Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities.

Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage-dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A- and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A-mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death.

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal ß-cell lines and human islet ß-cells but not in α-cells. In human islets, glutamate-induced ß-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of ß-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced ß-cell death, whereas GLT1 up-regulation promoted ß-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that ß-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

Bone marrow-derived cell regulation of skeletal muscle regeneration.

Limb regeneration requires the coordination of multiple stem cell populations to recapitulate the process of tissue formation. Therefore, bone marrow (BM) -derived cell regulation of skeletal muscle regeneration was examined in mice lacking the CC chemokine receptor 2 (CCR2). Myofiber size, numbers of myogenic progenitor cells (MPCs), and recruitment of BM-derived cells and macrophages were assessed after cardiotoxin-induced injury of chimeric mice produced by transplanting BM from wild-type (WT) or CCR2(-/-) mice into irradiated WT or CCR2(-/-) host mice. Regardless of the host genotype, muscle regeneration and recruitment of BM-derived cells and macrophages were similar in mice replenished with WT BM, whereas BM-derived cells and macrophage accumulation were decreased and muscle regeneration was impaired in all animals receiving CCR2(-/-) BM. Furthermore, numbers of MPCs (CD34(+)/Sca-1(-)/CD45(-) cells) were significantly increased in mice receiving CCR2(-/-) BM despite the decreased size of regenerated myofibers. Thus, the expression of CCR2 on BM-derived cells regulated macrophage recruitment into injured muscle, numbers of MPC, and the extent of regenerated myofiber size, all of which were independent of CCR2 expression on host-derived cells. Future studies in regenerative medicine must include consideration of the role of BM-derived cells, possibly macrophages, in CCR2-dependent events that regulate effective skeletal muscle regeneration.

An unexpected protein interaction promotes drug resistance in leukemia.

The overall survival of patients with acute myeloid leukemia (AML) is poor and identification of new disease-related therapeutic targets remains a major goal for this disease. Here we show that expression of MPP1, a PDZ-domain-containing protein, highly correlated with ABCC4 in AML, is associated with worse overall survival in AML. Murine hematopoietic progenitor cells overexpressing MPP1 acquired the ability to serially replate in methylcellulose culture, a property crucially dependent upon ABCC4. The highly conserved PDZ-binding motif of ABCC4 is required for ABCC4 and MPP1 to form a protein complex, which increased ABCC4 membrane localization and retention, to enhance drug resistance. Specific disruption of this protein complex, either genetically or chemically, removed ABCC4 from the plasma membrane, increased drug sensitivity, and abrogated MPP1-dependent hematopoietic progenitor cell replating in methylcellulose. High-throughput screening identified Antimycin A as a small molecule that disrupted the ABCC4-MPP1 protein complex and reversed drug resistance in AML cell lines and in primary patient AML cells. In all, targeting the ABCC4-MPP1 protein complex can lead to new therapies to improve treatment outcome of AML, a disease where the long-term prognosis is poor.

Role of the promyelocytic leukemia body in the dynamic interaction between the androgen receptor and steroid receptor coactivator-1 in living cells.

The dynamic interaction between the androgen receptor (AR) and steroid receptor coactivator-1 (SRC-1) was explored in living cells expressing chimeric forms of the receptor and the coactivator containing two spectral variants of jellyfish fluorescent protein. Laser scanning confocal imaging of transfected cells expressing fluorescently labeled SRC-1 revealed that in an unsynchronized cell population, the coactivator is distributed in approximately 40% cells as nuclear bodies of 0.2-1.0 microm in diameter. Immunostaining of cyan fluorescent protein-labeled SRC-1 (CFP-SRC1)-expressing cells with antibody to promyelocytic leukemia (PML) protein showed significant overlap of the CFP fluorescence with the antibody stain. Cotransfection of cells with a plasmid expressing the CFP conjugate of Sp100 (another marker protein for the PML nuclear body) also showed colocalization of the yellow fluorescent protein (YFP)-SRC1 containing nuclear foci with the PML bodies in living cells. Analysis of the three-dimensional structure revealed that the PML bodies are round to elliptical in shape with multiple satellite bodies on their surface. Some of these satellite bodies contain the SRC-1. Activation and nuclear import of CFP-AR by the agonistic ligand 5alpha-dihydrotestosterone, but not by the antagonist casodex, transferred YFP-SRC1 from the PML bodies to an interlacing filamentous structure. In a single living cell, agonist-activated AR caused a time-dependent movement of YFP-SRC1 from the PML bodies to this filamentous structure. Additionally, coexpression of a constitutively active mutant of AR (AR-deltaligand binding domain) also displaced YFP-SRC1 from the PML bodies to this intranuclear filamentous structure. The fluorescence recovery after photobleaching approach was used to examine changes in the kinetics of movement of YFP-SRC1 during its mobilization from the PML bodies to the intranuclear filamentous structure by the agonist-activated AR. Results of the relative half-times (t(1/2)) of replacement of YFP-SRC1 within the photobleached region of a single PML body from its surrounding nuclear space supported the conclusion that SRC-1 is actively transported from the PML bodies to the intranuclear filamentous structure by the ligand-activated AR. This observation also suggests an interaction between AR and SRC-1 before its binding to the target gene. The PML bodies have been implicated as a cross-road for multiple regulatory pathways that control cell proliferation, cellular senescence, and apoptosis. Our present results along with other recent reports expand the role of this subnuclear structure to include the regulation of steroid hormone action.

Increased human AP endonuclease 1 level confers protection against the paternal age effect in mice.

Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.

Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia.

Alterations of IKZF1, encoding the lymphoid transcription factor IKAROS, are a hallmark of high-risk acute lymphoblastic leukemia (ALL), however the role of IKZF1 alterations in ALL pathogenesis is poorly understood. Here, we show that in mouse models of BCR-ABL1 leukemia, Ikzf1 and Arf alterations synergistically promote the development of an aggressive lymphoid leukemia. Ikzf1 alterations result in acquisition of stem cell-like features, including self-renewal and increased bone marrow stromal adhesion. Retinoid receptor agonists reversed this phenotype, partly by inducing expression of IKZF1, resulting in abrogation of adhesion and self-renewal, cell cycle arrest, and attenuation of proliferation without direct cytotoxicity. Retinoids potentiated the activity of dasatinib in mouse and human BCR-ABL1 ALL, providing an additional therapeutic option in IKZF1-mutated ALL.

Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3.

HEK-293T cells transiently transfected with ovine (o) GH receptor (GHR) and prolactin receptor (PRLR) constructs respectively tagged downstream with cyan or yellow fluorescent proteins were used to study ovine placental lactogen (oPL)-stimulated heterodimerization by fluorescence resonance energy transfer (FRET) microscopy. The oPL-stimulated transient heterodimerization of GHR and PRLR had a peak occurring 2.5-3 min after oPL application, whereas oGH or oPRL had no effect at all. The results indicate none or only little dimerization occurring before the hormonal stimulation. The effect of heterodimerization was studied by comparing activation of Janus kinase 2, signal transducer and activator of transcription (STAT)1, STAT3, STAT5, and MAPK in Chinese hamster ovary cells stably transfected with chimeric genes encoding receptors consisting of cytosolic and transmembrane parts of oGHR and oPRLR, extracellular domains of human granulocyte and macrophage colony-stimulating factor (hGM-CSF) receptor alpha or beta, and cells transfected with the two forms (alpha or beta) of PRLR and GHR. Functionality of those proteins was verified by hGM-CSF-induced phosphorylation of both intracellular PRLR and GHR domains and hGM-CSF-induced heterodimerization was documented by chimeric receptor coimmunoprecipitation. Homodimerization or heterodimerization of PRLRs and GHRs had no differential effect on activation of STAT5 and MAPK. However, heterodimerization resulted in a prolonged phosphorylation of STAT1 and in particular STAT3, suggesting that the heterodimerization of alpha-oGHR and beta-oPRLR is able to transduce a signal, which is distinct from that occurring on homodimeric associations.

Fluorescence Resonance Energy Transfer (FRET) as a method to calculate the dimerization strength of basic Helix-Loop-Helix (bHLH) proteins.

Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, we show that HAND1 is phosphorylated during the trophoblast giant cell differentiation on residues residing in Helix I of the bHLH domain. Our hypothesis is that these modifications result in changes in HAND1 dimerization affinities with other bHLH factors. To test this idea, we employed FRET to measure the protein-protein interactions of HAND1 and HAND1 point mutants in HEK293 cells using YFP and CFP fusion proteins and laser scanning confocal microscopy.

An in vitro model to study the pathogenesis of the early endometriosis lesion.

OBJECTIVE: To determine if whole fragments of endometrium can adhere to peritoneum with intact mesothelium. DESIGN: Tissue culture and immunohistochemical study. SETTING: University Medical Center. PATIENTS: Reproductive-age women undergoing surgery for benign conditions. INTERVENTIONS: Whole explants of human peritoneum from the anterior abdominal wall and the posterior surface of the uterus were cultured with whole fragments of mechanically dispersed endometrium. MAIN OUTCOME MEASURES: Adhesion of endometrial fragments to the surface of the peritoneum was evaluated. Adherent fragments of endometrium were identified using the dissecting microscope and by performing serial sections of the peritoneum explants for light and confocal laser-scanning microscopy. Immunohistochemical staining of the mesothelium with antibodies to cytokeratin and vimentin was used to ensure an intact layer of mesothelium beneath the endometrial implants. Transmission electron microscopy was also used to evaluate the adhesion of endometrium to the mesothelium. RESULTS: Endometrium was identified attached to the surface of the peritoneum. After 18-24 hours of culture, the majority of implants did not have identifiable mesothelium beneath them, but most had intact mesothelium running up to the point of attachment. Approximately 10% of the endometrial implants had intact mesothelium at the site of attachment. After 1 hour of culture, both endometrial stromal and epithelial cells were attached to intact mesothelium in nearly all cases. Early transmesothelial invasion involves endometrial stromal cells. CONCLUSIONS: Endometrial stromal and epithelial cells can attach to the intact mesothelial surface of the peritoneum. Endometrial stromal cell invasion through the mesothelium occurs in less than 18-24 hours.

Time series analysis of transmesothelial invasion by endometrial stromal and epithelial cells using three-dimensional confocal microscopy.

OBJECTIVE: To evaluate endometrial adhesion and invasion of peritoneal mesothelium. DESIGN: Descriptive study using confocal laser-scanning microscopy. SETTING: University-based laboratory. PATIENT(S): Women undergoing surgery for benign conditions. INTERVENTION(S): Fluorescence-labeled peritoneal mesothelial cells (PMCs) were grown on coverslips. Fluorescence-labeled endometrial stromal cells (ESCs) and epithelial cells (EECs) and myometrial cells (Myos) were plated on the PMCs. Cultures were examined at 1, 6, 12, and 24-27 hours with differential interference contrast and confocal laser-scanning microscopy. MAIN OUTCOME MEASURE(S): Demonstration of adherence and invasion of endometrial cells through peritoneal mesothelium. RESULT(S): At 1 hour, there was adherence of the ESCs, EECs, and Myos on the perimeter of PMCs. There was no invasion by the Myos. By 6 hours, ESCs and EECs spread over the surface of the PMCs and extended cell processes through PMC junctions. Extension of pseudopodia under the PMCs followed. By 12 hours, there was vacuolization and lifting of PMCs that had been undermined by endometrial cells. CONCLUSION(S): This is the first time-phase study to demonstrate adherence and the process of invasion of endometrial cells through the mesothelium. The application of three-dimensional confocal laser-scanning microscopy is a novel technique that can be used to further examine mechanisms involved in the pathogenesis of the early endometriotic lesion.

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm).

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH(m)). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.

Microscopic analysis of fluorescence resonance energy transfer (FRET).

We describe here detailed practical procedures for implementing various approaches of fluorescence resonance energy transfer (FRET) in microscope-based measurements. A comprehensive theoretical formalism is developed and the different experimental procedures are outlined. A step-by-step protocol is provided for preparing the specimens for FRET measurements, data acquisition procedures, analysis, and quantification. Particular emphasis is given to exemplify the FRET applications in the study of protein-protein interactions.

Pathogenesis of endometriosis--current research.

Proliferative, secretory and menstrual endometrial cells of both the stroma and epithelium adhere to intact peritoneal mesothelium and mesothelial monolayers. Endometrial attachment to the mesothelium appears to occur rapidly (within 1 h) and transmesothelial invasion occurs between 1 and 18-24 h. These results demonstrate that the mesothelium is not a 'no-stick' surface and indicates that molecules present at the surface of the mesothelium are involved in the pathogenesis of the early endometriotic lesion. The inhibition of endometrial cell adherence to peritoneal mesothelium by hyaluronidase indicates that CD44-hyaluronan binding is at least one of the mechanisms involved in the pathogenesis of endometriosis. We believe that investigation of mesothelial cell adhesion molecules is central to understanding the pathogenesis of endometriosis.

Effect of short-term free Fatty acids elevation on mitochondrial function in skeletal muscle of healthy individuals.

CONTEXT: Mitochondrial dysfunction has been proposed as an underlying mechanism in the pathogenesis of insulin resistance and type 2 diabetes mellitus. OBJECTIVE: To determine whether mitochondrial dysfunction plays a role in the free fatty acid (FFA)-induced impairment in insulin action in skeletal muscle of healthy subjects. DESIGN: Eleven lean normal glucose tolerant individuals received 8 h lipid and saline infusion on separate days with a euglycemic insulin clamp during the last 2 h. Vastus lateralis muscle biopsies were performed at baseline and after 6 h lipid or saline infusion. Inner mitochondrial membrane potential (Psi(m)) and mitochondrial mass were determined ex vivo by confocal microscopy. RESULTS: Compared with saline infusion, lipid infusion reduced whole-body glucose uptake by 22% (P < 0.05). Psi(m) decreased by 33% (P < 0.005) after lipid infusion and the decrement in Psi(m) correlated with change in plasma FFA after lipid infusion (r = 0.753; P < 0.005). Mitochondrial content and morphology did not change after lipid infusion. No significant changes in genes expression, citrate synthase activity, and total ATP content were observed after either lipid or saline infusion. CONCLUSIONS: Short-term physiological increase in plasma FFA concentration in lean normal glucose tolerant subjects induces insulin resistance and impairs mitochondrial membrane potential but has no significant effects on mitochondrial content, gene expression, ATP content, or citrate synthase activity.

High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction.

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor kappaB kinases (IKK)alpha, beta, and gamma] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKbeta in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKbeta interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 +/- 0.65-fold) and translational (1.97 +/- 0.17-fold) expression as well as the catalytic activity of IKKbeta (2.45 +/- 0.4-fold). Both IKKbeta and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 microM) or Radicicol (20 microM) mitigated (0.45 +/- 0.04-fold and 0.93 +/- 0.16-fold, respectively) HG induced-IKKbeta activity (2.5 +/- 0.42-fold). Blocking of IKKbeta expression by IKK inhibitor II (15 microM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization.

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Spectral kinetics ratiometry: a simple approach for real-time monitoring of fluorophore distributions in living cells.

BACKGROUND: Spectral Imaging Microscopy is gaining attention in biological research. Most of the commercial systems in vogue employ linear spectral un-mixing algorithms and/or spectral profile matching algorithms to extract the component spectral information from the measured specimen spectra. The need to accurately deconvolve multiple spectra with minimal cross-contamination is always accompanied by an increase in system complexity and cost. METHODS: We describe here a variant of the spectral waveform cross-correlation analysis (SWCCA) method where the master reference spectral library is constructed by composite spectra with varying ratios of component spectra, unlike the conventional spectral library where pure spectra form the components. We demonstrate that this spectral kinetics ratiometric approach gives realistic estimates of fluorophore distribution in living cells with a better spectral correlation as compared with pure component spectral libraries. RESULTS: Biological applications demonstrated in this article include acceptor photobleaching FRET, caspase activity during cell death and mitochondrial membrane polarization kinetics during substrate metabolism. CONCLUSIONS: Beyond the representative applications presented in this article, we think the proposed approach can be valuable in dynamic studies of a variety of other cellular processes such as pH oscillations, photobleaching and quenching kinetics. Besides giving better spectral correlation and real-time monitoring of biophysical processes in living cells, this method can serve as an economical solution for high-throughput spectral classification requirements.