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PubMed2005

Estrogen receptor alpha and beta mRNA expressions by proliferating and differentiating cells in the adult rat dentate gyrus and subventricular zone.

Numerous factors modulate neurogenesis in the adult dentate gyrus and subventricular zone, but it is often not clear if the modulation is mediated by direct effects on the proliferating and differentiating cells or secondary to effects on other cells. Also, while some factors selectively affect neurogenesis in one of the neurogenetic zones, it is not clear how selectivity is achieved. Estrogen is a hormonal modulator of neurogenesis. To address the issues of direct versus indirect control and regional specificity we investigated the colocalization of immunoreactivity for a proliferating cell marker, Ki-67, and a marker for migrating and differentiating cells with a neuronal phenotype, doublecortin, with the expressions of mRNA for estrogen receptors alpha and beta. We found an extensive colocalization of estrogen receptor alpha with both markers in the dentate gyrus and only with Ki-67 in the subventricular zone. An extensive colocalization of estrogen receptor beta with both markers was found in the dentate gyrus, but only a few Ki-67-immunoreactive and no doublecortin-immunoreactive cells of the subventricular zone expressed estrogen receptor beta mRNA. Estrogen receptor alpha and beta mRNAs were not expressed in other telencephalic Ki-67-immunoreactive cells or in constitutively doublecortin-immunoreactive cells of the piriform cortex. The extensive colocalization of immunoreactive markers for cell proliferation and differentiation with mRNAs for estrogen receptor alpha and estrogen receptor beta points to the direct modulation of dentate cell proliferation, differentiation and survival by estrogen, while direct effects of estrogen in the subventricular zone appear restricted to estrogen receptor alpha-mediated effects operating at the time of cell proliferation.

Animals↗

PubMed2006

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest.

Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G(1)/S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the mature centriole present at microtubule foci, indicates that cyclin G2 resides primarily on the mother centriole. Copurification of cyclin G2 and PP2A subunits with microtubules and centrosomes, together with the effects of ectopic cyclin G2 on cell cycle progression, nuclear morphology and microtubule growth and stability, suggests that cyclin G2 may modulate the cell cycle and cellular division processes through modulation of PP2A and centrosomal associated activities.

A Kinase Anchor Proteins↗

PubMed2001

cis Interaction of the cell adhesion molecule CEACAM1 with integrin beta(3).

CEACAM1 is a cell adhesion molecule that has been implicated in a number of physiological processes (eg, tumor suppressor in epithelial tissues, potent angiogenic factor in microvessel formation, microbial receptor in human granulocytes and epithelial cells). The mechanism of CEACAM1 action is still largely unresolved but recent findings demonstrated that the cytoplasmic CEACAM1 domain is linked indirectly to the actin-based cytoskeleton. We have isolated integrin beta(3) as an associated protein using CEACAM1 tail affinity purification. This association depends on phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain. Confocal laser scanning microscopy confirmed in vivo colocalization of both molecules in human granulocytes and epithelial cells. Furthermore, the concentrated colocalization at the tumor-stroma interface of invading melanoma masses suggests a functional role of CEACAM1-integrin beta(3) interaction in melanoma invasion. Moreover, colocalization of the two adhesion molecules is also found at the apical surface of glandular cells of pregnancy endometrium. Colocalization of CEACAM1 and integrin beta(3) at the transitional zone from proliferative to invasive extravillous trophoblast of the maternal-fetal interface supports a role for CEACAM1/integrin beta(3) complexes in cell invasion.

Antigens, CD↗

PubMed2003

Immunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala.

Specific neuronal populations in the basolateral amygdala (ABL) exhibit immunoreactivity for distinct neuropeptides and calcium-binding proteins. In the present study, immunohistochemical techniques were used to analyze neurons in the rat ABL that contain cholecystokinin (CCK). Some pyramidal projection neurons in the anterior subdivision of the basolateral nucleus exhibited low levels of CCK immunoreactivity in rats that received injections of colchicine to interrupt axonal transport; staining was concentrated in the axon initial segments of these cells. High levels of CCK immunoreactivity were observed in two subpopulations of nonpyramidal interneurons in all nuclei of the ABL: (1) type L neurons (characterized by large somata and thick dendrites), and (2) type S neurons (characterized by small somata and thin dendrites). Dual-labeling immunofluorescence studies using confocal laser scanning microscopy revealed that many (30-40%) type L CCK+ interneurons exhibited immunoreactivity for calbindin (CB), but not for parvalbumin (PV), calretinin (CR), or vasoactive intestinal polypeptide (VIP). In contrast, there was extensive colocalization of CR and VIP with CCK in type S neurons, but no significant colocalization with CB or PV. In addition, the majority of CR and VIP interneurons exhibited colocalization of both neurochemicals. Collectively, the results of this and previous studies indicate that there are at least four distinct interneuronal subpopulations in the ABL: (1) PV+ neurons (the great majority of which are CB+); (2) SOM+ neurons (many of which are CB+ and NPY+); (3) large CCK+ neurons (some of which are CB+); and (4) small bipolar/bitufted neurons that exhibit various amounts of colocalization of CCK, VIP, and CR.

Amygdala↗

PubMed1997

Calretinin in neurochemically well-defined cell populations of rabbit retina.

In the rabbit retina, parvalbumin has been localized selectively to AII amacrine cells, while 28 kDa calbindin could be detected in horizontal cells, in one type of depolarizing cone bipolar cell and a population of wide-field amacrine cells. The distribution of the third neuronal calcium binding protein, calretinin, however, has not been studied to date in detail in the rabbit retina. Therefore in this study we aimed to describe the overall distribution of calretinin in the different retinal layers and the possible colocalization pattern with other neurochemical marker molecules. A few cone photoreceptor cells were found to be labeled, whereas the outer plexiform layer was free from immunoreactive elements. In the most proximal row of the inner nuclear layer amacrine cells were labeled, while more distally a few cells emitted beaded axon-like processes toward the outer retina. There were large (18-28 microm in diameter) cells labeled in the ganglion cell layer, of which many apparently had their axon stained. Some of the calretinin immunoreactive amacrine cells (the AII neurons) also contained parvalbumin. Colocalization of calretinin and 28 kDa calbindin could not be ascertained in the same amacrine cell populations, nor was tyrosine hydroxylase present in calretinin-containing cells. There was partial colocalization of calretinin in the gamma-aminobutyric acid-positive amacrine cell population. Parvalbumin containing ganglion cells were also positive for calretinin; however, the calretinin-positive ganglion cells were more numerous. gamma-Aminobutyric acid could be colocalized in some calretinin-positive neurons of the ganglion cell layer.

Animals↗

PubMed1998

Coexistence of s100beta and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat.

The coexistence of S100beta with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100beta immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100beta-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100beta-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100beta-ir. Many neurons in the nodose ganglion colocalized S100beta-ir and NADPH-d activity, whereas S100beta-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100beta- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100beta and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.

Animals↗

PubMed1998

Neurochemical characterization and distribution of enteric GABAergic neurons and nerve fibres in the human colon.

GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide (NO) is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase (GABA-T) immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: (i) neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, (ii) neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and (iii) neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis.

4-Aminobutyrate Transaminase↗

PubMed1998

Lipoprotein(a) and inflammation in human coronary atheroma: association with the severity of clinical presentation.

OBJECTIVES: The purpose of this study was the investigation of the in vivo role of lipoprotein(a) [Lp(a)] and inflammatory infiltrates in the human coronary atherosclerotic plaque and their correlation with the clinical syndrome of presentation. BACKGROUND: Lipoprotein(a) is an atherogenic and thrombogenic lipoprotein, and has been implicated in the pathogenesis of acute coronary syndromes. Lipoprotein(a) induces monocyte chemoattraction and smooth muscle cell activation in vitro. Macrophage infiltration is considered one of the mechanisms of plaque rupture. METHODS: This study of atherectomy specimens investigated the in vivo role of Lp(a) at different stages of the atherogenic process, and its relationship with macrophage infiltration. We examined coronary atheroma removed from 72 patients with stable or unstable angina. Specimens were stained with antibodies specific for Lp(a), macrophages (KP-1), and smooth muscle cells (alpha-actin). Morphometric analysis was used to quantify the plaque areas occupied by each of the three antigens, and their colocalization. RESULTS: All specimens had localized Lp(a) staining; the mean fractional area was 58.2%. Ninety percent of the macrophage areas colocalized with Lp(a) positive areas, whereas 31.3% of the smooth muscle cell areas colocalized with Lp(a) positive areas. Patients with unstable angina (n = 46) had specimens with larger mean plaque Lp(a) areas than specimens from stable angina patients (n = 26): 64.4% versus 47.7% (p = 0.004). Unstable angina patients with rest pain (n = 28) had greater mean plaque Lp(a) area than unstable angina patients with crescendo exertional pain (n = 18): 71.1% versus 52.4% (p < 0.001). Mean KP-1 area was 31.2% in unstable rest angina versus 18.3% in stable angina (p = 0.05); alpha-actin area was greater in stable (48.5%) and crescendo exertional angina (48.8%) than in rest angina (30.4%). The strongest correlation between plaque KP-1 and Lp(a) area was in unstable rest angina (r = 0.88, p < 0.001), and between alpha-actin and Lp(a) areas in the crescendo exertional angina (r = 0.62, p < 0.01). CONCLUSIONS: Lipoprotein(a) is ubiquitous in human coronary atheroma. It is detected in larger amounts in tissue from culprit lesions in patients with unstable compared to stable syndromes, and has significant colocalization with plaque macrophages. A correlation of plaque alpha-actin and Lp(a) area suggests a role of Lp(a) in plaque growth.

Angina Pectoris↗

PubMed1999

mRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes.

Subcellular localization signals for several mRNAs are positioned in their 3' untranslated regions (UTR). We have utilized the human alpha- and beta-actin 3' UTRs as signals for colocalizing hammerhead ribozymes with a lacZtarget mRNA. Ribozyme and target genes containing matched or unmatched 3' UTRs were cotransfected into 12-day-old chicken embryonic myoblast and fibroblast (CEMF) cultures and assayed by in situ hybridization (ISH) using a dual label, antibody sandwich procedure, and dual fluorescence microscopy to monitor intracellular colocalization. Beta-galactosidase localization in transfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl beta-D-galactopyranoside (ONPG) assay. We found that the percentage of colocalization using the matched alpha- or beta-actin 3' UTR (alpha-alpha or beta-beta) was enhanced approximately threefold relative to unmatched 3' UTRs. The increase in ribozyme-mediated inhibition of beta-galactosidase activity observed when matched 3' UTRs were used was consistent with the observed percentage of colocalization. These results represent the first direct demonstration that mRNA localization signals (zipcodes) can be utilized to enhance intracellular ribozyme efficacy.

3' Untranslated Regions↗

PubMed2004

The lupus band: do the autoantibodies target collagen VII?

BACKGROUND: Circulating autoantibodies directed against basement membrane zone (BMZ) components from patients with bullous pemphigoid and epidermolysis bullosa acquisita have been used to identify their target antigen in the skin and to confirm pathogenicity. Although the pattern of immunofluorescence in those diseases is similar to the lupus band, little is known about the origin and pathogenesis of the lupus band. Identifying the binding sites of the lupus band could provide a clue to the nature of the autoantigen that stimulates autoantibody formation in the skin of patients with systemic lupus erythematosus (SLE) and might provide valuable insight into the factors that influence the localization and pathogenicity of the lupus band. OBJECTIVES: To investigate the relation between the lupus band and the main BMZ components and to identify the target epitopes of autoantibodies deposited in the skin of patients with SLE. METHODS: Colocalization of the main components of the skin BMZ in nonlesional SLE skin with the lupus band was investigated using conventional immunofluorescence and confocal laser scanning microscopy. The effect of collagenase and pepsin on the expression of the lupus band was correlated with the differential sensitivity of these proteases on the collagenous and noncollagenous (NC) domains of collagen VII. Reactivity of sera from patients with SLE to a complete recombinant human NC1 domain of type VII collagen was then investigated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Near complete colocalization of the lupus band with collagen VII was found in this study, and chemical degradation of the skin attenuated the expression of the lupus band. Collectively, the NC1 domain of collagen VII was suggested as the target antigen of the lupus band, but none of the sera from patients with SLE reacted with recombinant NC1 domain-coated ELISA plates. Alternative explanations for the results of the colocalization of the lupus band with collagen VII are discussed. CONCLUSIONS: The lupus band colocalized with collagen type VII. The findings of this study ruled out the NC1 domain of collagen VII as a target antigen for circulating autoantibodies in SLE patients with no clinical evidence of blistering. Further studies are required to determine if other regions of collagen VII or another BMZ component is the target antigen for the immunoglobulins of the lupus band.

Autoantibodies↗

PubMed2003

Leptin-target neurones of the rat hypothalamus express somatostatin receptors.

Hypothalamic leptinoceptive neurones can be visualized by histochemical demonstration of leptin-induced nuclear translocation of the signalling molecule STAT3. We investigated the relationship of the leptinoceptive neurones to the somatostatin signalling system. With double-labelling immunohistochemistry, we studied the colocalization of leptin-activated transcription factor, STAT3, with somatostatin receptor subtypes, sst1, sst2A, sst2B, sst3 and sst4, or the neuropeptide itself, in the rat hypothalamus. Immunoreactivity for all the entities was widely distributed throughout the entire hypothalamus. Despite the wide distribution, only few cases of colocalization of somatostatin with leptin-activated STAT3 were detected in the paraventricular, arcuate and dorsomedial nuclei. A moderate to high degree of colocalization of nuclear STAT3 and all investigated subtypes of somatostatin receptors was found in the lateral and dorsal hypothalamic areas and in the dorsomedial hypothalamic nucleus. Immunoreactivity for sst1, sst2B and sst4 was present in STAT3-containing nuclei of the paraventricular, periventricular, arcuate and ventromedial hypothalamic neurones, as well as in the retrochiasmatic and posterior hypothalamic areas. Despite the wide distribution of sst2A in the rat hypothalamus, few events of colocalization with leptin-activated STAT3 were observed in the dorsomedial nucleus and in the lateral and dorsal hypothalamic areas only. Many leptin-responsive neurones of the dorsal, lateral, periarcuate, perifornical and posterior hypothalamic areas, as well as in the ventromedial and dorsomedial hypothalamic nuclei, displayed sst3 immunoreactivity at their neuronal cilia. These results provide strong anatomical evidence for the direct interaction of leptin and the somatostatin systems in neuroendocrine control loops such as the energy homeostasis, growth or stress response.

Animals↗

PubMed1999

Confocal laser scanning analysis of the association of fibulin-2 with fibrillin-1 and fibronectin define different stages of skin regeneration.

The fibulins represent a novel family of extracellular matrix proteins. We report the temporo-spatial expression of fibulin-2 in skin regenerating from keratinocyte autografts. In normal dermis, fibulin-2 was associated with the fibrillin-containing microfibrillar apparatus, except for the portion immediately adjacent to the dermo-epidermal junction. In contrast, early regenerating dermis showed numerous fusiform fibrillin-microfibrils along the basement membrane, whereas fibulin-2 was present in a distinct and separate layer below. Both proteins formed independent fibrillar systems also in the reticular dermis without significant colocalization; however, over time both fibril systems became congruent: after 4 mo there was extensive colocalization of fibulin-2/fibrillin in the reticular dermis, after 17 and 24 mo this also occurred in the papillary dermis. Simultaneous visualization of fibulin-2 and fibronectin revealed an inverse pattern: complete colocalization at 7 d and discordant distribution 17-24 mo after grafting. In particular, the fibrillar fibronectin pattern at early time points changed into a faint granular distribution throughout the dermis and along the subbasement membrane region as in normal skin. Dermal fibroblast cultures showed that fibrillin and fibronectin participated in distinct fibrillar systems; however, fibulin-2 colocalized with either protein. We propose that, in regenerating skin, fibulin-2 is a late component of the cutaneous microfibrillar apparatus with an earlier existence in a fibrillar matrix mediated by fibronectin. This suggests interaction of fibulin-2 with both fibronectin fibrils and fibrillin microfibrils, and is consistent with in vitro binding data.

Adult↗

PubMed2005

The association of epsin with ubiquitinated cargo along the endocytic pathway is negatively regulated by its interaction with clathrin.

Monoubiquitination of plasma membrane proteins is a mechanism to control their endocytic trafficking by promoting their interaction with cytosolic adaptor proteins that contain ubiquitin (Ub)-binding domains. Epsin, which contains Ub interaction motifs (UIMs), as well as binding sites for the clathrin coat and clathrin accessory factors, is thought to function as one of such adaptors. The importance of clathrin in the internalization of ubiquitinated cargo, however, has been questioned. Here, we show that a GFP-Ub chimera directly targeted to the plasma membrane via a lipid-based interaction is efficiently taken up by endocytosis and delivered to the same endosomes that accumulate internalized EGF. Internalization of the chimera requires integrity of the UIM binding interface of Ub, but does not require clathrin. Surprisingly, WT epsin showed little colocalization with this chimera, whereas UIM-containing epsin constructs that lack the clathrin and AP2 binding region, strikingly colocalized with this chimera on endocytic vacuoles. In addition, extensive colocalization of WT epsin with the chimera on endocytic structures could be observed in cells where clathrin levels were drastically reduced by RNA interference. Our results reveal an important regulatory mechanism in epsin function. The mutually exclusive colocalization of epsin with membrane-bound Ub or clathrin may play a role in controlling the endocytic route taken by ubiquitinated cargo.

Adaptor Protein Complex 2↗

PubMed2000

Intracellular localization of processing events in human surfactant protein B biosynthesis.

Surfactant protein B (SP-B) is essential to the function of pulmonary surfactant and to alveolar type 2 cell phenotype. Human SP-B is the 79-amino acid product of extensive post-translational processing of a 381-amino acid preproprotein. Processing involves modification of the primary translation product from 39 to 42 kDa and at least 3 subsequent proteolytic cleavages to produce the mature 8-kDa SP-B. To examine the intracellular sites of SP-B processing, we carried out immunofluorescence cytochemistry and inhibitor studies on human fetal lung in explant culture and isolated type 2 cells in monolayer culture using polyclonal antibodies to human SP-B(8) (Phe(201)-Met(279)) and specific epitopes within the N- (NFProx, Ser(145)-Leu(160); NFlank Gln(186)-Gln(200)) and C-terminal (CFlank, Gly(284)-Ser(304)) propeptides of pro-SP-B. Fluorescence immunocytochemistry using epitope-specific antisera showed colocalization of pro-SP-B with the endoplasmic reticulum resident protein BiP. The 25-kDa intermediate was partially endo H-sensitive, colocalized with the medial Golgi resident protein MG160, and shifted into the endoplasmic reticulum in the presence of brefeldin A, which interferes with anterograde transport from endoplasmic reticulum to Golgi. The 9-kDa intermediate colocalized in part with MG160 but not with Lamp-1, a transmembrane protein resident in late endosomes and lamellar bodies. Brefeldin A induced a loss of colocalization between MG160 and NFlank, shifting NFlank immunostaining to a juxtanuclear tubular array. In pulse-chase studies, brefeldin A blocked all processing of 42-kDa pro-SP-B whereas similar studies using monensin blocked the final N-terminal processing event of 9 to 8 kDa SP-B. We conclude that: 1) the first enzymatic cleavage of pro-SP-B to the 25-kDa intermediate is in the brefeldin A-sensitive, medial Golgi; 2) cleavage of the 25-kDa intermediate to a 9-kDa form is a trans-Golgi event that is slowed but not blocked by monensin; 3) the final cleavage of 9 to 8 kDa SP-B is a monensin-sensitive, post-Golgi event occurring prior to transfer of SP-B to lamellar bodies.

Antibody Specificity↗

PubMed2005

Intracellular trafficking and secretion of adiponectin is dependent on GGA-coated vesicles.

Adiponectin (Acrp30) is an insulin-sensitizing hormone produced and secreted exclusively by adipose tissue. Confocal fluorescent microscopy demonstrated the colocalization of adiponectin with the Golgi membrane markers p115, beta-COP, and the trans-Golgi network marker, syntaxin 6. Treatment of cells with brefeldin A redistributed adiponectin to the endoplasmic reticulum where it colocalized with the chaperone protein BIP and inhibited secretion of adiponectin demonstrating a requirement for a functional Golgi apparatus for adiponectin release. Confocal fluorescent microscopy also demonstrated a colocalization of endogenous adiponectin with that of expressed GGA1myc (Golgi-localizing gamma-adaptin ear homology ARF-binding protein) but with no significant overlap between adiponectin and the GGA2myc or GGA3myc isoforms. Consistent with confocal fluorescent microscopy, transmission electron microscopy demonstrated the colocalization of GGA1 with adiponectin. Although GGA1 did not directly interact with the adiponectin protein, the adiponectin enriched membrane compartments of adipocyte were precipitated by a GST-GGA1 cargo binding domain (VHS) fusion protein but not with a GST-GGA2 VHS or GST-GGA3 VHS fusion proteins. Moreover, co-expression of adiponectin with a GGA1 dominant-interfering mutant (GGA1-VHS GAT domain) resulted in a marked inhibition of adiponectin secretion in both 3T3L1 adipocytes and HEK293 cells, whereas no inhibition was detected with the truncated mutants GGA2-VHSGAT or GGA3-VHSGAT. Moreover, co-expression of wild type GGA1 with adiponectin enhanced secretion of adiponectin. Interestingly, leptin secretion was unaffected by neither the wild type form or GGA1 mutant. Taken together these data demonstrate that the trafficking of adiponectin through its secretory pathway is dependent on GGA-coated vesicles.

3T3-L1 Cells↗

PubMed1989

Agrin-related molecules are concentrated at acetylcholine receptor clusters in normal and aneural developing muscle.

Agrin induces the clustering of acetylcholine receptors (AchRs) and other postsynaptic components on the surface of cultured muscle cells. Molecules closely related if not identical to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a key part in orchestrating synapse regeneration. Agrin or agrin-related molecules are thus likely to play a role in directing the differentiation of the postsynaptic apparatus at the regenerating neuromuscular junction. The present studies are aimed at understanding the role of agrin at developing synapses. We have used anti-agrin monoclonal antibodies combined with alpha-bungarotoxin labeling to establish the localization and time of appearance of agrin-related molecules in muscles of the chick hindlimb. Agrinlike immunoreactivity was observed in premuscle masses from as early as stage 23. AchR clusters were first detected late in stage 25, coincident with the entry of axons into the limb. At this and all subsequent stages examined, greater than 95% of the AchR clusters colocalized with agrin-related molecules. This colocalization was also observed in unpermeabilized whole mount preparations, indicating that the agrin-related molecules were disposed on the external surface of the cells. Agrin-related molecules were also detected in regions of low AchR density on the muscle cell surface. To examine the role of innervation in the expression of agrin-related molecules, aneural limbs were generated by two methods. Examination of these limbs revealed that agrin-related molecules were expressed in the aneural muscle and they colocalized with AchR clusters. Thus, in developing muscle, agrin or a closely related molecule (a) is expressed before AchR clusters are detected; (b) is colocalized with the earliest AchR clusters formed; and (c) can be expressed in muscle and at sites of high AchR density independently of innervation. These results indicate that agrin or a related molecule is likely to play a role in synapse development and suggest that the muscle cell may be at least one source of this molecule.

Agrin↗

PubMed1990

Echistatin is a potent inhibitor of bone resorption in culture.

The venom protein, s-echistatin, originally derived from the saw-scaled viper Echis carinatus, was found to be a potent inhibitor of bone resorption by isolated osteoclasts. This Arg24-Gly25-Asp26-(RGD)-containing protein inhibited the excavation of bone slices by rat osteoclasts (IC50 = 0.1 nM). It also inhibited the release of [3H]proline from labeled bone particles by chicken osteoclasts (IC50 = 100 nM). By comparison, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited resorption by rat or chicken osteoclasts with an IC50 of 0.1 mM while ala24-echistatin was inactive. Video microscopy showed that rat osteoclast attachment to substrate was more sensitive to s-echistatin than was the attachment of mononuclear cells or chicken osteoclasts. The difference in sensitivity of rat and chicken osteoclasts to s-echistatin may be due to differences between receptors on rat and chicken osteoclasts for s-echistatin. Antibody localization of echistatin on these cells showed much greater echistatin binding to rat osteoclasts than to chicken osteoclasts. Laser scanning confocal microscopy after immunohistochemical staining showed that s-echistatin binds to osteoclasts, that s-echistatin receptors are most abundant at the osteoclast/glass interface, and that s-echistatin colocalizes with vinculin. Confocal interference reflection microscopy of osteoclasts incubated with s-echistatin, demonstrated colocalization of s-echistatin with the outer edges of clusters of grey contacts at the tips of some lamellipodia. Identification of the echistatin receptor as an integrin was confirmed by colocalization of echistatin fluorescence with staining for an alpha-like subunit. Attachment of bone particles labeled with [3H]proline to chicken osteoclasts confirmed that the mechanism of action of echistatin was to inhibit osteoclast binding to bone presumably by disrupting adhesion structures. These data demonstrate that osteoclasts bind to bone via an RGD-sequence as an obligatory step in bone resorption, that this RGD-binding integrin is at adhesion structures, and that it colocalizes with vinculin and has an alpha-like subunit.

Amino Acid Sequence↗

PubMed1990

Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits.

The distribution of subunits of the basement membrane proteins laminin and merosin in human and rabbit tissue was studied by immunofluorescence using monoclonal antibodies. The laminin A chain is present in epithelial, endothelial, and smooth muscle basement membranes. Merosin, as defined by its heavy chain M, is present in striated muscle and peripheral nerve. The A subunit colocalizes with at least two B subunits: B2 plus either B1 or the recently discovered B1 homologue S. The M subunit most often colocalizes with B1 and B2. Exceptions include the myotendinous junction, where M colocalizes with S, and the trophoblast basement membrane, where the M subunit colocalizes with S as well as B1. The presence of all five known subunits of the laminin family in placenta allowed isolation of their parent molecules in native form by the use of monoclonal antibodies in affinity chromatography. Four different heterotrimeric proteins could be identified: B1 chain-containing laminin (A-B1-B2), S chain-containing laminin (A-S-B2), B1-containing merosin (M-B1-B2), and S-containing merosin (M-S-B2). The data show that the proteins in the laminin family are heterotrimers composed of one heavy and two light chains; that most basement membranes contain predominantly one protein of the laminin family; and that laminin, as defined by the A subunit, has a much more restricted distribution than previously thought.

Animals↗