The Single-Molecule Approach to Membrane Protein Stoichiometry.

The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background levels. Their fluorescence decreases in discrete equal-amplitude steps, consistent with the photobleaching of single fluorophores. We count the number of steps required to photobleach each molecule. The molecular stoichiometry is then deduced using a binomial model.

Circadian Pattern of Melatonin MT1 and MT2 Receptor Localization in the Rat Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker. The pineal hormone melatonin is involved in the regulation of circadian phase. As a part of the circadian system, its synthesis and secretion is under SCN control. On the other hand, melatonin feeds back on the SCN to regulate its function. Melatonin has two specific windows of time at which it regulates SCN function, namely dusk and dawn. It has been suggested that melatonin exerts its effect on the SCN during that specific window of time via one or both of its specific receptors, MT1 or MT2. The hypothesis that the density of these receptors varies across the circadian cycle was tested. Using immunohistochemistry with receptor-specific antibodies, the localization and distribution of melatonin receptors MT1 and MT2 was studied in the SCN at different Zeitgeber times (ZT): ZT 11-13 (dusk), 23-01 (dawn), 5-7 (mid-day), and 17-19 (midnight). Our results show that MT1 receptor density significantly increased at dusk relative to dawn and midnight (p<0.01 and p<0.001 respectively). Although MT1 receptors were widespread in the SCN and parts of the optic chiasm at dusk, they were restricted to the SCN during the mid-day period. MT2 receptors were not detected in the SCN. Thus, we find that melatonin receptor MT1 density and distribution varies with circadian time. This creates a time window during which melatonin can affect the operation of the SCN. We also find that melatonin regulates SCN function via MT1 receptors with a minimal role for MT2.

Use of a novel cell adhesion method and digital measurement to show stimulus-dependent variation in somatic and oral ciliary beat frequency in Paramecium.

When Paramecium encounters positive stimuli, the membrane hyperpolarizes and ciliary beat frequency increases. We adapted an established immobilization protocol using a biological adhesive and a novel digital analysis system to quantify beat frequency in immobilized Paramecium. Cells showed low mortality and demonstrated beat frequencies consistent with previous studies. Chemoattractant molecules, reduction in external potassium, and posterior stimulation all increased somatic beat frequency. In all cases, the oral groove cilia maintained a higher beat frequency than mid-body cilia, but only oral cilia from cells stimulated with chemoattactants showed an increase from basal levels.

The conserved tetrameric subunit stoichiometry of Slc26 proteins.

The Slc26 family proteins, with one possible exception, transport anions across membranes in a wide variety of tissues in vertebrates, invertebrates, and plants. Mutations in human members of the family are a significant cause of disease. Slc26 family proteins are thought to be oligomers, but their stoichiometry of association is in dispute. A recent study, using sequential bleaching of single fluorophore-coupled molecules in membrane fragments, demonstrated that mammalian Slc26a5 (prestin) is a tetramer. In this article, the stoichiometry of two nonmammalian prestins and three human SLC26 proteins has been analyzed by the same method, including the evolutionarily-distant SLC26A11. The analysis showed that tetramerization is common and likely to be ubiquitous among Slc26 proteins, at least in vertebrates. The implication of the findings is that tetramerization is present for functional reasons.

Single molecule imaging approach to membrane protein stoichiometry.

Recent technical advances have enabled the imaging of single fluorescent molecules. The application of single molecule visualization techniques has opened up new avenues of experimentation in biology at the molecular level. In this article, we review the application of single fluorescent molecule visualization and analysis to an important problem, that of subunit stoichiometry in membrane proteins, with particular emphasis on our approach. Single fluorescent molecules, coupled to fluorescent proteins, are localized in the membranes of cells. The molecules are then exposed to continuous low-level excitation until their fluorescent emissions reach background levels. The high sensitivity of modern instrumentation has enabled direct observations of discrete step decreases in the fluorescence of single molecules, which represent the bleaching of single fluorophores. By counting the number of steps over a large number of single molecules, an average step count is determined from which the stoichiometry is deduced using a binomial model. We examined the stoichiometry of a protein, prestin, that is central to mammalian hearing. We discuss how we prepared, identified, and imaged single molecules of prestin. The methodological considerations behind our approach are described and compared to similar procedures in other laboratories.

Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging.

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells.

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.

Prestin in HEK cells is an obligate tetramer.

The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiometry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.

The roles of conserved and nonconserved cysteinyl residues in the oligomerization and function of mammalian prestin.

The creation of several prestin knockout and knockin mouse lines has demonstrated the importance of the intrinsic outer hair cell membrane protein prestin to mammalian hearing. However, the structure of prestin remains largely unknown, with even its major features in dispute. Several studies have suggested that prestin forms homo-oligomers that may be stabilized by disulfide bonds. Our phylogenetic analysis of prestin sequences across chordate classes suggested that the cysteinyl residues could be divided into three groups, depending on the extent of their conservation between prestin orthologs and paralogs or homologs. An alanine scan functional analysis was performed of all nine cysteinyl positions in mammalian prestin. Prestin function was assayed by measurement of prestin-associated nonlinear capacitance. Of the nine cysteine-alanine substitution mutations, all were properly membrane targeted and all demonstrated nonlinear capacitance. Four mutations (C124A, C192A, C260A, and C415A), all in nonconserved cysteinyl residues, significantly differed in their nonlinear capacitance properties compared with wild-type prestin. In the two most severely disrupted mutations, substitution of the polar residue seryl for cysteinyl restored normal function in one (C415S) but not the other (C124S). We assessed the relationship of prestin oligomerization to cysteine position using fluorescence resonance energy transfer. With one exception, cysteine-alanine substitutions did not significantly alter prestin-prestin interactions. The exception was C415A, one of the two nonconserved cysteinyl residues whose mutation to alanine caused the most disruption in function. We suggest that no disulfide bond is essential for prestin function. However, C415 likely participates by hydrogen bonding in both nonlinear capacitance and oligomerization.

Metabolic imaging of the organ of corti--a window on cochlea bioenergetics.

Hair cell loss is a major cause of sensorineural hearing loss. We have developed a method to examine metabolic events in hair cells in response to stimuli known to cause hair cell loss, such as acoustic trauma and aminoglycoside administration. The method employs two-photon excitation of the metabolic intermediate, reduced nicotinamide adenine dinucleotide (NADH), in hair cell mitochondria in an explanted mouse cochlea. Using this method, we show evidence that the aminoglycoside gentamicin selectively affects the level of mitochondrial NADH in outer hair cells, but not inner hair cells, within minutes of administration.

Fluorescence microscopy methods in the study of protein structure and function.

As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we describe two fluorescence microscopic methodologies applied to the outer hair cell-specific membrane protein prestin: the intensity and fluorescence lifetime (FLIM) variants of FRET (Fluorescence Resonance Energy Transfer), used in the study of protein-protein interactions, and the Scanning Cysteine Accessibility Method (SCAM), used for the determination of protein conformation. The methods are readily adaptable to other proteins.

Evolutionary insights into the unique electromotility motor of mammalian outer hair cells.

Prestin (SLC26A5) is the molecular motor responsible for cochlear amplification by mammalian cochlea outer hair cells and has the unique combined properties of energy-independent motility, voltage sensitivity, and speed of cellular shape change. The ion transporter capability, typical of SLC26A members, was exchanged for electromotility function and is a newly derived feature of the therian cochlea. A putative minimal essential motif for the electromotility motor (meEM) was identified through the amalgamation of comparative genomic, evolution, and structural diversification approaches. Comparisons were done among nonmammalian vertebrates, eutherian mammalian species, and the opossum and platypus. The opossum and platypus SLC26A5 proteins were comparable to the eutherian consensus sequence. Suggested from the point-accepted mutation analysis, the meEM motif spans all the transmembrane segments and represented residues 66-503. Within the eutherian clade, the meEM was highly conserved with a substitution frequency of only 39/7497 (0.5%) residues, compared with 5.7% in SLC26A4 and 12.8% in SLC26A6 genes. Clade-specific substitutions were not observed and there was no sequence correlation with low or high hearing frequency specialists. We were able to identify that within the highly conserved meEM motif two regions, which are unique to all therian species, appear to be the most derived features in the SLC26A5 peptide.

Localization of betav tubulin in the cochlea and cultured cells with a novel monoclonal antibody.

Tubulin, the dimeric structural protein of microtubules, is a heterodimer of alpha and beta subunits; both alpha and beta exist as numerous isotypes encoded by different genes. In vertebrates the sequence differences among the beta(I), beta(II), beta(III), beta(IV) and beta(V) isotypes are highly conserved in evolution, implying that the isotypes may have functional significance. Isotype-specific monoclonal antibodies have been useful in determining the cellular and sub-cellular distributions and possible functions of the beta(I), beta(II), beta(III), and beta(IV) isotypes; however, little is known about the beta(V) isotype. We here report the creation and purification of a monoclonal antibody (SHM.12G11) specific for beta(V). The antibody was designed to be specific for the C-terminal sequence EEEINE, which is unique to rodent and chicken beta(V). The antibody was found to bind specifically to the C-terminal peptide EEEINE, and does not cross-react with the carboxy-termini of either alpha-tubulin or the other beta-tubulin isotypes. However, the antibody also binds to the peptide EEEVNE, but not to the peptide EEEIDG, corresponding respectively to the C-terminal peptides of bovine and human beta(V). Immunofluorescence analysis indicates that beta(V) is found in microtubules of both the interphase network and the mitotic spindle. In gerbils, beta(V) also occurs in the cochlea where it is found largely in the specialized cells that are unique in containing bundled microtubules with 15 protofilaments.

Absence of voltage-dependent compliance in high-frequency cochlear outer hair cells.

Cochlear outer hair cells are the key element in a mechanical amplification process that enhances auditory sensitivity and tuning in the mammalian inner ear. The electromotility of outer hair cells, that is, their ability to extend or contract at acoustic frequencies, is proposed to be the source of the mechanical amplification. For amplification to take place, some stiffness is required for outer hair cells to communicate force to the organ of Corti, the sensory epithelium of the inner ear. Modulation of this stiffness would be expected to have a significant effect on inner ear function. Outer hair cell compressive stiffness has recently been shown to be dependent on membrane potential, but this has only been demonstrated for cells originating in the apical, low-frequency segment of the cochlea, whereas cochlear amplification is arguably more important in the more basal high-frequency segment. The voltage-dependent compliance (the reciprocal of stiffness) of high-frequency outer hair cells was investigated by two methods in cells isolated from the basal turns of the guinea pig cochlea. In contrast to previous findings, no evidence was found for voltage-dependent changes in compliance. The results call into question the importance of outer hair cell voltage-dependent compliance as a component of cochlear amplification.

CD4(+)CD25(-) effector T-cells inhibit hippocampal long-term potentiation in vitro.

During neuroinflammation T-cells invade the CNS, and may lead to the development and progression of several pathologies, of which multiple sclerosis is the most common. In these pathologies neuroinflammation is often associated with cognitive dysfunction. Using mouse hippocampal slices, we show here that CD4(+)CD25(-) T-cells inhibit long-term potentiation (LTP) induced by high-frequency stimulation. The T-cell-mediated inhibition of LTP can be prevented by blockade of gamma-aminobutyric acid (GABA)(A) receptors. These findings provide additional insight into the multiple functions of T-cells in CNS pathologies.

Mammalian cilia function is independent of the polymeric state of tubulin glycylation.

Polyglycylation is a polymeric post-translational modification of tubulin that is ubiquitous and widely present in cilia and flagella. It consists of the addition of highly variable numbers of glycyl residues as side chains onto the gamma carboxyl group of specific glutamyl residues at the C-termini of alpha- and beta-tubulin. The function of polyglycylation is poorly understood, however, studies in Tetrahymena have shown that the mutation of polyglycylation sites in beta-tubulin resulted in axonemal abnormality or lethality. This suggests that polyglycylation is functionally essential in protists. We hypothesize that polyglycylation is also essential in mammalian cilia and that the extent of polyglycylation has functional significance. In this study, we examined polyglycylation states in ciliated tissues and in mouse tracheal epithelial cell cultures. We utilized two antibodies, TAP 952 and AXO 49, which recognize glutamyl sites possessing monomeric glycylation sites and glutamyl sites possessing polymeric glycylation sites, respectively. Monomeric glycylation sites were observed in cilia of all the ciliated tissues examined but were invariably excluded from the distal tips. In contrast, polymeric glycylation sites were rare, but when observed, they were localized at the bases of cilia. During ciliogenesis, in epithelial cell cultures, monomeric glycylation sites were observed, but the extent of polymeric glycylation sites were variable and were only observed during the early stages of the cultures. Our observations suggest that while monomeric glycylation sites are universal and likely essential in mammalian cilia, polymeric glycylation sites are not required for ciliary beating. Rather, our observations suggest that the number of added glycyl residues increases progressively from the tips of cilia toward their bases.

Lateral wall protein content mediates alterations in cochlear outer hair cell mechanics before and after hearing onset.

Specialized outer hair cells (OHCs) housed within the mammalian cochlea exhibit active, nonlinear, mechanical responses to auditory stimulation termed electromotility. The extraordinary frequency resolution capacity of the cochlea requires an exquisitely equilibrated mechanical system of sensory and supporting cells. OHC electromotile length change, stiffness, and force generation are responsible for a 100-fold increase in hearing sensitivity by augmenting vibrational input to non-motile sensory inner hair cells. Characterization of OHC mechanics is crucial for understanding and ultimately preventing permanent functional deficits due to overstimulation or as a consequence of various cochlear pathologies. The OHCs' major structural assembly is a highly-specialized lateral wall. The lateral wall consists of three structures; a plasma membrane highly-enriched with the motor-protein prestin, an actin-spectrin cortical lattice, and one or more layers of subsurface cisternae. Technical difficulties in independently manipulating each lateral wall constituent have constrained previous attempts to analyze the determinants of OHCs' mechanical properties. Temporal separations in the accumulation of each lateral wall constituent during postnatal development permit associations between lateral wall structure and OHC mechanics. We compared developing and adult gerbil OHC axial stiffness using calibrated glass fibers. Alterations in each lateral wall component and OHC stiffness were correlated as a function of age. Reduced F-actin labeling was correlated with reduced OHC stiffness before hearing onset. Prestin incorporation into the PM was correlated with increased OHC stiffness at hearing onset. Our data indicate lateral wall F-actin and prestin are the primary determinants of OHC mechanical properties before and after hearing onset, respectively.

Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy.

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence. This fluorescence is characterized and shown to be consistent with a mixture of oxidized flavoproteins (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The location of the fluorescence within hair cells is also consistent with the different mitochondrial distributions in these cell types. Treatments with cyanide and mitochondrial uncouplers show that hair cells are metabolically active. Both NADH and Fp in inner hair cells gradually become completely oxidized within 50 min from the time of death of the animal. Outer hair cells show similar trends but are found to have greater variability. We show that it is possible to use two-photon metabolic imaging to assess metabolism in the mouse organ of Corti.