R-3 Repository :: Browsing by Author "Raphael, Robert M."

Browsing by Author "Raphael, Robert M."

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3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography
(Frontiers, 2019) Triffo, William Jeffrey; Palsdottir, Hildur; Song, Junha; Morgan, David Gene; McDonald, Kent L.; Auer, Manfred; Raphael, Robert M.
Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features.
A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis
(Nature Publishing Group, 2013) Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T.C.; Tseng, Hubert; Souza, Glauco R.
There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.
A Novel Statistical Potential for Protein Beta-Sheets Prediction
(2014-04-25) Yu, Linglin; Ma, Jianpeng; Nordlander, Peter J.; Raphael, Robert M.
One of the most long-term challenging problems in biophysics studies for both computational scientists and experimentalists is protein structure prediction, whose goal is to obtain three-dimensional native protein structure from one-dimensional sequence. In protein structure prediction problems, a fundamental problem is Beta-sheets structure prediction. Though more than 85% of experimentally solved proteins contain Beta-sheet structures, limited methods have been found to rapidly and accurately predict the folded conformations. In this study, we proposed a novel statistical potential, named NP-Beta, to predict the protein Beta-sheet structure only based on the sequence information. We included three kinds of potential terms in NP-Beta, i.e. the self-packing term, the pair interacting term and the lattice term. The number of hydrogen bonds in Beta-sheets is also considered as a potential component, corresponding to a global penalty of the potential function. Computational tests show that the new statistical potential has an outstanding performance on native structure recognition from decoys comparing to the Beta-sheet specific potentials in literature. We will apply the potential to improve the prediction of Beta-strand arrangement and registration for beta proteins.
A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging
(Springer Nature, 2015) Tseng, Hubert; Gage, Jacob A.; Shen, Tsaiwei; Haisler, William L.; Neeley, Shane K.; Shiao, Sue; Chen, Jianbo; Desai, Pujan K.; Liao, Angela; Hebel, Chris; Raphael, Robert M.; Becker, Jeanne L.; Souza, Glauco R.
An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.
A thermodynamic analysis of tethers formed from lipid bilayers: Influence of electromechanical phenomena and entropically-driven tensions
(2006) Glassinger, Emily Elizabeth; Raphael, Robert M.
The material properties of biomembranes can be measured by forming a tether, a thin bilayer tube that extends from the membrane surface. The force required to maintain a tether at a given length depends upon both membrane properties as well as the mechanical, chemical and electrical environment. To characterize membrane material properties and responses, the influences of electromechanical energy, interfacial phenomena, and thermally-driven entropic tensions are considered in separate, thermodynamic models of tether formation. To determine how electric fields influence tether behavior, the energetic contributions arising from Maxwell stresses as well as from flexoelectric and piezoelectric coupling are included in an analysis of tether formation from an aspirated vesicle. For typical membrane elctromechanical coefficients, flexoelectric coupling alters the force required to form a tether of a given length, while piezoelectric coupling and Maxwell forces do not greatly change the force vs. tether length behavior. Given recent experiments demonstrate tethers formed from cellular membranes are sensitive to the transmembrane potential, an analysis is developed to characterize the electromechanical properties of unaspirated cellular membranes. Both flexoelectric and piezoelectric coupling energies as well as the voltage sensitivity of the interfacial tension and bending stiffness are included in the analysis. For typical membrane charge densities, small changes in tether force are calculated for the contributions of the interfacial phenomena. Inclusion of the electromechanical coupling energies leads to experimentally observable changes in tether force. These analyses are applied to determine the mechanism by which the motor protein prestin confers electromotility to the outer hair cell (OHC) of the mammalian cochlea. For the values of electromechanical coupling coefficients obtained from OHC deformation models, the tether force increases with depolarization for flexoelectric coupling and decreases for piezoelectric coupling. Since cellular membranes are typically under small entropically-driven tensions, the influence of thermally driven surface undulations on tether conformation is considered. By fitting the model to experimental tether data, the tension of a vesicle can be determined. The analyses developed in this thesis provide novel methods to determine many otherwise difficult to characterize membrane material properties and, thus, help to deepen understanding of the behavior of cellular membranes.
Advanced Computational Methods for Macromolecular Modeling and Structure Determination
(2013-12-05) Zhang, Chong; Ma, Jianpeng; Nordlander, Peter J.; Kiang, Ching-Hwa; Raphael, Robert M.
As volume and complexity of macromolecules increase, theories and algorithms that deal with structure determination at low X-ray resolution are of particular importance. With limited diffraction data in hand, experimentalists rely on advanced computational tools to extract and utilize useful information, seeking to determinate a three dimensional model that best fits the experiment data. Success of further studies on the property and function of a specific molecule - the key to practical applications - is therefore heavily dependent on the validity and accuracy of the solved structure. In this thesis I propose Deformable Complex Network (DCN) and introduce Normal Mode Analysis (NMA), which are designed to model the average coordinates of atoms and associated fluctuations, respectively. Their applications on structure determination target two major branches ? the positional refinement and temperature factor refinement. I demonstrate their remarkable performance in structure improvements based on several criteria, such as the free R value, overfitting effect and Ramachandran Statistics, with tests carried out across a broad range of real systems for generality and consistency.
Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation
(Liebert, 2013) Tseng, Hubert; Gage, Jacob A.; Raphael, Robert M.; Moore, Robert H.; Killian, Thomas C.; Grande-Allen, K. Jane; Souza, Glauco R.
A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle a-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.
Biophysical Interactions of the OHC Motor Protein Prestin: A Study at the Single Molecule Level
(2011) Kamar, Ramsey I.; Raphael, Robert M.
The exquisite frequency selectivity and amplification characteristics of mammalian hearing intimately depend on the fast electromechanical motion of the outer hair cells in the cochlea. This membrane based process, termed electromotility, is driven by the protein prestin which is uniquely present in the OHC lateral wall. Voltage dependent motility, in OHCs and mammalian cells expressing prestin, is accompanied by intramembranous charge movement which is widely considered a signature of electromotility and prestin function. How prestin converts changes in membrane potential into axial length changes of OHCs is currently not understood at the molecular level. Many electromotility models predict that prestin conformational changes are the underlying mechanism connecting charge movement and motility. Currently, however, only indirect evidence for a prestin conformational change is available. Various experiments have indicated that the oligomeric states of prestin may be an important determinant of function. Numerous reports have provided varying estimates of prestin oligomeric state. However, estimates have been based on measurements performed outside the membrane making, firm biophysical conclusions difficult. Biophysical studies of prestin function have demonstrated its dependence on membrane properties. Alterations of membrane cholesterol affect voltage dependence of charge movement and motility. In addition cholesterol manipulations cause spatial redistribution of prestin and possibly change prestin oligomeric state. However, the underlying cause for prestin sensitivity to cholesterol and its relation to membrane distribution is unknown. We have applied single molecule fluorescence (SMF) imaging, single particle tracking (SPT), and Förster resonance energy transfer (FRET) to investigate prestin interactions at the molecular level. The results of our SMF experiments have suggested that prestin forms mainly tetramers and dimers in the cell membrane. Using SPT to map the trajectories of prestin in the membrane, we have found that prestin undergoes diffusion in and hops between membrane confinements of varying size. In addition, we have found that cholesterol affects the size and confinement strength of the compartments but does not affect the diffusivity within the compartments. Finally, using a combination of electrophysiology and FRET we have demonstrated that prestin undergoes voltage dependent structural changes. In total, our results refine our molecular understanding of prestin function.
Characterization of Proteins Involved in Membrane Fusion- Atlastin and Munc18c
(2013-09-16) Verma, Avani; McNew, James A.; Braam, Janet; Raphael, Robert M.; Shamoo, Yousif; Wagner, Daniel S.
Membranes provide a barrier to cells and organelles, and allow the selective transport of molecules between compartments. Membrane fusion is essential for organelle biogenesis as well as trafficking of molecules between cellular compartments. Membrane fusion is also required for the formation of the branched network of tubules that make up the Endoplasmic Reticulum (ER). One protein implicated in ER fusion is Atlastin, a dynamin like GTPase. Mutations in Atlastin-1, among others, cause Hereditary Spastic Paraplegias (HSP), a group of neurological disorders that cause progressive weakness of lower extremities. We have shown that the C-terminal tail of atlastin is necessary for membrane fusion. The requirement of the C-terminal tail can be partially abrogated in an unstable lipid environment. This implies that the C-terminal tail of Atlastin plays a role in perturbing the lipid bilayer to allow membrane fusion. Understanding the molecular details of how Atlastin drives membrane fusion may help elucidate the pathogenesis of HSP. Intracellular fusion at the plasma membrane is SNARE mediated and regulated by Sec1p/Munc18 (SM) proteins. Increased rate of glucose transport into fat and muscles cells by translocation of glucose transporter GLUT4 in response to insulin is a SNARE regulated fusion process. Recent reports have linked Munc18c and Syntaxin4 with obesity and Type 2 diabetes. We characterized the function of Munc18c, an SM protein, in regulating GLUT-4 containing vesicle fusion with the plasma membrane. We have shown that Munc18c directly inhibits membrane fusion by interacting with its cognate SNARE complexes. Characterization of membrane fusion in a minimal system as the in vitro liposome fusion assay offers a powerful tool with which to finely dissect the mechanistic basis of SM protein function.
Coarse-grained Direct Phasing Method for Protein X-ray Crystallography
(2013-11-04) Chen, Dong; Ma, Jianpeng; Nordlander, Peter J.; Raphael, Robert M.
X-ray crystallography is the most powerful method to obtain the structural of biological molecules if the “phase problem” can be solved for the molecules under study. The phase problem arises from the loss of phase information in diffraction experiment. In all the solutions of the phase problem, the direct method is the only one that does not require additional experimental data or knowledge of homologous structures. It can determine the phase information directly from the observed structure factor magnitudes or intensities. However, the direct phasing method has limitations when applying to macromolecule. It is only applicable in molecules with up to about 1000 non-H atoms and requires ultra-high resolution (the Sheldrick's 1.2 Å rule) diffraction data that is not available in most protein crystallography experiments. To overcome the two limitations, here we propose a coarse-grained direct phasing method. This thesis will focus on how to break the 1.2 Å resolution requirement.
Computational modeling of potassium transport in the inner ear
(2008) Quraishi, Imran Habib; Raphael, Robert M.
In this thesis, I construct and evaluate biophysically realistic mathematical models of potassium transport in two inner ear epithelia. Deficits in potassium transport cause deafness and imbalance, because proper hearing and balance require the presence of an electrochemical potassium gradient. The first tissue considered is the cochlear stria vascularis, which produces the high endolymphatic potassium and endocochlear potential (EP). The prevailing theory for the operation of this epithelium is that one layer of cells provides a large current, which creates a low intrastrial potassium concentration that allows the EP to develop across the membranes of a second layer. The second tissue is the vestibular sensory epithelium, in which local ion accumulation in calyx-type synapses has been proposed to enhance synaptic signaling. I use computational models to study the behavior of both systems. For the stria vascularis, I applied compartmental analysis with the addition of equations for volume and electrical potentials. For the calyx synapse, I derive a distributed circuit model with Nernst-Planck electrodiffusion of potassium and a stochastic description of quantal transmission. Both models are based upon experimentally derived channel and transporter kinetics. The model of the stria vascularis accurately reproduces experimental measurements and confirms that the two-layer theory of EP generation is feasible with known channels and transporters. I can also estimate the potassium in the intrastrial space, which has not been accurately measured. Using the calyx model, I demonstrate nonquantal transmission in spite of high transporter densities, primarily by potassium accumulation and in a small part by ephaptic transmission. In addition to a direct effect on the afferent neuron, potassium increases the effective input resistance of the model hair cell, increasing quantal release. The model also exhibits retrograde transmission of afferent action potentials, which may be an observable measure of ephaptic transmission. These models provide information about variables that cannot easily be measured, such as the intrastrial and calyceal cleft potassium concentrations. Over time this work could grow into a complete multiscale model of inner ear ion transport, allowing virtual experiments of loss of function and restorative therapies.
Diflunisal inhibits prestin by chloride-dependent mechanism
(Public Library of Science, 2017) Duret, Guillaume; Pereira, Fred A.; Raphael, Robert M.
The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.
Effect of nonsteroidal anti-inflammatory drugs on the mechanical and electrical stability of phospholipid membranes
(2007) Zhou, Yong; Raphael, Robert M.
Non-steroidal anti-inflammatory drugs (NSAIDs) display powerful anti-inflammatory, analgesic and anti-pyretic activities. Serious side effects of NSAIDs, such as gastrointestinal (GI) bleeding and peptic ulcer disease, cause hospitalization and even death of many patients who take these drugs. This is thought to result from the ability of NSAIDs to induce a back-diffusion of luminal acids into GI tissues. NSAID-induced back-diffusion of luminal acids has both biochemical and biophysical aspects. The biochemical aspect of NSAID activity, i.e. the ability of NSAIDs to inhibit the enzyme cyclo-oxygenase, has been examined extensively. However, the biophysical aspect of NSAID cyto-toxicity, i.e. the ability of NSAIDs to directly induce proton permeation across a phospholipid layer covering the GI tract, is not well understood. In order to gain a deeper understanding of the cyto-toxicity of NSAIDs, the biophysical effect of NSAIDs on lipid membranes must be examined. The proton permeability of a phospholipid membrane depends on several factors: the packing of lipids, the membrane rigidity and deformability, the tendency for a membrane to form pores and the electrostatic properties of the membrane. We utilized a variety of experimental techniques, including micropipette aspiration of giant unilamellar vesicles, fluorescent spectroscopy and electrophoretic motility, to quantitatively characterize the effect of the sodium salt of salicylic acid, a classic anti-inflammatory agent, on mechanical and electrostatic properties of phospholipid membranes. We found that at near neutral pH, salicylate/salicylic acid decreases the bending stiffness of membranes composed of 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC) and induces pore formation. As the solution pH was lowered to simulate the condition in the GI tract, the packing stability of SOPC vesicles exposed to sodium salicylate was seriously disrupted and the membrane dipole potential was decreased significantly. Salicylate anions also alter the membrane interfacial charge distribution by partitioning into the membrane. Thus, both salicylic acid and salicylate anion partition into membranes and affect membrane mesoscopic properties that determine membrane ion permeability. The ability of salicylate/salicylic acid to induce membrane pores suggests that this anti-inflammatory agent increases the probability of protons permeating across a phospholipid layer in the GI tract. The larger membrane damage caused by salicylate/salicylic acid at acidic pHs correlates well with clinical findings of higher toxicity of these drugs at low stomach pHs. It further confirms that the NSAID-lipid interaction is crucial the integrity of the mucosa. Moreover, since membranes are intimately involved in many important cell functions, effects of salicylate/salicylic acid on membranes observed in this thesis have relevance beyond the GI tract.
Enhanced Sampling Method in Statistical Physics and Large-Scale Molecular Simulation of Complex Systems
(2014-04-25) Zang, Tianwu; Ma, Jianpeng; Kiang, Ching-Hwa; Raphael, Robert M.
In large-scale complex systems, traditional computational methods in equilibrium statistical mechanics such as Monte Carlo simulation and molecular dynamics in canonical ensemble often face the broken ergodicity issue, which highly reduces the performance and accuracy of simulation. The past decades have witnessed the development of generalized ensemble, which has significantly enhanced the efficiency of molecular simulation. In this thesis, we get a review of typical generalized ensembles, such as multi-canonical ensemble, parallel tempering, simulating tempering and continuous simulated tempering (CST). We also present a method called parallel continuous simulated tempering(PCST) for enhanced sampling in studying large complex. It mainly inherits and CST method in previous work, while adopts the spirit of parallel tempering, by employing multiple copies with different temperature distributions. The sampling efficiency of PCST was tested in two-dimensional Ising model, Lennard-Jones liquid and all-atom folding simulation of a small globular protein trp-cage in explicit solvent. The results demonstrate that the PCST method has significantly improved sampling efficiency compared with other methods and it is particularly effective in simulating systems with long relaxation time or correlation time.
Imaging and vibrometry of the mouse cochlear apex using spectral domain optical coherence tomography
(2014-03-13) Gao, Simon; Raphael, Robert M.; Oghalai, John S; Baraniuk, Richard G.; Brownell, William; Saggau, Peter; Tkaczyk, Tomasz S.
Hearing loss affects millions of Americans and is of increasing concern to an aging population. It can occur as a result of congenital malformations or damage to the functional soft tissues within the hearing organ, the cochlea. Current clinical imaging modalities such as magnetic resonance imaging and computed tomography do not have the necessary resolution to detect such changes and, furthermore, provide no functional information. As well, research into how the intracochlear tissues vibrate and thus transduce sound pressure waves into neural signals has stagnated because of the limitations inherent to currently available technologies. To address these challenges, we developed a spectral domain optical coherence tomography system to visualize and measure nanoscale vibrations of intracochlear structures. Using this system and mouse models, we first imaged excised cochlea from a transgenic mouse model of human hearing loss with an altered tectorial membrane. The soft tissue structures and expected anatomical variations were visible using OCT, and quantitative measurements confirmed the ability to detect critical changes relevant to hearing. We then compared the vibratory patterns of the intracochlear structures of live and dead normal hearing mice and found that active force generation by outer hair cells produced larger displacements of the tectorial membrane than any other structure, including the basilar membrane. As well, there was a traveling wave that emanated from the point of outer hair cell attachment and moved radially. This presumably propels fluid and drives the stimulation of inner hair cell stereociliary bundles. Because inner hair cells provide the majority of the afferent auditory input, these nanoscale movements thus describe how the forces produced by outer hair cells improve the auditory sensitivity and frequency selectivity of mammalian hearing.
Improved Biomolecular Crystallography at Low Resolution with the Deformable Complex Network Approach
(2013-07-24) Zhang, Chong; Ma, Jianpeng; Huang, Huey W.; Raphael, Robert M.
It is often a challenge to atomically determine the structure of large macromolecular assemblies, even if successfully crystallized, due to their weak diffraction of X-rays. Refinement algorithms that work with low-resolution diffraction data are necessary for researchers to obtain a picture of the structure from limited experimental information. Relationship between the structure and function of proteins implies that a refinement approach delivering accurate structures could considerably facilitate further research on their function and other related applications such as drug design. Here a refinement algorithm called the Deformable Complex Network is presented. Computation results revealed that, significant improvement was observed over the conventional refinement and DEN refinement, across a wide range of test systems from the Protein Data Bank, indicated by multiple criteria, including the free R value, the Ramachandran Statistics, the GDT (<1Å) score, TM-score as well as associated electron density map.
Investigating the lateral mobility of outer hair cell plasma membrane constituents by fluorescence recovery after photobleaching
(2008) Organ, Louise E.; Raphael, Robert M.
Mammalian hearing exhibits exquisite sensitivity and frequency selectivity attributed to the unique properties of cochlear outer hair cells (OHCs). These sensory epithelial cells are electro-mechanical transducers, capable of converting sound-induced electrical signals into mechanical forces which provide feedback via a mechanism known as the cochlear amplifier. In a process aptly termed electromotility, electro-mechanical transduction manifests as whole-cell axial length changes in OHCs that occur in response to changes in the transmembrane potential. The polytopic motor protein prestin functions as the voltage sensor and molecular motor, both in OHCs and when expressed in heterologous systems. As the molecular mechanism(s) of electromotility remain unknown, examining the structure and function of prestin is a major focus of ongoing research. Since changes in membrane composition and biophysical properties affect protein function and organization, we are particularly interested in membrane-protein interactions. Recent studies suggest that manipulations in membrane cholesterol levels reversibly shift the membrane microdomain distribution of prestin, modulate prestin oligomerization states, and alter prestin function, thus regulating electromotility through membrane-protein interactions. Measurements of protein and lipid lateral mobility provide a powerful tool to dynamically examine such interactions. We hypothesize that OHC plasma membrane cholesterol levels affect electromotility either through microdomain-mediated mechanisms that cluster or segregate prestin molecules or via alterations in the material properties of the membrane, which in turn affect the resident proteins. Using fluorescence recovery after photobleaching (FRAP), we evaluated the lateral mobility of both protein and lipid components of the OHC. Then we showed that the diffusion of both prestin in HEK cells and lipids in OHCs is altered in response to changes in membrane cholesterol concentration. Cumulatively, this work demonstrates the complexity of prestin-membrane interactions and highlights the importance of their inclusion in current models of prestin function and electromotility.
Low Resolution ab initio Phasing Method by Modification of Density and Phase in Real and Reciprocal Space
(2013-09-30) Liao, Yunxiang; Ma, Jianpeng; Raphael, Robert M.; Kiang, Ching-Hwa
Phasing problem in X-ray structure determination can be challenging. Several methods, such as Isomorphous Replacement and Molecule Replacement, are frequently used. But their success relies on the availability of either an isomorphous heavy-atom derivative or a high identity homologous model. In this thesis, a low resolution ab initio phasing method is proposed. A large number of trial phases value and their corresponding high density masks are generated and modified alternately in reciprocal and real space. Then, the output phase sets are averaged to give the estimated phases and figure of merit which are capable of capturing key feature of the molecules’ low resolution envelope by Fourier synthesis. Smoothed particle hydrodynamics is employed to animate the high density masks’ modification process. The method is tested and compared with Lunin’s connectivity-based phasing method which also takes advantage of geometric properties of high density masks.
Nonquantal transmission at the vestibular hair cell–calyx synapse: KLV currents modulate fast electrical and slow K+ potentials
(PNAS, 2023) Govindaraju, Aravind Chenrayan; Quraishi, Imran H.; Lysakowski, Anna; Eatock, Ruth Anne; Raphael, Robert M.
Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal called a calyx. The vestibular hair cell–calyx synapse supports a mysterious form of electrical transmission that does not involve gap junctions, termed nonquantal transmission (NQT). The NQT mechanism is thought to involve the flow of ions from the presynaptic hair cell to the postsynaptic calyx through low-voltage-activated channels driven by changes in cleft [K+] as K+ exits the hair cell. However, this hypothesis has not been tested with a quantitative model and the possible role of an electrical potential in the cleft has remained speculative. Here, we present a computational model that captures experimental observations of NQT and identifies features that support the existence of an electrical potential (ϕ) in the synaptic cleft. We show that changes in cleft ϕ reduce transmission latency and illustrate the relative contributions of both cleft [K+] and ϕ to the gain and phase of NQT. We further demonstrate that the magnitude and speed of NQT depend on calyx morphology and that increasing calyx height reduces action potential latency in the calyx afferent. These predictions are consistent with the idea that the calyx evolved to enhance NQT and speed up vestibular signals that drive neural circuits controlling gaze, balance, and orientation.
Quantitative FLIM-FRET Measurement of Voltage Dependent Prestin Conformational Changes
(2013-09-16) Mooney, Chance; Raphael, Robert M.; Diehl, Michael R.; Mittleman, Daniel M.
The transmembrane protein prestin forms an integral part of the mammalian sense of hearing by providing the driving force for the electromotility of the outer hair cell, a specialized cell that resides within the cochlea. This provides the cochlea with an ability to amplify mechanical vibrations, allowing for a high degree of sensitivity and selectivity in auditory transduction. The phenomenon, driven by changes in the transmembrane potential, is thought to be the result of conformational changes in self-associating prestin oligomers. We have previously utilized Forster resonance energy transfer (FRET), by both sensitized emission and acceptor photobleach methods, to detect prestin self -association. While these methods can qualitatively confirm prestin-prestin association, determining nanoscale changes in prestin organization requires greater accuracy than either technique provides. In this thesis, a FRET methodology based on fluorescence lifetime imaging (FLIM), detected by time correlated single photon counting (TCSPC), is implemented and utilized to quantitatively measure conformational changes within prestin-prestin oligomers in response to voltage stimulus.

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