Robert M. Raphael - US grants

The objective of the research in this proposal is to understand the molecular mechanism of outer hair cell electromotility. This unique form of cellular motility is responsible for the remarkable sensitivity and exquisite frequency selectivity of mammalian hearing. Loss of electromotility is believed to be responsible for sensioneural hearing loss. Until we understand the molecular mechanism of electromotility, we will be powerless to undertake any clinical efforts to treat people suffering from hearing impairment. The research in this proposal plans to test the novel hypothesis that changes in membrane curvature are responsible for the electromechanical coupling process. This will be accomplished experimentally by testing the effects of various agents known to affect the ability of a membrane to bend on the outer hair cell's voltage dependent capacitance and motility. A theoretical model of electromotility will be developed which includes an analytic expression for the curvature elastic energy. This model will be used to predict the shift in the nonlinear capacitance with membrane tension. The model will also be used to predict the capacitance change expected from membrane bending which will be calculated from the Poisson- Boltzmann equation and membrane electrostatic theory.

0321275
Raphael
The ability to label individual molecules inside of living cells with fluorescent probes has revolutionized our understanding of basic cell biology and opened new opportunities to bioengineer living cells. Laser scanning confocal microscopy has proved to be a powerful and versatile technique for studying fluorescent molecules in cells. Rice University recognized the importance of this technique, and purchased a Zeiss LSM 410 confocal microscope in 1993. This microscope has supported numerous research projects in cell biology and bioengineering. However, there are serious limitations in the current technology, including limited ability to resolve molecules with overlapping emission spectra and the damage to cells that occurs with laser excitation. The importance and magnitude of these issues led Carl Zeiss, Inc. to expend a significant amount of time and resources to develop a user-friendly system that can overcome these problems. Zeiss recently integrated powerful new technologies, including novel algorithms for resolving overlapping emission spectra and the ability to use multi-photon laser excitation, into their new confocal microscope. The new system is called the 510 LSM META/NLO.

This Nanotechnology in Undergraduate Education (NUE) award to Rice University supports Dr. Kristen M. Kulinowski, Department of Chemistry, for her work to develop and implement a new course on nanotechnology. The course, entitled "Nanotechnology: Content and Context," aims to introduce the essential scientific and technical content of nanotechnology and the essential ethical, political and social contexts of nanotechnology research through a one-semester multi-disciplinary undergraduate course. Science/engineering majors and social science/humanities majors alike will benefit by learning about both the specific technology and the social impacts and cultural meaning of science and technology in the same class. Students will engage directly with both working scientists and with the heated public debate over its potential impacts on society. They will be asked to acquire a technical understanding of nanotechnology (e.g., the methods of visualization, experimentation, manufacture, and the evaluation of what is and is not technically feasible) as well as a nuanced understanding of scientific and technical research as a social and political process (issues of ethics, regulation, risk assessment, history, funding, intellectual property, controversy and conflict). By combining these skills in the classroom the proposed work will cultivate a critical and civil discussion of science and technology in an emerging field amongst a younger generation. Dr. Kulinowski is joined in this work by Professor Chris Kelty in the Anthropology department and several other faculty members from the schools of science, engineering and social science.

The proposal for this award was received in response to the Nanoscale Science and Engineering Education announcement, NSF 03-444, category NUE and was jointly funded by the Divison of Engineering Education and Centers (EEC) in the Directorate for Engineering (ENG) and the Division of Social and Economic Sciences (SES) in the Directorate for Social, Behavioral and Economic Sciences (SBE).

0449379
Raphael




The proposed research is in the field of membrane-based bionanotechnology with a focus on prestin, a protein found on outer hair cell membrane and responsible for hair cell electromobility and non-linear capacitance. The objective of the proposed project is to characterize the electromechanical characteristics of membranes containing prestin and to lay the foundation for bionanotechnological devices based on prestin.

[unreadable] DESCRIPTION (provided by applicant): Five years ago, the protein prestin (SLC26A5) was identified as the motor protein that drives electromechanical transduction in cochlear outer hair cells. Subsequent experiments have confirmed prestin is essential for both outer hair cell (OHC) electromotility and normal auditory function. This unique polytopic membrane protein contributes to the voltage sensor that detects changes in the transmembrane potential and to the motor mechanism of OHC electromotility. Prestin is a member of the SLC26A family of anion transporters which play critical roles in ion and fluid homeostasis as well as pH and cell volume regulation. The molecular basis of prestin motor function is presently unknown. The objective of this proposal is to advance our understanding of prestin function. In particular, we will test the hypothesis that prestin molecules self-associate to form functional complexes. The C-terminus of SLC26 family members contains a domain that is highly conserved throughout prokaryotes and eukaryotes, referred to as the STAS domain. Mutations in the STAS domain of several SLC26A proteins are responsible for a variety of human diseases including Pendred syndrome (SLC26A4), congenital chloride diarrhea (SLC26A3), and diastrophic dysplasia (SLC26A2). STAS domains from lower phyla have demonstrated interactions with the membrane. Based on these and other findings, we hypothesize that the STAS domain of prestin mediates prestin-prestin interactions and interactions between prestin and the membrane. Importantly, our preliminary data demonstrates that alterations in the membrane microenvironment alter prestin function. In this proposal, we will use biochemical, cellular, biophysical and optical (FRET and FRAP) approaches to probe the molecular interactions between prestin, prestin and the membrane, and the STAS domain and the membrane. Additionally, we will determine if SLC26A STAS domains can be functionally exchanged. The results from our studies will lead to a deeper understanding, not only of prestin function and the molecular basis of electromotility, but will also provide insights into the function of STAS domains from the SLC26A family. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cochlear outer hair cells enhance the sensitivity of mammalian hearing through active mechanical feedback powered by the membrane protein prestin. The long-term goal of our research efforts is to understand the mechanism by which prestin interacts with the membrane and responds to changes in the transmembrane potential. Preliminary research has established that prestin molecules self-associate, and that changes in prestin-prestin interactions alter the function and lateral organization of prestin in the membrane. We propose to extend these studies and further elucidate the relationship between the function of prestin and the formation of prestin complexes in the membrane. The first specific aim will be to determine the relationship between prestin oligomerization and function and establish if a particular oligomeric state represents the functional unit of prestin. To accomplish this aim we will utilize advanced optical imaging techniques, including fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), fluorescence recovery after photobleaching (FRAP) and single molecule imaging. The function of prestin will be assayed by measuring the nonlinear capacitance. Native and mutated forms of prestin will be studied in order to delineate the molecular motifs that mediate prestin-prestin and prestin-membrane interactions. One focus of the mutational studies will be on regions in the C-terminal STAS domain that are predicted to participate in protein-protein interactions. In a second specific aim, we will determine whether prestin-prestin and prestin-membrane interactions depend on voltage. This will be accomplished by performing FRET, FRAP and FLIM experiments in voltage-clamped cells. The successful completion of these aims will represent the first direct determination of voltage-induced molecular changes in prestin organization and contribute a powerful new tool to prestin research. We will also investigate the effect of agents that perturb prestin function and alter membrane properties on prestin-prestin and prestin-membrane interactions, including non-steroidal anti-inflammatory agents (NSAIDs), which are known to induce hearing loss and tinnitus. The sensitivity of human hearing depends on the proper function of a motor protein called prestin located in sensory outer hair cells. The malfunction or absence of prestin, as may occur through altered prestin-prestin interactions by ototoxic compounds, results in high-frequency hearing loss. Because of the conservation of the part of the prestin molecule we will study, our research also has relevance for understanding the molecular basis of other diseases such as congenital diarrhea and skeletal abnormalities including dwarfism.

This Integrative Graduate Education and Research Traineeship (IGERT) award provides Ph.D. students at Rice University with innovative training in neuroengineering, spanning the disciplines of neuroscience, electrical engineering, mechanical engineering, and bioengineering. In collaboration with Baylor College of Medicine and seven other universities and participating organizations, Rice University trainees are developing the tools to understand, interface with, model, and manipulate the nervous system.

Intellectual Merit: Due to improved technologies that enable neuroscientists to interact with brain cells, and due to the increasing types of neuroscientific data collected through electrical and optical methods, the neuroengineers who create and work with these complex data sets require highly specialized training. This program trains students in three specific areas: (1) cellular systems neuroengineering, which studies the nervous system?s signaling processes at the molecular and cellular levels; (2) engineering multi-neuron circuits, which involves collecting and analyzing data from groups of brain cells and devising methods to induce them to produce new functional responses; and (3) translational neuroengineering, which develops systematic approaches to improve clinical devices such as prosthetics and deep brain stimulators. Trainees in this program are learning to be technologically innovative; to be aware of social, cultural, and ethical aspects of neuroengineering; to communicate their work effectively to a wide variety of audiences; and to understand the pathways to commercialize their discoveries.

Broader Impacts: As this program trains neuroengineers to develop advanced solutions to functional and structural problems in the brain, a new problem-based learning curriculum will result and will be shared with the public through open education resources. By cultivating relationships with biomedical device companies, international researchers, and collaborators within two minority-serving institutions in Texas, students in this program are expanding the applications of their training and increasing participation in their research.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and leadership skills needed for the career demands of the future. The program is intended to establish new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries, and to engage students in understanding the processes by which research is translated into innovations that benefit society.

The Planning Grants for Engineering Research Centers competition was run as a pilot solicitation within the ERC program. Planning grants are not required as part of the full ERC competition, but intended to build capacity among teams to plan for convergent, center-scale engineering research.

The sense of hearing is a critically important ability that enables an individual to relate to the external world. Loss of hearing affects an individual?s health, function, quality of life and mortality. Current estimates are that hearing loss results in excess medical costs ranging from $3.3 to $12.8B in the United States, and over $750B worldwide. A recent report from the National Academies highlighted the need to recognize hearing loss as a major public health concern. This planning grant brings together a diverse team of researchers from Rice University, Baylor College of Medicine, the University of Southern California and Oregon Health Science University. This team will have complementary expertise in auditory neuroscience, cochlear implants, speech perception and devices that connect directly to neurons. This team will engage in a series of meeting and activities that will lay the foundation for an Engineering Research Center in Auditory Bioengineering. The proposed Center will work to develop new technologies, expand the engineering workforce and foster a culture of diversity and inclusion by supporting the deaf and hard of hearing, especially members of underserved populations. At the scientific level, the engineering advances in technologies for precise electrical stimulation of neurons will be broadly applicable to devices such as retinal implants, auditory implants and deep brain stimulators.

Recognizing the limitations of hearing aids and cochlear implants, the planning team will seek to revolutionize current hearing rehabilitative technology through transformative engineering enabled by a convergent approach involving several engineering disciplines. The major scientific goal will be to develop a next generation auditory implant that more intimately connects to spiral ganglion cells, more faithfully encodes sound, is smaller and easier to use, and is more economical. During the course of this grant, the planning team will critically evaluate the potential of recent technological advances to improve hearing health care and identify additional participants to perform center-scale research. This will be accomplished through a series of meetings held in in Houston, TX, Los Angeles, CA and Portland, OR. The team will engage stakeholders - including individuals with cochlear implants, hearing health care professionals, policy makers in hearing health care, and low-income individuals whose hearing health care needs are currently unmet. The team will used evidence-based strategies to recruit and develop the center leadership team. The team will also engage major cochlear implant manufacturers and identify other industrial partners.
The intellectual focus of this proposal will reside in a critical evaluation of the potential for the following thrust areas to serve as a foundation for convergent research in Auditory Bioengineering. Thrust 1: Enabling Technologies for Next Generation Auditory Implants. In this thrust, recent advances in engineering technologies such as carbon nanofiber electrodes for neural interfacing will be integrated into biomedical microsystems to produce an innovative design for a next generation auditory implant. Thrust 2: Speech Perception for Auditory Bioengineering. In this thrust, recent advances the cognitive aspects of speech perception will be harnessed to determine the relevant information that needs to be passed to auditory implants and how best to provide that information based on what is known about the neural code, with consideration that different languages are tuned to different acoustic features. Thrust 3: Global Health and Disparities Research in Auditory Rehabilitation. This thrust will seek to investigate and evaluate ways to expand access to auditory rehabilitation with hearing aids and cochlear implants, especially for underserved population.
The goal will be to develop an ERC in Auditory Bioengineering that will serve a national resource for auditory researchers, neuroengineers, audiologists and clinicians. With a central location in Houston, TX, the center will have national outreach and serve as a way to pool patient populations for studies on outcomes and disparities in auditory rehabilitation. This proposal will fill a key need in workforce development in training more students at the interface of hearing and speech research, who are prepared to employ modern engineering approaches for auditory rehabilitation.

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.

Project Summary/Abstract The vestibular inner ear supplies information about head motion and position to the brain, driving powerful reflexes that stabilize gaze and posture during head motions, and contributing to our sense of heading and orientation as we move through the world. Although we are not normally aware of these functions, their loss severely affects mobility by destabilizes vision and causes vertigo. Loss of vestibular function often originates in damage to hair cells and their synapses with the afferent vestibular nerve fibers that project to the brain. These hair cells, synapses, and afferent fibers have striking properties that are only partly understood. The longterm goal of this program of research is to build a comprehensive understanding of how vestibular information is generated and encoded in the inner ear. The current proposal focuses on the synaptic transfer of head motion signals from hair cells to primary vestibular neurons (Aim 1) and the subsequent initiation of action potentials (spikes) (Aim 2) in the mouse utricle, a model preparation for genetic, developmental and physiological studies. Principal methods are whole-cell patch clamping of hair cells and afferent neurons; immunolocalization of voltage-gated ion channels, pumps and synaptic markers; and computational modeling of the hair cells, synapses and afferent nerve fibers, incorporating current information on ion channels, pumps, and morphology. Vestibular afferent neurons make conventional bouton synaptic terminals on type II hair cells and unique calyceal contacts on type I hair cells. At both boutons and calyces, hair cells release vesicles of glutamate (?quantal? synaptic transmission) into the synaptic cleft, activating glutamate receptor-channels in the postsynaptic membrane to produce excitatory postsynaptic potentials and initiate spikes. At calyceal contacts, an additional ?non-quantal? transmission mechanism depends not on vesicular release or gap junctions, but rather on flow of ions from the hair cell through ion channels into the synaptic cleft and into the calyx through different ion channels. Postsynaptic responses to controlled stimulation of individual hair bundles show that quantal and non-quantal transmission modes can occur at the same calyceal synapse and that the non-quantal mode provides a fast signal that may be important for high-speed vestibular reflexes. Proposed experiments and modeling will investigate the impact of key hair cell ion channels on non-quantal transmission and delineate how quantal and non-quantal transmission are integrated in individual calyces and afferent nerve fibers. Other experiments will test how specific voltage-gated potassium and sodium channels in calyces and boutons shape the postsynaptic voltage response and spikes in the axonal initial segment. Immunolocalization has revealed remarkable concentrations of ion channels in microdomains of the calyx ending and nearby spike initiation zone. Experiments focus on channels with the potential to shape salient differences in response dynamics and spike timing between afferents of different connectivity (hair cell inputs) and different zones of the sensory epithelium.

Optical tweezers use light to non-invasively manipulate tiny objects (typically smaller than can be seen with the naked eye) including cells, nanoparticles, and individual molecules. The ability to hold, move, and stretch microscale objects with nanometer precision while simultaneously measuring fluorescence signals enables new scientific discoveries in biology, chemistry, and engineering. The Lumicks C-Trap system is state-of-the art optical tweezer system will support advanced experiments by a variety of researchers including single-molecule manipulation, mechanical properties of proteins and lipid membranes, and the study of forces governing colloidal and polymer assemblies. The knowledge obtained from optical tweezer studies have the potential to impact applications of national interest, ranging from new therapeutics and clinical treatments for various diseases to advanced materials for energy and sustainability needs.

Widescale adoption of optical tweezers has been limited due to the difficulty in constructing and maintaining a robust optical tweezer system that can correctly measure nanoscale forces. The objective of this MRI proposal is to acquire a Lumicks C-Trap to add optical tweezer capabilities to the shared research instrumentation facility at Rice University. The Lumicks C-Trap is a user-friendly optical tweezer instrument that combines wide-field and total internal reflection fluorescence microscopy to provide state-of-the-art imaging with simultaneous single-molecule manipulation. Additionally, the Lumicks C-Trap incorporates a novel microfluidic accessory that streamlines workflow and enables experiments to be performed under laminar flow in a temperature-controlled environment. Proposed experiments include tracking cellular processes, measuring protein and polymer surface interactions, probing the mechanical properties of biological membranes, and designing novel colloidal molecules. With the Lumicks C-Trap system, researchers and students will be trained to make sensitive force measurements, probe single-molecules, and manipulate materials with nanoscale precision. Acquisition of the C-Trap will advance the growing field of soft condensed matter and biophysical research at Rice University and lead to collaborative research with institutions in the greater Houston region, including the Texas Medical Center.

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.