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PubMed2003

Differential colocalization of Islet-1 and estrogen receptor alpha in the murine preoptic area and hypothalamus during development.

Estrogen receptor (ER) expression and regulation is vital to the correct functioning of the neuroendocrine brain. Islet-1 (Isl-1) is a LIM homeodomain-containing transcription factor that has been implicated in neuronal differentiation, is located in the hypothalamus, and can alter ER function in vitro. We have determined that Isl-1 is localized in several regions of the hypothalamus, including the ER rich areas of the ventromedial nucleus (VMH), the preoptic area, and the anterior hypothalamus. Using double-label immunocytochemistry, we examined the overlap between immunoreactive ERalpha and Isl-1 in these different hypothalamic brain regions. In the developing brain, almost 100% of VMH cells that contain immunoreactive ERalpha also contain Isl-1. However, in older animals, the percentage of double-label cells decreased below 70%. This change is due to a decrease in the number of cells containing Isl-1, because there was no difference in the number of ERalpha-containing cells. By contrast, in more anterior regions of the hypothalamus, cells containing both Isl-1 and ERalpha were less common, with the two populations adjacent to each other, rather than overlapping. These data suggest that, although Isl-1 and ERalpha can interact, they are not always found in the same cells and that regulation of ERalpha function is not under the same control in the VMH, preoptic area, and the anterior hypothalamus.

Age Factors↗

PubMed1988

Distribution of progestin-concentrating cells in rat brain: colocalization of [3H]ORG.2058, a synthetic progestin, and antibodies to tyrosine hydroxylase in hypothalamus by combined autoradiography and immunocytochemistry.

The anatomical distribution of progestin-concentrating cells in rat brain was investigated by thaw-mount autoradiography. Fifteen 23-day-old ovariectomized and adrenalectomized female Holtzman rats were injected sc with 5 micrograms 17 beta-estradiol in oil/100 g BW daily for 4 days. On the fifth day, all animals were injected iv with 1 microgram/100 g BW [3H]ORG.2058, a synthetic progestin. The animals were killed after 30 or 60 min, and the brains were frozen and processed for autoradiography. Nuclear concentration of radioactivity was found in certain cells of the forebrain and midbrain and was prevented by prior injection (15 min) of excess unlabeled ORG.2058. In the preoptic-septal region, progestin-labeled neurons were observed in the nucleus (n.) septi lateralis, n. interstitialis striae terminalis, n. preopticus medialis, n. preopticus lateralis, n. preopticus periventricularis, and n. preopticus suprachiasmaticus. In the hypothalamus, labeled neurons were seen in n. periventricularis hypothalami, n. arcuatus hypothalami, n. ventromedialis hypothalami, n. dorsomedialis hypothalami, n. premammillaris ventralis, n. premammillaris dorsalis, and lateral hypothalamus. In the extrahypothalamic region, a few labeled cells were found in the central, medial, and cortical nuclei of the amygdala, the organum subforniculare, the lateral geniculate nucleus, the parietal and entorhinal cortex, and the central gray of the midbrain. Some of the progestin-concentrating cells were characterized with combined autoradiography and immunocytochemistry. Tyrosine hydroxylase-containing cells of the arcuate nucleus and the hypothalamic periventricular nucleus (group A12) showed nuclear concentration of radioactivity. Tyrosine hydroxylase cells in groups A11, A13, and A14; the substantia nigra (group A9); and the ventral tegmental area (group A10) did not show nuclear concentration of [3H]ORG.2058. The autoradiographic study demonstrates specific progestin-binding sites in nuclei of neurons in select areas of the rat brain. The results of combined autoradiography and immunocytochemistry suggest, for the first time, a direct action of progestin on tuberoinfundibular dopaminergic neurons.

Animals↗

PubMed1991

Subcellular distribution of low molecular weight guanosine triphosphate-binding proteins in adipocytes: colocalization with the glucose transporter Glut 4.

Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Since low mol wt GTP-binding proteins (LMW-GTP-binding proteins) have been implicated in the regulation of vesicular trafficking, we have analyzed these proteins in adipocytes. Isolated adipocytes were incubated in the absence or presence of insulin before separation of plasma membranes (PM) and low density microsomes (LDM). [alpha-32P]GTP binding to proteins transferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific and distinct subsets of proteins in the PM and LDM; those proteins were more abundant in PM than in LDM. [alpha-32P]GTP binding to these proteins was specific for the guanylnucleotides, since it was competed for by GTP and guanosine 5'-O-(3-thiotriphosphate), but not by ATP or adenosine 5'-O-(3-thiotriphosphate). The LMW-GTP-binding proteins were tightly associated with the membranes, as treatment with 1.5 M KCl did not modify this association. The distribution of the LMW-GTP-binding proteins in the fractions and their affinity for guanylnucleotides were the same in control and insulin-treated adipocytes. When the presence of Gi alpha subunits was looked for with a specific antibody, Gi alpha 1 and Gi alpha 2 were found almost exclusively in PM. By contrast, the same antibody revealed the presence of a 100 kDa band in the LDM. Insulin treatment of adipocytes did not modify the amounts of those G-proteins in PM or LDM fractions, although it promoted the translocation of Glut 4 proteins from LDM to PM. LDM fractions contain a specific subset of vesicles markedly enriched in Glut 4 molecules. When those vesicles were isolated from the total LDM fraction by immunoadsorption on highly specific antibodies to Glut 4 protein, LMW-GTP-binding proteins were found in the immune pellet. Those proteins were absent when immunoprecipitation was performed after solubilization of the vesicles with 1% Triton X-100. Our results strongly suggest that the vesicles containing the Glut 4 protein also contained LMW-GTP-binding proteins and indicate that these GTP-binding proteins could play a role in the exocytosis of the Glut 4-containing vesicles.

Adipose Tissue↗

PubMed1992

Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat brain and its colocalization in hypothalamic somatostatin neurons.

The activity of both somatostatin (SS) and GH-releasing hormone (GHRH) neurons within several hypothalamic nuclei is regulated, in part, by the feedback effects of GH. However, whether GH, or its intermediate, insulin-like growth factor I, acts on these neurons to alter the synthesis and release of SS and GHRH is unknown. We argued that if GH itself acts directly on the brain to govern its own secretion, then regions of the brain containing SS and GHRH neurons may express the GH receptor gene. We tested this hypothesis by performing in situ hybridization for GH receptor messenger RNA (mRNA) and mapping its distribution in the brain. We observed GH receptor mRNA-containing cells in various brain regions including the thalamus, septal region, hippocampus, dentate gyrus, amygdala, and hypothalamus. Next we sought evidence for expression of the GH receptor mRNA by SS neurons in the hypothalamus. We addressed this by performing a double-label in situ hybridization to identify neurons expressing both SS mRNA and GH receptor mRNA. We report that SS neurons in the periventricular nucleus and in the paraventricular nucleus coexpress the GH receptor gene, whereas few, if any, of the SS neurons in the cortex express detectable amounts of the GH receptor mRNA. These findings suggest that GH acts directly on the brain and participates in the regulation of its own secretion through a direct action on hypothalamic SS neurons.

Animals↗

PubMed1993

Basic fibroblast growth factor (bFGF) receptors decrease with luteal age in rat ovarian luteal cells: colocalization of bFGF receptors and bFGF in luteal cells.

Ovarian growth factors have been implicated in the development and differentiation of corpus luteum. We have characterized both high and low affinity receptors for basic fibroblast growth factor (bFGF) in luteal cells and tissue throughout the life span of corpus luteum using gonadotropin-treated rat luteinized ovaries. Additionally, we determined bFGF location in luteal tissue. High affinity (Kd, approximately 0.2 nM) and low capacity (approximately 500-6000 sites/cell, depending on luteal ages) [125I] bFGF-binding sites (mol wt, 140 kilodaltons, determined by affinity labeling) were found on luteal cells. [125I]bFGF binding to luteal cells and corpus luteum membranes progressively decreased in binding capacity without affecting binding affinity as the age of corpus luteum advanced. bFGF receptor (flg) mRNA in luteinized ovaries decreased with the luteal age similar to the [125I]bFGF binding. In contrast, low affinity binding sites, identified as heparan sulfate proteoglycans, which were immunohistochemically visualized around luteal cells, did not appear to change with luteal age. The signals for heparan sulfate proteoglycans that were found only in granulosa, but not theca-interstitial, cells of follicles became intense during folliculogenesis. The functionality of the bFGF receptors was shown by tyrosine phosphorylation of 16.5- and 18-kilodalton proteins in luteal cells with the antiphosphotyrosine antibody. Immunohistochemistry localized bFGF to steroidogenic luteal cells, and immunoblotting displayed larger molecular forms of bFGF in luteal cells. These results suggest that the bFGF receptors may be associated with the development and differentiation of corpus luteum in an autocrine manner.

Animals↗

PubMed1994

Colocalization of NADPH-diaphorase and estrogen receptor immunoreactivity in the rat ventromedial hypothalamic nucleus: stimulatory effect of estrogen on NADPH-diaphorase activity.

NADPH-diaphorase (NADPH-d) histochemistry was combined with estrogen receptor (ER) immunohistochemistry in order to study effect of estrogen on NADPH-d activity and establish an anatomical relationship between NADPH-d and ER-immunoreactivity (ir) containing neurons in the ventromedial hypothalamic nucleus (VMN). Gonadectomized female and male rats received either oil (control group) or 10 micrograms of estradiol benzoate (EB) for two successive days and were sacrificed on day 4. While NADPH-d histochemistry stained a specific subpopulation of neurons in the ventrolateral portion of the VMN of all groups, there was a marked sex difference in the effect of EB treatment. EB elevated the number of NADPH-d positive neurons in females but had no significant effect in males. Double-labeling histochemistry revealed that more than 70% of the NADPH-d positive neurons contained ER-ir in the VMN of all groups, with EB-treated females having a significantly higher frequency than all other groups. Since neuronal NADPH-d is nitric oxide synthase (NOS), these results provide anatomical evidence for an association of estrogen with NOS in the rat VMN and suggest that NOS activity can be modulated by estradiol in estrogen sensitive neurons.

Animals↗

PubMed1995

Thyrotropin-releasing hormone and c-fos/c-jun genes are colocalized in rat anterior pituitary cells: stimulation of transcription by glucocorticoids.

We recently reported that glucocorticoids (GC) enhance the level of TRH peptide and messenger RNA in anterior pituitary cells. However, the regulating mechanism is as yet unclear. The protooncogenes c-fos/c-jun belong to the class of immediate early genes that are activated in neurons by a variety of stimuli, including GC. Fos protein acts as an intracellular third messenger, regulating gene transcription of neurotransmitters. To determine whether c-fos/c-jun are involved in regulating the effect of GC on TRH in rat anterior pituitary cells, the coexpression and nuclear transcription activity of TRH and c-fos/c-jun after dexamethasone (DEX) stimulation (7 days) were investigated. The double labeled in situ hybridization results demonstrated that TRH and c-fos/c-jun are coexpressed in anterior pituitary cells and that DEX (10(-8) M) enhanced the cell intensity for TRH and c-fos/c-jun. The mean cell intensity of treatment vs. control was 2.4-fold for TRH, 1.4-fold for c-fos, and 1.4-fold for c-jun (n = 24; P < 0.01). The Northern blot analysis also showed that DEX increased the messenger RNA level of TRH 5.1-fold (n = 4; P < 0.01), that of c-fos 1.8-fold (n = 5; P < 0.01), and that of c-jun 4.2-fold (n = 4; P < 0.01). The nuclear run-on analysis indicated that DEX increased the nuclear transcription activity of TRH 3.3-fold, that of c-jun 3.2-fold, and that of c-fos 3-fold (n = 3; P < 0.01) vs. the control value. The coexpression of TRH and c-fos/c-jun in anterior pituitary cells as well as the enhancement of transcription after DEX treatment raise the possibility that c-fos/c-jun could mediate the effect of GC on TRH gene transcriptional activity.

Animals↗

PubMed1998

The V1a and V1b, but not V2, vasopressin receptor genes are expressed in the supraoptic nucleus of the rat hypothalamus, and the transcripts are essentially colocalized in the vasopressinergic magnocellular neurons.

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.

Animals↗

PubMed1998

Evidence for the colocalization of estrogen receptor-beta mRNA and estrogen receptor-alpha immunoreactivity in neurons of the rat forebrain.

Estrogen receptor beta (ER beta) mRNA is expressed in several rat brain regions where ER alpha is abundant. In vitro studies have shown that ER alpha and ER beta can heterodimerize and that the activity of this complex may be different than an ER alpha or ER beta homodimer complex. The purpose of the present study was to ascertain if ER alpha and ER beta are co-expressed by certain neuronal populations using a double label in situ hybridization/immunocytochemistry method. The results revealed that neurons in the bed nucleus of the stria terminalis, medial amygdala and preoptic area contain both ERs, with the vast majority of the neurons being double labeled. In other brain regions including the arcuate nucleus, cortical amygdaloid nuclei and ventromedial nucleus, only a few double-labeled cells were detected, while neurons in the paraventricular nucleus, supraoptic nucleus, and cerebral cortex expressed only ER beta mRNA. The results of these double label experiments provide the first evidence that ER alpha and ER beta coexist in neurons under in vivo conditions and suggest that estrogens may differentially modulate the activity of certain neuronal populations depending on whether the cells expresses ER alpha, ER beta or both ERs.

Animals↗

PubMed2001

Unmasking the progesterone receptor in the preoptic area and hypothalamus of the ewe: no colocalization with gonadotropin-releasing neurons.

Progesterone powerfully inhibits GnRH secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Visualization of the neural ovine progesterone receptor has proved elusive but, using a high temperature antigen unmasking technique, the progesterone receptor was revealed in the ewe brain. Progesterone receptors were located in the preoptic-hypothalamic continuum, especially in the preoptic area, ventrolateral region of the ventromedial nucleus and the arcuate nucleus. This study also suggests that the inhibitory action of progesterone on GnRH release is not transduced directly through the GnRH neurons as a single GnRH perikaryon of 732 was immunoreactive for the progesterone receptor.

Acrolein↗

PubMed2002

Distribution, neuronal colocalization, and 17beta-E2 modulation of small conductance calcium-activated K(+) channel (SK3) mRNA in the guinea pig brain.

Molecular cloning has revealed the existence of three distinct small conductance (SK1-3) Ca(2+)-activated K(+) channels. Because SK channels underlie the afterhyperpolarization (AHP) that is critical for sculpturing phasic firing in hypothalamic neurons, we investigated the distribution of these channels in the female guinea pig. Both SK1 and SK3 cDNA fragments were cloned using PCR, and ribonuclease protection assay as well as in situ hybridization analysis illustrated that the SK3 channel was the predominant subtype expressed in the guinea pig hypothalamus. Combined in situ hybridization and fluorescence immunocytochemistry revealed that SK3 mRNA was expressed in GnRH, dopamine, and vasopressin neurons, and all of these neurons exhibited an AHP current. Moreover, SK3 mRNA was found in other brain areas, including the septum, bed nucleus, amygdala, thalamus, midbrain, and hippocampus. Using quantitative ribonuclease protection assay, the rank order of SK3 mRNA expression was septum >or= midbrain > rostral thalamus >or= rostral basal hypothalamus >or= caudal thalamus >or= preoptic area >> caudal basal hypothalamus >or= hippocampus. Moreover, 17beta-E2 treatment, which reduces plasma LH during the negative feedback phase, significantly increased SK3 mRNA levels in the rostral basal hypothalamus (P < 0.05; n = 6). Therefore, these findings suggest that estrogen increases the mRNA expression of SK3 channels, which may represent a mechanism by which estrogen regulates hypothalamic neuronal excitability during negative feedback.

Animals↗

PubMed1995

Colocalization of vascular endothelial growth factor (vascular permeability factor) and insulin in pancreatic islet cells.

Previously, we found that the islet of pancreas stained with a antibody against the vascular permeability factor (VPF; also known as vascular endothelial growth factor, VEGF) protein. To determine how common this reaction was and whether it was a specific reaction for the islet, we examined its expression and specific cellular localization. Two different antibodies directed against VPF/VEGF peptide revealed an intense reaction for beta-cells in the human islets of Langerhans, and several human beta-cell tumors (insulinomas), but no reaction, were detectable in the vascular endothelium. In the fetal pancreas (second and third trimesters), the VPF/VEGF peptide was detected in immature islets. Northern blot analysis of cell lines derived from rodent insulinomas revealed expression of VPF/VEGF messenger ribonucleic acid. Western blot analysis of conditioned medium from one of these cell lines showed the presence of the released VPF/VEGF protein. These findings indicate that beta-cells have a specific role other than endocrine function in the pancreas. VPF/VEGF in beta-cells may be involved in the maintenance and control of permeability within the islet capillary system.

Adult↗

PubMed1999

Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle.

The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, that function as ligand-activated transcription factors. In vitro studies suggest that the isoforms differ functionally and that the relative levels in a target cell may determine the nature and magnitude of response to progesterone. However, it is not known whether the two isoforms are normally coexpressed in vivo. To understand the functional significance of relative PR isoform expression in normal physiology, it is essential to determine whether PRA and PRB are coexpressed in the same cell. This study reports the development of a dual immunofluorescent staining technique to demonstrate PRA and PRB proteins by single cell analysis in the same tissue section of human endometrium during the menstrual cycle. PRA and PRB are coexpressed in target cells of the human uterus. In the glands, PRA and PRB were expressed before subnuclear vacuole formation and glycogenolysis, implicating both isoforms in this process, whereas persistence of PRB during the midsecretory phase suggested its significance in glandular secretion. In the stroma, the predominance of PRA throughout the cycle implicates this isoform in post-ovulatory progesterone-mediated events. These results support the view that PRA and PRB mediate distinct pathways of progesterone action in the glandular epithelium and stroma of the human uterus throughout the menstrual cycle.

Cell Nucleus↗

PubMed2006

Signaling complexes associated with the type I gonadotropin-releasing hormone (GnRH) receptor: colocalization of extracellularly regulated kinase 2 and GnRH receptor within membrane rafts.

Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in alphaT3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G(alphaq), c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in alphaT3-1 cells and in whole mouse pituitary. Cholesterol depletion of alphaT3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In alphaT3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.

Animals↗

PubMed1991

Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2003

Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage.

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. We have characterized a null allele of maelstrom (mael) and shown that Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, we have defined maelstrom as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, we have determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena, aubergine and spindle-E (spn-E), for its nuage localization. Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway.

Active Transport, Cell Nucleus↗

PubMed2003

Observation of keratin particles showing fast bidirectional movement colocalized with microtubules.

Keratin intermediate filament networks were observed in living cultured epithelial cells using the incorporation of fluorescently tagged keratin from a transfected enhanced green fluorescent protein (EGFP) construct. In steady-state conditions EGFP-keratin exists not only as readily detectable intermediate filaments, but also as small particles, of which there are two types: a less mobile population (slow or static S particles) and a highly dynamic one (fast or F particles). The dynamic F particles move around the cell very fast and in a non-random way. Their movement is composed of a series of steps, giving an overall characteristic zig-zag trajectory. The keratin particles are found all over the cell and their movement is aligned with microtubules; treatment of cells with nocodazole has an inhibitory effect on keratin particle movement, suggesting the involvement of microtubule motor proteins. Double-transfection experiments to visualize tubulin and keratin together suggest that the movement of keratin particles can be bidirectional, as particles are seen moving both towards and away from the centrosome area. Using field emission scanning and transmission electron microscopy combined with immunogold labelling, we also detected particulate keratin structures in untransfected epithelial cells, suggesting that keratin particles may be a natural component of keratin filament dynamics in living cells.

Cell Line↗

PubMed2006

Versican-thrombospondin-1 binding in vitro and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells.

We identified a specific interaction between two secreted proteins, thrombospondin-1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. Thrombospondin-1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of thrombospondin-1 but only weakly with the corresponding N-terminal region of thrombospondin-2. The G1 domain of versican contains two Link modules, which are known to mediate TNFalpha-stimulated gene-6 protein binding to thrombospondin-1, and the related G1 domain of aggrecan is also recognized by thrombospondin-1. Therefore, thrombospondin-1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added thrombospondin-1 incorporates into these structures. Binding of exogenous thrombospondin-1 to these structures, to purified versican and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous thrombospondin-1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that thrombospondin-1 negatively modulates this component of a vascular smooth muscle inflammatory response.

Aggrecans↗