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Results for “colocalization”

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PubMed1997

Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy.

Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure-function relationships, including elucidation of protein-protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two fluorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA) (parasite)/protein4.1(host) and P. falciparum histidine rich protein (PfHRP1) (parasite)/protein4.1(host) showed good real-space correlation for the MESA/protein4.1 pair, but relatively poor correlation for the PfHRP1/protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in biological membranes. This method of detecting colocalization of proteins in cellular structures may have general applicability in many areas of current biological research.

Cells, Cultured↗

PubMed1997

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.

Animals↗

PubMed2001

Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes.

The effectiveness of catalytic RNAs (ribozymes) should be increased when they are colocalized to the same intracellular compartment as their RNA targets. We colocalized ribozymes with their mRNA targets in an animal model by using the discrete RNA localization signals present in the 3' untranslated regions (UTRs) of Drosophila bicoid and oskar mRNAs. These signals have been fused to a lacZ mRNA target and hammerhead ribozymes targeted against lacZ. Ribozyme efficacy was first assessed by an oligodeoxyribonucleotide-based assay to identify the most accessible sites for ribozyme interaction on native lacZ transcripts in ovary extracts. The most accessible sequence was used for the design and in vivo testing of a hammerhead ribozyme. When the ribozyme and target with synonymous 3' UTRs were expressed in the same ovaries, colocalization could be indirectly demonstrated by in situ hybridization. Colocalized ribozyme and target mRNAs resulted in a two- to threefold enhancement of ribozyme function compared with noncolocalized transcripts. This study provides the first demonstration of functional ribozyme target colocalization in an animal model.

3' Untranslated Regions↗

PubMed1997

Nitric oxide synthase in neurons of the human gall-bladder and its colocalization with neuropeptides.

The distributions of nerve cells and fibres that are immunoreactive for nitric oxide synthase (NOS) have been investigated in the human gall-bladder. In addition, the colocalization of NOS immunoreactivity (IR) with neuropeptide Y (NPY), pituitary adenylyl cyclase activating peptide (PACAP), somatostatin (SOM), substance P (SP), tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP)-IR was determined. Nitric oxide synthase-IR nerve cell bodies comprised 13 and 30% of nerve cells in ganglia of the fibromuscular and subepithelial layers, respectively. To determine these percentages, neuron-specific enolase-IR was used as a marker for all nerve cells. Although SOM- and VIP-IR nerve cell bodies were found in both ganglia, they rarely contained NOS-IR. In the fibromuscular layer, NOS-IR nerve fibres were abundant and most PACAP-, SOM- and VIP-IR fibres and many NPY-IR fibres were also NOS positive. No colocalization was observed between NOS- and SP- or TH-IR. In the mucosal layer, moderate numbers of NOS-IR fibres were found and the degree of colocalization of NOS-IR with each of NPY-, PACAP-, SOM-, SP- and VIP-IR were as follows: PACAP and NPY > VIP > SOM and SP. Nitric oxide synthase and TH were not colocalized in mucosal fibres. These results suggest that nerve fibres in the fibromuscular layer in the human gall-bladder with the chemical coding NOS/NPY/PACAP/SOM/VIP are axons of inhibitory motor neurons. Nitric oxide synthase-IR fibres in the mucosal layer that contained NPY, PACAP, SOM, SP and VIP with various degrees of colocalization probably contribute to the control of epithelial secretion or absorption.

Gallbladder↗

PubMed1992

High-Resolution Light and Electron Microscopic Immunocytochemistry of Colocalized GABA and Calbindin D-28k in Somata and Double Bouquet Cell Axons of Monkey Somatosensory Cortex.

A simple method for high-resolution immunocytochemical colocalization of different antigens in semithin sections 1 - 3 microm thick was used to study the colocalization of the calcium binding protein calbindin D-28k (calbindin) with gamma-aminobutyric acid (GABA) in double bouquet cells of monkey (Macaca fuscata) somatosensory cortex. Double bouquet cells were first visualized in vibratome sections by pre-embedding immunocytochemical staining for calbindin. Sections containing calbindin-immunoreactive somata and double bouquet cell axons were then osmicated, embedded in Araldite, resectioned at 1 - 3 microm and stained for GABA by postembedding immunocytochemistry after elution of the bound anti-calbindin antibodies. Other semithin sections adjacent to those eluted and still containing calbindin immunoreactive somata and processes were resectioned at 60 - 70 nm for electron microscopy and stained immunocytochemically for GABA by the postembedding immunogold procedure. Calbindin-positive cells are most numerous in layer II and upper layer III, where they outnumber those in all other layers combined. In layers II and upper III, approximately 30% of the stained cells are pyramidal and do not colocalize GABA. Only approximately two-thirds of the calbindin-stained nonpyramidal cells in these layers colocalize GABA, but among these virtually all the calbindin-positive double bouquet cells and their axons are GABA-immunoreactive. In deeper layers all calbindin-positive cells are nonpyramidal and all colocalize GABA. At the electron microscopic level, however, significant numbers of calbindin-positive axon terminals making symmetrical synapses are not GABA-immunoreactive. These results suggest the calbindin cells of monkey somatosensory cortex are a heterogeneous population that includes GABAergic and non-GABAergic cell types.

Journal Article↗

PubMed2002

Mammalian reovirus nonstructural protein microNS forms large inclusions and colocalizes with reovirus microtubule-associated protein micro2 in transfected cells.

Cells infected with mammalian orthoreoviruses contain large cytoplasmic phase-dense inclusions believed to be the sites of viral replication and assembly, but the morphogenesis, structure, and specific functions of these "viral factories" are poorly understood. Using immunofluorescence microscopy, we found that reovirus nonstructural protein microNS expressed in transfected cells forms inclusions that resemble the globular viral factories formed in cells infected with reovirus strain type 3 Dearing from our laboratory (T3D(N)). In the transfected cells, the formation of microNS large globular perinuclear inclusions was dependent on the microtubule network, as demonstrated by the appearance of many smaller microNS globular inclusions dispersed throughout the cytoplasm after treatment with the microtubule-depolymerizing drug nocodazole. Coexpression of microNS and reovirus protein micro2 from a different strain, type 1 Lang (T1L), which forms filamentous viral factories, altered the distributions of both proteins. In cotransfected cells, the two proteins colocalized in thick filamentous structures. After nocodazole treatment, many small dispersed globular inclusions containing microNS and micro2 were seen, demonstrating that the microtubule network is required for the formation of the filamentous structures. When coexpressed, the micro2 protein from T3D(N) also colocalized with microNS, but in globular inclusions rather than filamentous structures. The morphology difference between the globular inclusions containing microNS and micro2 protein from T3D(N) and the filamentous structures containing microNS and micro2 protein from T1L in cotransfected cells mimicked the morphology difference between globular and filamentous factories in reovirus-infected cells, which is determined by the micro2-encoding M1 genome segment. We found that the first 40 amino acids of microNS are required for colocalization with micro2 but not for inclusion formation. Similarly, a fusion of microNS amino acids 1 to 41 to green fluorescent protein was sufficient for colocalization with the micro2 protein from T1L but not for inclusion formation. These observations suggest a functional difference between microNS and microNSC, a smaller form of the protein that is present in infected cells and that is missing amino acids from the amino terminus of microNS. The capacity of microNS to form inclusions and to colocalize with micro2 in transfected cells suggests a key role for microNS in forming viral factories in reovirus-infected cells.

Animals↗

PubMed2005

Colocalization of the tetraspanins, CO-029 and CD151, with integrins in human pancreatic adenocarcinoma: impact on cell motility.

PURPOSE: Patients with pancreatic adenocarcinoma have a poor prognosis due to the extraordinary high invasive capacity of this tumor. Altered integrin and tetraspanin expression is suggested to be an important factor. We recently reported that after protein kinase C activation, colocalization of alpha6beta4 with the tetraspanin CO-029 strongly supports migration of a rat pancreatic adenocarcinoma. The finding led us to explore whether and which integrin-tetraspanin complexes influence the motility of human pancreatic tumors. EXPERIMENTAL DESIGN: Integrin and tetraspanin expression of pancreatic and colorectal adenocarcinoma was evaluated with emphasis on colocalization and the impact of integrin-tetraspanin associations on tumor cell motility. RESULTS: The majority of pancreatic and colorectal tumors expressed the alpha2, alpha3, alpha6, beta1, and beta4 integrins and the tetraspanins CD9, CD63, CD81, CD151, and CO-029. Expression of alpha6beta4 and CO-029 was restricted to tumor cells, whereas alpha1, alpha2, alpha3, alpha6, beta1, and CD9, CD81, CD151 were also expressed by the surrounding stroma. CD63, CD81, and beta1 expression was observed at comparably high levels in healthy pancreatic tissue. alpha3beta1 frequently colocalized and coimmunoprecipitated with CD9, CD81, and CD151, whereas alpha6beta4 colocalized and coimmunoprecipitated mostly with CD151 and CO-029. Notably, protein kinase C activation strengthened only the colocalization of CD151 and CO-029 with beta4 and was accompanied by internalization of the integrin-tetraspanin complex, decreased laminin 5 adhesion, and increased cell migration. CONCLUSION: alpha6beta4 is selectively up-regulated in pancreatic and colorectal cancer. The association of alpha6beta4 with CD151 and CO-029 correlates with increased tumor cell motility.

Adenocarcinoma↗

PubMed2005

Cloning, expression, and localization of MNAR/PELP1 in rodent brain: colocalization in estrogen receptor-alpha- but not in gonadotropin-releasing hormone-positive neurons.

MNAR/PELP1 is a recently identified scaffold protein in the human that modulates the nongenomic activity of estrogen receptors by facilitating linkage/cross talk with the Src/Erk activation cascade. We report herein the cloning of rat MNAR/PELP1 and provide new information concerning its distribution in the female rat brain and its degree of colocalization with estrogen receptor-alpha (ER-alpha) and GnRH. PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence and retained all the key binding motifs (PXXP, LXXLL, and glutamic acid clusters) in its primary structure that are known to be critical for its interaction with Src and steroid receptors. RT-PCR revealed that the MNAR/PELP1 transcript is expressed in many regions of the brain, and immunohistochemistry studies showed intense MNAR/PELP1 immunoreactivity (MNAR/PELP1-ir) in areas such as the hypothalamus, cerebral cortex, hippocampus, amygdala, and cerebellum. MNAR/PELP1-ir principally localized in the nucleus, but some cytoplasmic and plasma membrane-associated staining was also observed. MNAR/PELP1-ir was also primarily neuronal, although some localization in glia cells was observed in select brain regions. Colocalization studies revealed that a majority of ER-alpha-positive cells in the brain colocalized MNAR/PELP1-ir. In contrast, MNAR/PELP1-ir rarely colocalized in GnRH neurons. In conclusion, the current study provides evidence that MNAR/PELP1 is expressed in key neural tissues of the rat brain that are known targets of steroid action, that its expression is primarily neuronal, and that MNAR/PELP1-ir is strongly colocalized in ER-alpha, but not GnRH neurons in the rodent brain.

Animals↗

PubMedDate not supplied

Novel complex integrating mitochondria and the microtubular cytoskeleton with chromosome remodeling and tumor suppressor RASSF1 deduced by in silico homology analysis, interaction cloning in yeast, and colocalization in cultured cells.

Availability of the complete sequence of the human genome and sequence homology analysis has accelerated new protein discovery and clues to protein function. Protein-protein interaction cloning suggests multisubunit complexes and pathways. Here, we combine these molecular approaches with cultured cell colocalization analysis to suggest a novel complex and a pathway that integrate the mitochondrial location and the microtubular cytoskeleton with chromosome remodeling, apoptosis, and tumor suppression based on a novel leucine-rich pentatricopeptide repeat-motif-containing protein (LRPPRC) that copurified with the fibroblast growth factor receptor complex. One round of interaction cloning and sequence homology analysis defined a primary LRPPRC complex with novel subunits cat eye syndrome chromosome region candidate 2 (CECR2), ubiquitously expressed transcript (UXT), and chromosome 19 open reading frames 5 (C19ORF5) but still of unknown function. Immuno, deoxyribonucleic acid (DNA), and green fluorescent protein (GFP) tag colocalization analyses revealed that LRPPRC appears in both cytosol and nuclei of cultured cells, colocalizes with mitochondria and beta-tubulin rather than with alpha-actin in the cytosol of interphase cells, and exhibits phase-dependent organization around separating chromosomes in mitotic cells. GFP-tagged CECR2B was strictly nuclear and colocalized with condensed DNA in apoptotic cells. GFP-tagged UXT and GFP-tagged C19ORF5 appeared in both cytosol and nuclei and colocalized with LRPPRC and beta-tubulin. Cells exhibiting nuclear C19ORF5 were apoptotic. Screening for interactive substrates with the primary LRPPRC substrates in the human liver complementary DNA library revealed that CECR2B interacted with chromatin-associated TFIID-associated protein TAFII30 and ribonucleic acid splicing factor SRP40, UXT bridged to CBP/p300-binding factor CITED2 and kinetochore-associated factor BUB3, and C19ORF5 complexed with mitochondria-associated NADH dehydrogenase I and cytochrome c oxidase I. C19ORF5 also interacted with RASSF1, providing a bridge to apoptosis and tumor suppression.

Carcinoma, Hepatocellular↗

PubMed2005

New methods to evaluate colocalization of fluorophores in immunocytochemical preparations as exemplified by a study on A2A and D2 receptors in Chinese hamster ovary cells.

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A2A and dopamine D2 receptors belonging to the G protein-coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor-receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.

Adenosine A2 Receptor Agonists↗

PubMed2025

Potential therapeutic targets for ovarian hyperstimulation syndrome revealed by proteome-wide mendelian randomization and colocalization analysis.

Ovarian hyperstimulation syndrome (OHSS) is a severe complication associated with assisted reproductive technologies, characterized by metabolic, immune and vascular disorders. Understanding the molecular mechanisms underlying OHSS could reveal potential therapeutic targets and improve patient outcomes. In this study, We aimed to utilize proteome-wide Mendelian randomization (MR) and colocalization analysis to identify plasma proteins associated with OHSS and evaluate their potential as therapeutic targets through druggability assessment. We employed proteome-wide MR analysis summary data-based Mendelian randomization (SMR) analysis and phenome-wide association study (PheWAS) analysis to establish causal relationships between plasma proteins and OHSS. Colocalization analysis confirmed overlaps between proteins and genetic signals associated with OHSS. Pathway and network analyses were conducted to explore biological functions and protein interactions, while drug-target databases were queried for potential therapeutic interventions. Our results showed that 4 key proteins, including Suprabasin (SBSN), SLAMF4 (CD244), Enolase 3 (ENO3) and Thioredoxin domain-containing protein 12 (TXNDC12) were identified as significant contributors to OHSS. Pathway enrichment and interaction analyses further supported their involvement in metabolic, immune and structural pathways related to OHSS. Drug availability for colocalized proteins reveled potential drug targets for ENO3 (2-deoxy-D-glucose), CD244 (lenalidomide) and TXNDC12 (Auranofin), while no potential drug targets were identified for SBSN. Over all, our study identified15 plasma proteins, including SBSN, CD244, ENO3, and TXNDC12, as key contributors to the risk of OHSS through MR and colocalization analysis. These proteins were involved in metabolic regulation, immune response and antioxidant pathways, highlighting potential therapeutic targets and suggesting new directions for treatment strategies.

Humans↗

PubMed1996

Colocalization of calbindin D-28k, calretinin, and GABA immunoreactivities in neurons of the human temporal cortex.

The calcium-binding proteins calbindin D-28k (CalB) and calretinin (CalR) have been shown to be useful markers of neuronal subpopulations located mainly in layers II-III of the neocortex of a variety of species, including human. Double labeling immunocytochemical studies of CalB, CalR, and GABA in experimental animals have shown that CalB and CalR are present in separate subpopulations of neurons. However, there are no studies of colocalization of these calcium-binding proteins and GABA in the human neocortex. The principal goal of the present work was to investigate the degree of colocalization of these substances in layers II-III of the human temporal neocortex, using a postembedding immunocytochemical method. The patterns of staining for CalB, CalR, and GABA in the human cortex were similar to those found in monkey neocortex. However, the degree of colocalization for certain combinations was different from that reported in the monkey and other experimental animals. A relatively large proportion of CalB- and CalR-immunoreactive cells (approximately 71% and 74%, respectively) were found to be immunoreactive for GABA. However, the degree of colocalization of CalB with CalR was low (between 4% and 6%). Thus, our quantitative and qualitative data suggest that these calcium-binding proteins are present in similar cortical circuits in all primates, but that in the human neocortex, there might be additional GABAergic and perhaps also non-GABAergic interneurons with unique chemical characteristics.

Adolescent↗

PubMed2000

Quantitative assessment of localization and colocalization of glutamate, aspartate, glycine, and GABA immunoreactivity in the chick retina.

We examined the posthatch chick retina for the frequency of occurrence of localization and colocalization of four amino acid transmitter candidates: glutamate (Glu), aspartate (Asp), gamma aminobutyric acid (GABA), and glycine (Gly) using postembedding methods. We support previous studies of Glu, Asp, GABA, and Gly localization in the direct and indirect functional pathways of the chick retina and extend these studies with new qualitative and quantitative observations. We found that photoreceptors show distinct cellular immunoreactivity for both Glu (Glu+) and Asp+, but not for Gly (Gly-) or GABA. Moreover, there is compartmentalization of Glu and Asp staining within the photoreceptors. All horizontal cells react strongly with Asp and Glu, about three-fourths are GABA+ and three-fourths of these are Gly+. Bipolar cells are uniformly Glu+, heterogeneously Asp+, occasionally Gly+, but GABA-. A majority of amacrine cells stain heterogeneously with all antibodies: 90% are Gly+, slightly more than half colocalize Glu, GABA, and Gly. Furthermore, amacrine cells in the outer two or three rows of cells are more likely to be stained by Gly than Glu, Asp, or GABA. Confirming previous studies, ganglion cells were mostly immunoreactive for Glu and Asp with fewer reactive for GABA and Gly. Strong and distinctly cellular immunoreactivity was found in both central and peripheral retina. Our findings show: 1) there is extensive colocalization of Glu, Asp, GABA, and Gly among most retinal neurons, including some cells that contain all four; 2) cells of the direct functional pathway tend to be labeled by Glu and Asp generally to the exclusion of GABA and Gly, while those of the indirect pathway tend to be labeled by GABA+ and/or Gly+ in addition to Glu+ and Asp+; 3) different cell body layers have distinct patterns of colocalization; and 4) there is no qualitative difference in staining patterns between peripheral and central retina.

Animals↗

PubMed2002

Colocalization of gamma-aminobutyric acid-like immunoreactivity and catecholamines in the feeding network of Aplysia californica.

Functional consequences of neurotransmitter coexistence and cotransmission can be readily studied in certain experimentally favorable invertebrate motor systems. In this study, whole-mount histochemical methods were used to identify neurons in which gamma-aminobutyric acid (GABA)-like immunoreactivity (GABAli) was colocalized with catecholamine histofluorescence (CAh; FaGlu method) and tyrosine hydroxylase (TH)-like immunoreactivity (THli) in the feeding motor circuitry (buccal and cerebral ganglia) of the marine mollusc Aplysia californica. In agreement with previous reports, five neurons in the buccal ganglia were found to exhibit CAh. These included the paired B20 buccal-cerebral interneurons (BCIs), the paired B65 buccal interneurons, and an unpaired cell with projections to both cerebral-buccal connectives (CBCs). Experiments in which the FaGlu method was combined with the immunohistochemical detection of GABA revealed double labeling of all five of these neurons. An antibody generated against TH, the rate-limiting enzyme in the biosynthesis of catecholamines, was used to obtain an independent determination of GABA-CA colocalization. Biocytin backfills of the CBC performed in conjunction with TH immunohistochemistry revealed labeling of the rostral B20 cell pair and the unpaired CBI near the caudal surface of the right hemiganglion. THli was also present in a prominent bilateral pair of caudal neurons that were not stained with CBC backfills. On the basis of their position, size, shape, and lack of CBC projections, the lateral THli neurons were identified as B65. Double-labeling immunohistochemical experiments revealed GABAli in all five buccal THli neurons. Finally, GABAli was observed in individual B20 and B65 neurons that were identified using electrophysiological criteria and injected with a marker (neurobiotin). Similar methods were used to demonstrate that a previously identified catecholaminergic cerebral-buccal interneuron (CBI) designated CBI-1 contained THli but did not contain GABAli. Although numerous THli and GABAli neurons and fibers were present in the cerebral and buccal ganglia, additional instances of their colocalization were not observed. These findings indicate that GABA and a catecholamine (probably dopamine) are colocalized in a limited number of interneurons within the central pattern generator circuits that control feeding-related behaviors in Aplysia.

Animals↗

PubMed2006

Distribution of MT1 melatonin receptor immunoreactivity in the human hypothalamus and pituitary gland: colocalization of MT1 with vasopressin, oxytocin, and corticotropin-releasing hormone.

Melatonin is implicated in numerous physiological processes, including circadian rhythms, stress, and reproduction, many of which are mediated by the hypothalamus and pituitary. The physiological actions of melatonin are mainly mediated by melatonin receptors. We here describe the distribution of the melatonin receptor MT1 in the human hypothalamus and pituitary by immunocytochemistry. MT1 immunoreactivity showed a widespread pattern in the hypothalamus. In addition to the area of the suprachiasmatic nucleus (SCN), a number of novel sites, including the paraventricular nucleus (PVN), periventricular nucleus, supraoptic nucleus (SON), sexually dimorphic nucleus, the diagonal band of Broca, the nucleus basalis of Meynert, infundibular nucleus, ventromedial and dorsomedial nucleus, tuberomamillary nucleus, mamillary body, and paraventricular thalamic nucleus were observed to have neuronal MT1 receptor expression. No staining was observed in the nucleus tuberalis lateralis and bed nucleus of the stria terminalis. The MT1 receptor was colocalized with some vasopressin (AVP) neurons in the SCN, colocalized with some parvocellular and magnocellular AVP and oxytocine (OXT) neurons in the PVN and SON, and colocalized with some parvocellular corticotropin-releasing hormone (CRH) neurons in the PVN. In the pituitary, strong MT1 expression was observed in the pars tuberalis, while a weak staining was found in the posterior and anterior pituitary. These findings provide a neurobiological basis for the participation of melatonin in the regulation of various hypothalamic and pituitary functions. The colocalization of MT1 and CRH suggests that melatonin might directly modulate the hypothalamus-pituitary-adrenal axis in the PVN, which may have implications for stress conditions such as depression.

Adult↗

PubMed1995

Colocalization of somatostatin, neuropeptide Y, and NADPH-diaphorase in the caudate-putamen of the rat.

Somatostatin, neuropeptide Y, and nicotinamide adenine dinucleotide phosphate-diaphorase are colocalized within a small population of medium aspiny neurons in the caudate-putamen of the rat. The extent of colocalization, however, appears to be in dispute. In order to examine the question of colocalization between these three neuroactive substances, a series of double-labelling experiments was performed. This was accomplished by combining immunocytochemistry for somatostatin or neuropeptide Y or enzyme histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase with in situ hybridization for somatostatin and/or neuropeptide Y mRNA. The results of such analysis indicate that nicotinamide adenine dinucleotide phosphate-diaphorase and somatostatin mRNA are 100% colocalized throughout the caudate-putamen, except for the area bordering the globus pallidus. All neurons that contain neuropeptide Y contain somatostatin message. Only 84% of the neurons that contain somatostatin mRNA, however, also contain neuropeptide Y. Neurons that contain somatostatin 28 but not neuropeptide Y are found throughout the caudate-putamen. These results indicate that the somatostatin neuron population in the rat caudate-putamen is not homogeneous. Instead, the medium aspiny neuron population is actually composed of several subpopulations based on the content of neuroactive substances.

Animals↗

PubMed2006

FRET and colocalization analyzer--a method to validate measurements of sensitized emission FRET acquired by confocal microscopy and available as an ImageJ Plug-in.

Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.

Arrestins↗

PubMed1995

Colocalization of bFGF and the myogenic regulatory gene myogenin in dystrophic mdx muscle precursors and young myotubes in vivo.

Tissue culture studies using muscle cell lines suggest that in addition to mitogenic effects, fibroblast growth factors (FGF) inhibit skeletal muscle differentiation and the expression of members of a family of muscle-specific regulatory genes including MyoD and myogenin. We examined the possible coexpression of bFGF and myogenin by tandem in situ hybridization (detecting mRNA) and immunocytochemistry studies (detecting protein) to determine whether myogenic cells in vivo endogenously produce bFGF. Mdx mouse muscle, which shows characteristic dystrophic damage and regeneration, demonstrated mononuclear cells containing myogenin and bFGF transcripts in similar regions of adjacent sections of focal degeneration and repair, particularly near recent segmental fiber damage. Using immunocytochemistry and in situ hybridization concurrently on the same sections, bFGF protein and myogenin mRNA were colocalized in both muscle precursors and new myotubes. The in vivo results were confirmed in vitro using primary explant cultures of mdx muscle. Approximately one-half of mononuclear cells in vivo were myogenic by the criterion of myogenin mRNA expression. Both myogenin and bFGF mRNAs were also colocalized with bFGF protein, indicating endogenous expression of bFGF in a subpopulation of myogenic cells. Small numbers of myogenic mononuclear cells were differentiated, as determined by the presence of developmental myosin heavy chain protein (DevMHC). These cells and new myotubes also colocalized myogenin, DevMHC, and bFGF. Since bFGF and myogenin are colocalized in mpec and myotubes in vivo and in vitro, endogenous expression of bFGF is not mutually exclusive of myogenic regulatory gene expression, either before or after differentiation of the skeletal muscle phenotype. Such features of coexpression suggest an important and complex role for bFGF in muscle regeneration in vivo.

Animals↗