanodine sensitive intracellular calcium stores via activate PI3 and/or PLC-g signaling cascades, which leading to an activation of CaMKII-dependent transmitter release.

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Graduate

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 163-177). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Virginia, 2002. / Includes bibliographical references (leaves 178-194). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 191-225). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 139-163). Available also in a digital version from Dissertation Abstracts.

Thesis (M.A.)--Central Connecticut State University, 1999. / Thesis advisor: Kathy Martin. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaves 42-44).