Christopher Turner - US grants

I have recently identified a novel vinculin-binding protein, paxillin. This proposal is aimed at further characterizing paxillin, its interaction with vinculin and at identifying other paxillin-binding proteins. These data are expected to contribute to our understanding of cytoskeletal- membrane interactions. The nucleotide sequence of paxillin will be obtained from paxillin cDNA. The resulting predicted amino acid sequence will be analyzed for potential homologies with other proteins and for suggestions of possible enzymatic or structural function. Appropriate biochemical assays will be performed on the purified protein to confirm/disprove such indications. The paxillin cDNA will be transfected in whole or in part into mammalian cells to determine which regions of the protein are required for focal adhesion localization. In parallel, partial or full length fusion proteins of paxillin and vinculin will be used in in vitro assays to map their binding sites. The interaction of these two proteins will also be examined at the electron microscope level. Following transformation by Rous sarcoma virus, paxillin contains high levels of phosphotyrosine. The role of paxillin phosphorylation in the loss of focal adhesion integrity during viral or chemically induced transformation will be investigated. The effects of paxillin phosphorylation on vinculin binding will be determined in vitro. In an attempt to identify novel paxillin-binding proteins I will isolate protein complexes containing paxillin from cells grown in culture possessing focal adhesions at their interface with the substratum. Cells will be gently permeabilized and, in some cases, the actin cytoskeleton stabilized with phalloidin. Proteins will be differentially extracted from these cells using changes in ionic strength, pH and divalent cation concentrations. Proteins released in conjunction with paxillin will be co-isolated by immunoprecipitation of paxillin under non-denaturing conditions. Where appropriate, experiments will be performed in the presence of chemical cross-linkers to stabilize interactions between paxillin and its associated proteins. Antibodies will be generated against any novel paxillin-binding proteins and these will be used to study further the proteins regarding their role in cytoskeletal membrane organization.

DESCRIPTION (provided by applicant): Cell migration is essential for normal embryonic development, tissue repair and immune surveillance, but is also a contributing factor in mental retardation, developmental defects, tumor cell invasion and tissue fibrosis. It is a highly dynamic process requiring exquisite spatial and temporal control of cell adhesion to the extracellular matrix (ECM) in coordination with remodeling of the actin cytoskeleton. The Rho family GTPases play a central role in this regulation but the mechanisms controlling the activity of their key regulators, the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) remain poorly understood. Paxillin is a multi-domain scaffold/adapter protein, which recruits numerous structural and signaling molecules to cell adhesion sites and thereby functions as a central hub in the regulation cell migration. Aim 1 of this proposal will test the hypothesis that paxillin coordinates the spatial-temporal regulation of Rho family GTPase signaling and focal adhesion dynamics by establishing a local signaling network comprising the ARF GAP PKL/GIT2, the guanine nucleotide exchange factor Vav2 and the tyrosine phosphatase PTP-PEST. Hic-5, a close relative of paxillin is upregulated during epithelial-mesenchymal transition to promote cell migration via Rho-ROCK signaling and is an important regulator of cell motility, as well as patho-physiologic matrix remodeling in myofibroblasts. In Aim 2, using 2D- and 3D-matrix model systems, we will dissect the mechanism through which Hic-5 controls cell migration and contractility and test the hypothesis that Hic-5 functions both independently and in conjunction with paxillin to regulate these processes. To accomplish these goals, we will suppress endogenous protein expression by RNA interference or express mutant proteins in fibroblasts and utilize confocal fluorescence time-lapse microscopy, combined with Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Resonance Energy Transfer (FRET) analysis to evaluate cell morphology, polarity and migration as well focal adhesion dynamics and spatial- temporal changes in protein- protein interactions and Rho family GTPase activity. This will be combined with biochemical analysis of changes in intracellular signaling to include GEF activity assays, protein phosphorylation profiling and protein- protein interactions. Completion of these Aims will elucidate the roles of paxillin and Hic-5 and their potential interactions in regulating cell migration vi modulation of the Rho GTPase system. PUBLIC HEALTH RELEVANCE: ll movement is essential for normal processes such as embryonic development and tissue repair but it is also a key factor in cancer progression, tissue fibrosis and several cardiovascular and neurodegenerative disorders. Information gained from the proposed study will contribute to our understanding of how the cell migration machinery is regulated and thereby will potentially identify novel targets for corrective therapies for migration-associated disorders.

DESCRIPTION: This project will identify the acoustic and articulatory features of the (mis)perceptions of speech sounds made by individuals with hearing loss who use hearing aids and cochlear implants. The project will also investigate the effects of signal modifications designed to overcome these misperceptions. Two procedures will be used to identify the frequency regions of and types of errors that these listeners make. The first procedure, the correlational method, is a new technique that yields rapid and reliable estimates of how listeners use various frequency regions and features of speech. This method holds considerable promise for use in clinical testing, the design and fitting of hearing aids, and also for evaluating and programming processors in cochlear implant patients. The second procedure, testing with filtered speech, is a more time-tested method, yielding much the same information, but in a longer and less efficient fashion. In the proposed experiments, these two methods will be used in conjunction to test the validity of each. In addition, both methods can be used to test hypotheses concerning the degree of hearing loss and the ability of patients to use audible speech information. If warranted, the correlational method will be further developed for clinical application. The information obtained from the correlational method applied to hearing-impaired patients will suggest some signal modifications and strategies for hearing aids and cochlear implants. Additional experiments will test the efficacy of these strategies upon speech recognition in listeners with sensorineural hearing loss.

DESCRIPTION (provided by applicant): Integrin-mediated interaction of cells with the extracellular matrix control a diverse set of physiologic processes including cell migration during embryogenesis and metastasis, cell proliferation, tissue maintenance, differentiation and repair. Protein complexes associated with the integrin cytoplasmic domains facilitate integrin signaling and interactions with the actin cytoskeleton. This proposal will focus on the importance of recently identified interactions between the integrin-linked kinase ILK, the actin binding protein actopaxin and the molecular adapter protein paxillin in the regulation of cell adhesion, motility, proliferation and differentiation. In Aim 1 epitope-tagged mutant cDNAs of ILK and actopaxin will be transfected into fibroblasts followed by co-precipitation experiments to further characterize the ILK and actopaxin binding domains. The importance of these domains for subcellular localization will be assessed by immunofluorescence microscopy. We have recently demonstrated that actopaxin mutants perturb cell adhesion/spreading on collagen. Experiments proposed in Aim 2 will identify the molecular basis for this defect by assaying for changes in integrin function as well as effect on downstream signaling events. The role of ILK and actopaxin in mediating cell motility will be addressed using time-lapse microscopy, Boyden chamber and wound assays. In Aim 3 we will investigate the importance of ILK-actopaxin associations in regulating cell proliferation and differentiation. Sites of actopaxin phosphorylation resulting from associated cyclinB/cdc2 kinase activity will be delineated. The role of phosphorylation of these sites in regulating protein-protein interactions and cytoskeleton changes associated with transition through mitosis will be examined by co-precipitation analysis and immunofluorescence microscopy. Potential effects on proliferation and cell survival will be examined. The involvement of ILK and actopaxin interactions in regulating cell differentiation will be examined in the context of skeletal muscle myoblast differentiation. Together these experiments will address evolutionarily conserved mechanisms of protein linkages between the extracellular matrix and the actin cytoskeleton that are of importance to the understanding of cardiovascular and musculoskeletal defects and metastatic transformation.

DESCRIPTION (provided by applicant): Three sets of experiments are proposed that address clinically-relevant questions for the management of hearing-impaired persons. The information obtained will assist in deciding which device (hearing aid, traditional cochlear implant, or short-electrode cochlear implant) is most appropriate for a particular patient. The project will also address the question of whether a device is performing adequately in the transmission of speech cues to the patient. Specifically, the project will 1) obtain speech recognition data from normal-hearing listeners using a simulation of the short-electrode cochlear implant to determine which types of hearing loss configurations might be appropriate candidates for this device; 2) to obtain speech recognition in noise data from listeners with cochlear implants and hearing aids to determine the potential advantages or disadvantages of a cochlear implant (both short- and long-electrode) for various degrees of hearing loss; and 3) to use a direct test of the ability to perceive the spectral and temporal cues of speech to predict the abilities of hearing aids and cochlear implants to recognize speech.

Experiments are proposed that address clinically relevant questions for the management of hearingimpaired patients. The information obtained will assist in deciding which approach for cochlear implantation is most appropriate for a particular patient. Considerable evidence is accumulating that for many patients the preservation of residual acoustic hearing, in addition to the stimulation provided by a cochlear implant, can provide important advantages compared to using only a hearing aid, or only a cochlear implant. One striking advantage is observed for understanding speech in background noise. In addition, this combined acoustic plus electric (A + E) hearing can expand the range of patients that can benefit from electrical stimulation, many of whom were unable to receive adequate benefit from a hearing aid. The proposed studies will continue our ongoing investigation of A + E hearing using the existing Hybrid (short-electrode) device as well as a new generation of longer, less-traumatic electrodes. The advantages and shortcomings of each approach will be measured in terms of preservation of hearing as well as speech understanding in quiet and in noise, for various patients. The goal is to develop eligibility criteria for the different device types, based upon the expected outcomes for various patient characteristics. Additional experiments are described that are designed to discover the mechanisms by which acoustic plus electric stimulation is integrated as an auditory sensation and what factors determine the success of the A + E approach in individual patients.

DESCRIPTION (provided by applicant): Previous research suggests that an approach that includes a combination of acoustic and electrical stimulation within the same ear can be particularly effective for this group of hearing impaired listeners (von Ilberg et al., 1999;Gantz and Turner, 2003;Turner et al., 2008). This approach has been termed "Combined Electric and Acoustic (A+E) Hearing" and requires implantation of a Hybrid cochlear implant (CI). Preliminary results with the Hybrid CI have been very encouraging. Hybrid CI users can outperform traditional CI users on many tasks, including perception of music and speech in noise. While such results are promising, many important questions remain relative to the most appropriate signal processing techniques to use to combine the acoustic and electric stimuli. This study addresses a very specific goal: to determine how best to divide the acoustic speech spectrum between the acoustic and electric stimulation modes. We hypothesize that manipulation of this variable will have a significant effect on performance, and that understanding more about how to combine the acoustic and electrical signals within the same ear will lead to significantly improved levels of performance with the CI. We propose to systematically vary the way the hearing aid and/or cochlear implant are programmed and to assess the impact of those changes on performance. The specific changes in programming will include varying the range of frequencies allotted for acoustic and electric stimulation modes as well as the degree of overlap between the two frequency ranges. We propose to evaluate the effectiveness of these different approaches to programming the two devices using a range of both speech perception tests as well as a series of electrophysiologic measures that may be more applicable for difficult-to-test or pediatric populations. Hybrid CIs are a new technology that addresses a very significant need for a relatively large population. The proposed research will determine how best to approach fitting the two devices for individual hearing impaired listeners in order to maximize the effectiveness of this A+E technology in a relatively short time. PUBLIC HEALTH RELEVANCE: Well over 28 million Americans suffer from some degree of hearing loss. Many, because of the specific configuration of their hearing loss, have too much hearing to be considered for a cochlear implant but because of the configuration of their hearing loss, are not able to benefit from conventional hearing aids. These hearing impaired individuals struggle tremendously - particularly in noisy listening environments - and the impact on their lives and the public as a whole is significant. Preliminary research suggests that a new type of cochlear implant (CI), the Nucleus Hybrid CI, that is designed to preserve acoustic low frequency hearing while allowing high frequency components in speech or other environmental sounds to be processed electrically, can result in substantially improved hearing. Importantly, the ability of these listeners to continue to use their residual low frequency hearing to process acoustic signals and to combine that with electrical hearing in the same ear has resulted in significantly improved performance on tests that measure understanding of speech in background noise and enhanced perception of music. Currently, however, the number of individuals who use this technology is small. There is still a great deal that is not known about how these individuals will perform relative to patients who use a traditional CI and importantly, the best way to program the hearing aid and/or speech processor of the cochlear implant to optimize hearing abilities in this subject group has not been determined. That is the specific challenge this proposal seeks to address. The goal is to determine how best to divide the acoustic signal between the cochlear implant and the hearing aid. Outcome will be assessed using both behavioral as well as electrophysiologic methods that will be needed when the candidacy criteria for the Hybrid device is expanded to include pediatric or otherwise difficult-to-test populations. Such information can reasonably be collected within the two year time period of this grant and has the potential to dramatically accelerate the acceptance of this strategy by the medical community for this relatively large but previously underserved population.

Project 1: Acoustic Plus Electric Hearing Preservation of residual acoustic hearing during cochlear implantation has become an important improvement in the performance of cochlear implants. Not only does it improve the performance of implants (particularly for noisy, real-world listening conditions) but also allows the treatment of patients with severe high-frequency hearing loss, who have substantial low-frequency hearing. This project proposes to continue this work on combining acoustic plus electric (A+E) hearing. In addition to the overall goal of improving patient care for hearing loss, several unique research opportunities arise from this work. The first opportunity arises because of the new population of patients that will be implanted with these A+E devices. Never before have patients with such high levels of pre-operative residual hearing been implanted in such a large- scale project. We are at the same time seeing levels of performance for the transmission of speech through the short electrode that are surprising in light of the previous literature. This will allow us to re-examine some of the commonly held beliefs about the limitations of electric stimulation due to channel interaction. The second opportunity arises because the short-electrode Hybrid implant assigns low- and mid-frequency speech bands to extreme basal locations in the cochlea. Thus we have a unique opportunity to study the effects of remapping, neural plasticity, and adaptation to highly-distorted place-frequency maps in the cochlea.

DESCRIPTION (provided by applicant): The migration of cancer cells away from the primary tumor mass and their subsequent metastasis to distant organs is regarded as a fatal step in cancer progression and is associated with the majority of cancer mortalities. Furthermore, individual cancer cells appear to be able to evade current pharmacologic intervention of invasion and metastasis by switching between mesenchymal and amoeboid modes of motility. The cellular mechanisms controlling this phenotypic plasticity are poorly understood. We have recently identified distinct functions for the closely related adhesion-associated scaffold proteins paxillin and Hic-5 in the regulation of tumor cell plasticity, invasion and metastasis. In this proposal, using established cancer cell lines, as well as cells isolated from primary tumors, we will apply state-of-the art real-time imaging techniques to track tumor cell morphology and migration as well as adhesion and cytoskeletal dynamics in 3D-extracellular matrix in vitro model systems. Xenograft studies in mice will be used to evaluate the relative impact of paxillin and Hic-5 signaling on tumor progression and metastasis in vivo. We will use RNA interference and mutant protein expression to dissect the respective roles for paxillin and Hic-5 in controlling the mode of tumor cell invasion and identify the pertinent functional domains and signaling pathways. We will use similar approaches to study a role for paxillin in the regulation of matrix metalloproteinase-2 (MMP-2) trafficking and secretion to control mesenchymal tumor invasion strategies. Hic-5 is upregulated during TGF-¿-induced epithelial mesenchymal transition. The role of Hic-5 in TGF-¿-dependent cell invasion, through the formation and function of matrix-degrading invadopodia, will also be examined. The proposed studies will provide insight into the underlying cellular mechanisms controlling tumor cell migration and invasion and the coordination of their phenotypic plasticity and may in the future suggest novel strategies for detection or treatment of metastatic cancers.