Human mesangial cells express inducible macrophage scavenger receptor.
(17/1146)
BACKGROUND: Type A scavenger receptors (Scr) mediate the uptake of modified low-density lipoproteins by macrophages. The accumulation of lipids via this process is thought to lead to foam cell formation in atherosclerotic plaques. Human mesangial cells (HMCs) have not been previously shown to express Scr in normal culture. We therefore investigated whether there is an inducible form of Scr in a human mesangial cell line (HMCL). METHODS: Scr activity was analyzed by cellular uptake of fluorescently labeled acetylated low-density lipoprotein using a flow cytometer. Scr mRNA expression was examined using reverse transcription-polymerase chain reaction, followed by Southern blotting. To investigate the molecular mechanism of Scr expression, several reporter gene constructs were designed. The first contained a full Scr promoter, the second a part of the Scr promoter that has both AP-1 and ets transcription factor binding sites. Other constructs were identical to the second, except that they contained either AP-1 or ets motif mutations. RESULTS: Phorbol 12-Myristate 13-acetate (PMA) and angiotensin II (Ang II) increased both the percentage of Scr-positive cells and the Scr mean fluorescence intensity. PMA and Ang II also increased Scr mRNA and promoter activity in a time- and dose-responsive manner. Protein kinase C and calmodulin transduction pathways were involved in Scr up-regulation induced by PMA and Ang II. Additionally, a serine/threonine kinase was involved in PMA stimulation. Functional analysis showed that both AP-1 and ets motifs were specific response elements to PMA stimulation in HMCLs. CONCLUSIONS: This study suggests that HMCs may express an inducible Scr, by which cells can acquire lipids and convert to foam cells in developing glomerulosclerosis. (+info)
Early neocortical regionalization in the absence of thalamic innervation.
(18/1146)
There is a long-standing controversy regarding the mechanisms that generate the functional subdivisions of the cerebral neocortex. One model proposes that thalamic axonal input specifies these subdivisions; the competing model postulates that patterning mechanisms intrinsic to the dorsal telencephalon generate neocortical regions. Gbx-2 mutant mice, whose thalamic differentiation is disrupted, were investigated. Despite the lack of cortical innervation by thalamic axons, neocortical region-specific gene expression (Cadherin-6, EphA-7, Id-2, and RZR-beta) developed normally. This provides evidence that patterning mechanisms intrinsic to the neocortex specify the basic organization of its functional subdivisions. (+info)
Transcriptional inhibition by interleukin-6 of the class A macrophage scavenger receptor in macrophages derived from human peripheral monocytes and the THP-1 monocytic cell line.
(19/1146)
Expression of the class A macrophage scavenger receptor (MSR) contributes to the uptake of modified low density lipoproteins (LDL) by macrophages and transformation of these cells into lipid-laden foam cells, which characterize atherosclerosis. Many environmental factors, in particular, proinflammatory cytokines and growth factors, can exert regulatory effects on MSR expression, whereas intracellular accumulation of cholesterol itself does not influence MSR levels to any considerable extent. In the present study, by using an in vitro model, we examined whether stimulation with interleukin-6 (IL-6), an immunoregulatory, multipotential cytokine, modulates the expression and activities of the MSR in macrophages. When treated with IL-6, macrophages derived from peripheral monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytic cells showed significantly reduced uptake and/or binding of the MSR ligand, acetylated LDL. This effect was paralleled by a reduction in the expression of MSR protein and mRNA. Analysis of MSR promoter activity in THP-1 cells transfected with an MSR promoter-reporter gene construct demonstrated decreased activity of the MSR promoter in IL-6-treated THP-1 macrophages. Electrophoretic mobility gel shift assay also showed a reduction in the binding of a transcription factor to the MSR promoter AP-1/ets elements in IL-6-treated cells. Thus, exposure to IL-6 may inhibit expression of the class A MSR in differentiated macrophages at transcriptional levels. This result suggests that this cytokine may modulate foam cell formation during atherogenesis. (+info)
Organisation of mitochondria in living sensory neurons.
(20/1146)
In this work, we have examined the mitochondrial organisation in living cultured primary dorsal root ganglion (DRG) neurons. Confocal microscopy and the mitochondrial potential-sensitive fluorescent dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo carbocyanine iodide (JC-1) were used to visualise intracellular structures with a high and low membrane potential. Three-dimensional reconstruction revealed a mitochondrial organisation featuring separate highly polarised mitochondria, clusters of mitochondria located mainly at the base of neurite hillocks and filamentous mitochondrial structures. Filamentous mitochondria were distributed along the cell body, especially between neurites. A functional integration between mitochondrial structures is proposed. (+info)
Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-Golgi transport complexes.
(21/1146)
Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in the selective packaging of anterograde cargo into coated transport vesicles budding from the ER [1]. In mammalian cells, these vesicles coalesce to form tubulo-vesicular transport complexes (TCs), which shuttle anterograde cargo from the ER to the Golgi complex [2] [3] [4]. In contrast, COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER [1] [5] [6] [7]. The binding of COPI to COPII-coated TCs [3] [8] [9], however, has led to the proposal that COPI binds to TCs and specifically packages recycling proteins into retrograde vesicles for return to the ER [3] [9]. To test this hypothesis, we tracked fluorescently tagged COPI and anterograde-transport markers simultaneously in living cells. COPI predominated on TCs shuttling anterograde cargo to the Golgi complex and was rarely observed on structures moving in directions consistent with retrograde transport. Furthermore, a progressive segregation of COPI-rich domains and anterograde-cargo-rich domains was observed in the TCs. This segregation and the directed motility of COPI-containing TCs were inhibited by antibodies that blocked COPI function. These observations, which are consistent with previous biochemical data [2] [9], suggest a role for COPI within TCs en route to the Golgi complex. By sequestering retrograde cargo in the anterograde-directed TCs, COPI couples the sorting of ER recycling proteins [10] to the transport of anterograde cargo. (+info)
The medial ganglionic eminence gives rise to a population of early neurons in the developing cerebral cortex.
(22/1146)
During development of the neocortex, the marginal zone (layer I) and the subplate (layer VII) are the first layers to form from a primordial plexiform neoropil. The cortical plate (layers II-VI) is subsequently established between these superficial and deep components of the primordial plexiform neuropil. Neurons in the early zones are thought to play important roles in the formation of the cortex: the Cajal-Retzius cells of the marginal zone are instrumental in neuronal migration and laminar formation, and cells of the subplate are involved in the formation of cortical connections. Using the fluorescent tracer 1,1'-dioctodecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), we have shown here that a substantial proportion of neurons of the marginal zone, including cells with features of Cajal-Retzius cells, and of the subplate and lower intermediate zone are not born in the ventricular neuroepithelium but instead originate in the medial ganglionic eminence (MGE), the pallidal primordium. These neurons follow a tangential migratory route to their positions in the developing cortex. They express the neurotransmitter GABA but seem to lack the calcium binding protein calretinin; some migrating cells found in the marginal zone express reelin. In addition, migrating cells express the LIM-homeobox gene Lhx6, a characteristic marker of the MGE. It is suggested that this gene uniquely or in combination with other transcription factors may be involved in the decision of MGE cells to differentiate in situ or migrate to the neocortex. (+info)
Cell membrane orientation visualized by polarized total internal reflection fluorescence.
(23/1146)
In living cells, variations in membrane orientation occur both in easily imaged large-scale morphological features, and also in less visualizable submicroscopic regions of activity such as endocytosis, exocytosis, and cell surface ruffling. A fluorescence microscopic method is introduced here to visualize such regions. The method is based on fluorescence of an oriented membrane probe excited by a polarized evanescent field created by total internal reflection (TIR) illumination. The fluorescent carbocyanine dye diI-C(18)-(3) (diI) has previously been shown to embed in the lipid bilayer of cell membranes with its transition dipoles oriented nearly in the plane of the membrane. The membrane-embedded diI near the cell-substrate interface can be fluorescently excited by evanescent field light polarized either perpendicular or parallel to the plane of the substrate coverslip. The excitation efficiency from each polarization depends on the membrane orientation, and thus the ratio of the observed fluorescence excited by these two polarizations vividly shows regions of microscopic and submicroscopic curvature of the membrane, and also gives information regarding the fraction of unoriented diI in the membrane. Both a theoretical background and experimental verification of the technique is presented for samples of 1) oriented diI in model lipid bilayer membranes, erythrocytes, and macrophages; and 2) randomly oriented fluorophores in rhodamine-labeled serum albumin adsorbed to glass, in rhodamine dextran solution, and in rhodamine dextran-loaded macrophages. Sequential digital images of the polarized TIR fluorescence ratios show spatially-resolved time-course maps of membrane orientations on diI-labeled macrophages from which low visibility membrane structures can be identified and quantified. To sharpen and contrast-enhance the TIR images, we deconvoluted them with an experimentally measured point spread function. Image deconvolution is especially effective and fast in our application because fluorescence in TIR emanates from a single focal plane. (+info)
The role of the 3-hydroxy 3-methylglutaryl coenzyme A reductase cytosolic domain in karmellae biogenesis.
(24/1146)
In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using beta-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain. (+info)