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Ammasi Periasamy

Publications and source records attributed to Ammasi Periasamy.

18 recordsLinked to original sources
PubMed2006

Angiotensin II type 2 receptor-bradykinin B2 receptor functional heterodimerization.

Angiotensin II type 2 (AT2R) or bradykinin B2 (B2R) receptor activation enhances NO production. Recently, we demonstrated enhancement of NO production when AT2R and B2R are simultaneously activated in vivo. However, the mechanism involved in this enhancement is unknown. Using confocal fluorescence resonance energy transfer microscopy, we report the distance between the AT2R and B2R in PC12W cell membranes to be 50+/-5 A, providing evidence and quantification of receptor heterodimerization as the mechanism for enhancing NO production. The rate of AT2R-B2R heterodimer formation is largely a function of the degree of AT2R-B2R expression. The physical association between the dimerized receptors initiates changes in intracellular phosphoprotein signaling activities leading to phosphorylation of c-Jun terminal kinase, phosphotyrosine phosphatase, inhibitory protein kappaBalpha, and activating transcription factor 2; dephosphorylation of p38 and p42/44 mitogen-activated protein kinase and signal transducer inhibitor of transcription 3; and enhancing production of NO and cGMP. Controlling the expression of AT2R-B2R, consequently influencing their biologically active dimerization, presents a potential therapeutic target for the treatment of hypertension and other cardiovascular and renal disorders.

Activating Transcription Factor 2↗
PubMed2006

Intensity range based quantitative FRET data analysis to localize protein molecules in live cell nuclei.

Förster (fluorescence) resonance energy transfer (FRET) is an ideal technique to estimate the distance between interacting protein molecules in live specimens using intensity-based microscopy. The spectral overlap of donor and acceptor- essential for FRET-also generates a contamination of the FRET signal. There are a number of algorithms available to remove this spectral bleedthrough (SBT) contamination and in this paper we compare two popular algorithms to estimate the SBT element and to calculate a more precise level of energy transfer efficiency, and with that a more accurate distance estimate.

Algorithms↗
PubMed2006

Issues in confocal microscopy for quantitative FRET analysis.

Previously, we have carried out extensive quantitative analysis of Förster (or fluorescence) resonance energy transfer (FRET) data to show that polymeric IgA receptors and their ligands cluster in endocytic membranes in the process of sorting and trafficking in polarized cells. Here, we use a similar technique to assay the organization and distribution of another membrane-bound receptor: transferrin receptor (TFR) and its ligand, holo-transferrin (Tfn), while explaining the step-by-step measures to be taken for successful quantitative analysis of the FRET data. In particular, methodological issues in FRET quantitative imaging, such as spectral bleed-through and background correction, optimal selection of regions of interest, how to deal with outliers and pooling data and statistical analysis of FRET data, are addressed. Our results indicating a clustered organization of TFR-Tfn complexes fit the well-known homodimeric structure of TFR. These quantitative approaches can be adapted for other biological applications of FRET.

Algorithms↗
PubMed2006

Monitoring dynamic protein interactions with photoquenching FRET.

The mammalian cell nucleus is a dynamic and highly organized structure. Most proteins are mobile within the nuclear compartment, and this mobility reflects transient interactions with chromatin, as well as network interactions with a variety of protein partners. To study these dynamic processes in living cells, we developed an imaging method that combines the photoactivated green fluorescent protein (PA-GFP) and fluorescence resonance energy transfer (FRET) microscopy. We used this new method, photoquenching FRET (PQ-FRET), to define the dynamic interactions of the heterochromatin protein-1 alpha (HP1alpha) and the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) in regions of centromeric heterochromatin in mouse pituitary cells. The advantage of the PQ-FRET assay is that it provides simultaneous measurement of a protein's mobility, its exchange within macromolecular complexes and its interactions with other proteins in the living cell without the need for corrections based on reference images acquired from control cells.

Animals↗
PubMed2005

Imaging protein molecules using FRET and FLIM microscopy.

Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have moved center stage and are increasingly forming part of multifaceted imaging approaches. They are complementary methodologies that can be applied to advanced quantitative analyses. The widening application of FRET and FLIM has been driven by the availability of suitable fluorophores, increasingly sophisticated microscopy systems, methodologies to correct spectral bleed-through, and the ease with which FRET can be combined with other techniques. FRET and FLIM have recently found use in several applications: in the analysis of protein-protein interactions with high spatial and temporal specificity (e.g. clustering), in the study of conformational changes, in the analysis of binding sequences, and in applications such as high-throughput screening.

Fluorescence Resonance Energy Transfer↗
PubMed2004

Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization.

Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.

Animals↗
PubMed2003

Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations.

The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications.

Animals↗
PubMed2003

Nitric-oxide-induced depolarization of neuronal mitochondria: implications for neuronal cell death.

Nitric oxide (NO(*)) has known toxic effects on central nervous system neurons. This study characterized the effect of NO(*) on mitochondrial membrane changes by exploring the relationship among NO(*), excitatory receptor activation, and the induction of peroxynitrite, a highly toxic NO(*) reactant, to neuronal injury. Cultured rat cortical neurons were exposed to the NO(*) generator, diethylenetriamine/nitric oxide adduct, and were examined for signs of cell death, mitochondrial membrane potential changes (Deltapsi(m)), and the induction of a mitochondrial permeability transition (MPT). Neurons were also examined for nitrotyrosine (NT) immunoreactivity, a marker of reactive nitrogen species (RNS) formation. Neurons exposed to NO(*) or to N-methyl-D-aspartate (NMDA) exhibited similar rapid depolarization of mitochondria, which was prevented by an NMDA receptor antagonist. Electrophysiological studies demonstrated NO(*) potentiation of NMDA-induced NMDA receptor currents. NO(*) and NMDA-treated neurons had evidence of mitochondrial-specific NT immunoreactivity that was prevented by a SOD/catalase mimetic (EUK-134). EUK-134 treatment reduced both NO(*) and NMDA-induced NT formation and neuronal cell death. EUK-134 did not prevent NO-induced Deltapsi(m) but partially prevented NMDA-induced Deltapsi(m) loss. Although NO(*) and NMDA both induced MPT and MPT inhibitors prevented NO-induced Deltapsi(m), they did not result in significant neuroprotection, in contrast to treatment designed to decrease peroxynitrite formation. These data suggest that NO-induced NMDA receptor activation is closely linked to intramitochondrial NO-peroxynitrite/RNS formation and thereby acts as a major mediator of neuronal cell death.

Animals↗
PubMed2003

Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes.

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.

Animals↗
PubMed2003

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy.

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.

Algorithms↗
PubMed2003

Protein localization in living cells and tissues using FRET and FLIM.

Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution. One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength. Mathematical algorithms are required to correct the spectral bleed-through signal in wide-field, confocal, and two-photon FRET microscopy. In contrast, spectral bleed-through is not an issue in FRET/FLIM imaging because only the donor fluorophore lifetime is measured; also, fluorescence lifetime imaging microscopy (FLIM) measurements are independent of excitation intensity or fluorophore concentration. The combination of FRET and FLIM provides high spatial (nanometer) and temporal (nanosecond) resolution when compared to intensity-based FRET imaging. In this paper, we describe various FRET microscopy techniques and its application to protein-protein interactions.

Animals↗
PubMed2003

Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy.

Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. One of the principal determinants of morbidity and mortality following TBI is traumatic axonal injury (TAI). Current hypotheses on the pathogenesis of TAI involve activation of apoptotic cascades secondary to TBI. While a number of studies have demonstrated direct evidence for the activation of apoptotic cascades in TAI, the precise pathway by which these cascades are initiated remains a subject of intense investigation. As axolemmal disruption with the subsequent intra-axonal influx of large molecular weight species has been demonstrated to occur in relation to local axonal breakdown, attention has focused on cascades that may occur as a result of loss of ionic homeostasis. One proposed pathway by which this has been hypothesized to occur is the Ca(2+)-mediated activation of calmodulin and subsequent activation of the phosphatase calcineurin with dephosphorylation of a protein known as BAD, leading to a proapoptotic interaction between BAD and the mitochondrial protein Bcl-xL. While this pathway is an intriguing route for traumatic axonal pathogenesis, neither conventional immunocytochemical/histochemical nor ultrastructural approaches have had the capacity to shed insight on whether BAD and Bcl-xL interact in TAI in vivo. We describe the implementation of confocal and two-photon excitation fluorescence resonance energy transfer (FRET) microscopy techniques through which we demonstrate interaction between the proapoptotic protein BAD and the prosurvival protein Bcl-xL within TAI following TBI. Further, we report on a method to reliably detect protein interactions within aldehyde fixed tissue sections through conventional immunohistochemical approaches.

Animals↗
PubMed2003

One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes.

One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover. Treatment with the PFRET correction algorithm results in increasing correction levels and comparable energy transfer efficiency (E%) values, thus validating our algorithm-based approach. The relationship between E% and acceptor and donor levels and donor:acceptor (D:A) ratio is used to characterize the distribution of receptor-ligand complexes in endocytic membranes. In addition to the standard test for clustering (E%'s independence from acceptor levels), we describe a second parameter: the negative dependence of E% on increasing donor levels and D:A ratio. A donor geometric exclusion hypothesis is proposed to explain this phenomenon. One- and two-photon FRET microscopy assays show that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized Madin-Darby canine kidney cells.

Algorithms↗
PubMed2002

Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus.

The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPalpha) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPalpha was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPalpha to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPalpha was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBPalpha transactivation domain disrupted the redistribution of C/EBPalpha by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPalpha, and the truncated C/EBPalpha still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPalpha that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPalpha to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBPalpha are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBPalpha at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.

Blotting, Western↗
PubMed2002

Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule.

Our senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Millions of people suffer from hearing and balance deficits caused by damage to hair cells as a result of exposure to noise, aminoglycoside antibiotics, and antitumor drugs. In some species such damage can be reversed through the production of new cells. This proliferative response is limited in mammals but it has been hypothesized that damaged hair cells might survive and undergo intracellular repair. We examined the fate of bullfrog saccular hair cells after exposure to a low dose of the aminoglycoside antibiotic gentamicin to determine whether hair cells could survive such treatment and subsequently be repaired. In organ cultures of the bullfrog saccule a combination of time-lapse video microscopy, two-photon microscopy, electron microscopy, and immunocytochemistry showed that hair cells can lose their hair bundle and survive as bundleless cells for at least 1 week. Time-lapse and electron microscopy revealed stages in the separation of the bundle from the cell body. Scanning electron microscopy (SEM) of cultures fixed 2, 4, and 7 days after antibiotic treatment showed that numerous new hair bundles were produced between 4 and 7 days of culture. Further examination revealed hair cells with small repaired hair bundles alongside damaged remnants of larger surviving bundles. The results indicate that sensory hair cells can undergo intracellular self-repair in the absence of mitosis, offering new possibilities for functional hair cell recovery and an explanation for non-proliferative recovery.

Animals↗