In epithelia, junction proteins are endocytosed for modulation of cell-cell adhesion and cell polarity. In response to growth factors, the cell-cell adhesion protein E-cadherin is internalized from the cell surface with degradation or recycling as potential fates. However, the cellular machinery involved in cadherin internalization and recycling remains controversial. Here we investigated EGF-induced E-cadherin internalization. EGF stimulation of MCF-7 cells resulted in Rac1-modulated macropinocytosis of the E-cadherin-catenin complex into endosomal compartments that colocalized with EEA1 and the sorting nexin, SNX1. Depletion of cellular SNX1 levels by siRNA resulted in increased intracellular accumulation and turnover of E-cadherin internalized from the cell surface in response to EGF. Moreover, SNX1 was also required for efficient recycling of internalized E-cadherin and re-establishment of epithelial adhesion. Together, these findings demonstrate a role for SNX1 in retrieval of E-cadherin from a degradative endosomal pathway and in membrane trafficking pathways that regulate E-cadherin recycling.
Activated macrophages secrete an array of proinflammatory cytokines, including tumor necrosis factor-α (TNFα) and interleukin 6 (IL-6), that are temporally secreted for sequential roles in inflammation. We have previously characterized aspects of the intracellular trafficking of membrane-bound TNFα and its delivery to the cell surface at the site of phagocytic cups for secretion (Murray, R.Z., J.G. Kay, D.G. Sangermani, and J.L. Stow. 2005. Science. 310:1492–1495). The trafficking pathway and surface delivery of IL-6, a soluble cytokine, were studied here using approaches such as live cell imaging of fluorescently tagged IL-6 and immunoelectron microscopy. Newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers either as the sole labeled cargo or together with TNFα, utilizing specific soluble NSF attachment protein receptor (SNARE) proteins to fuse with the recycling endosome. Within recycling endosomes, we demonstrate the compartmentalization of cargo proteins, wherein IL-6 is dynamically segregated from TNFα and from surface recycling transferrin. Thereafter, these cytokines are independently secreted, with TNFα delivered to phagocytic cups but not IL-6. Therefore, the recycling endosome has a central role in orchestrating the differential secretion of cytokines during inflammation.
E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membranebound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/ golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.
The transmembrane precursor of tumor necrosis factor-␣ (TNF) exits the trans-Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface; however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by >10-fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo, we generated retrogenic mice expressing a microRNAvector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery. intracellular trafficking ͉ RNAi transgenic mice ͉ TNF secretion ͉ inflammation ͉ membrane transport T umor necrosis factor-␣ (TNF) is the main proinflammatory cytokine made and secreted by inflammatory macrophages. Early release of TNF in response to LPS or other inflammatory signals enhances the activation and recruitment of T cells and ensures robust innate and acquired immune responses (1). The excessive secretion of TNF is also a prevalent and clinically significant problem in acute inflammation and in chronic inflammatory disease (2). Anti-TNF treatments have shown success in the treatment of rheumatoid arthritis, inflammatory bowel disease, and other conditions (3). Now improved antiTNF strategies that may offer more constrained or cell type specific control of TNF secretion are being sought. This requires identification of molecular mediators of TNF secretion, particularly those that can be targeted to block TNF release.We have characterized the secretory pathway for TNF in activated macrophages and identified key organelles and components of the trafficking machinery whose expression or function is up-regulated by LPS to support cytokine secretion (4-6). Among these are members of the SNARE family of membrane fusion proteins required at different points along this ...
Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit α1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses.
Vision plays a major role in the life of most teleosts, and is assumingly well adapted to each species ecology and behaviour. Using a multidisciplinary approach, we scrutinised several aspects of the visual system and ecology of the Great Barrier Reef anemonefish, Amphiprion akindynos, including its orange with white patterning, retinal anatomy and molecular biology, its symbiosis with anemones and sequential hermaphroditism. Amphiprion akindynos possesses spectrally distinct visual pigments and opsins: one rod opsin, RH1 (498 nm), and five cone opsins, SWS1 (370 nm), SWS2B (408 nm), RH2B (498 nm), RH2A (520 nm), and LWS (554 nm). Cones were arranged in a regular mosaic with each single cone surrounded by four double cones. Double cones mainly expressed RH2B (53%) in one member and RH2A (46%) in the other, matching the prevailing light. Single cones expressed SWS1 (89%), which may serve to detect zooplankton, conspecifics and the host anemone. Moreover, a segregated small fraction of single cones coexpressed SWS1 with SWS2B (11%). This novel visual specialisation falls within the region of highest acuity and is suggested to increase the chromatic contrast of Amphiprion akindynos colour patterns, which might improve detection of conspecifics.
1,25(OH)D (vitamin D) appears essential for the normal development of dopaminergic neurons. Vitamin D affects dopamine synthesis and metabolism as well as expression of glial cell line-derived neurotrophic factor (GDNF), which is crucial for the survival of dopaminergic neurons. We investigated the role of vitamin D on GDNF and its receptors protooncogene tyrosine-protein kinase receptor Ret (C-Ret) and GDNF family receptor alpha 1 (GFRα1) signaling. To this end, we used a developmental vitamin D-deficient rat model and SH-SY5Y cells transfected with vitamin D receptor (VDR). The absence of vitamin D ligand in gestation reduces C-Ret expression, but not GDNF and GFRα1, in embryo forebrains. Overexpression of VDR in SH-SY5Y in the absence of ligand (mimicking in vivo developmental vitamin D deficiency) also suppressed C-Ret mRNA levels. In the presence of vitamin D, C-Ret mRNA and protein expression were increased. The chromatin immunoprecipitation results suggested that C-Ret is directly regulated by vitamin D via VDR. GDNF was also increased by vitamin D in these cells. Our small interfering RNA studies showed that knocking down VDR leads to an increase in C-Ret in the absence of ligand. Finally, we confirmed the inverse relationship between GFRα1 and C-Ret, as knocking down C-Ret led to increases in GFRα1 expression. These data extend our knowledge of the diverse and important roles played by vitamin D in dopamine physiology.-Pertile, R. A. N., Cui, X., Hammond, L., Eyles, D. W. Vitamin D regulation of GDNF/Ret signaling in dopaminergic neurons.
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