Luke A. Hammond

EGF induces macropinocytosis and SNX1-modulated recycling of E-cadherin.

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.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by Golgin-97

E‐cadherin is a cell–cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane‐bound 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.

Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFα

Activated macrophages secrete an array of proinflammatory cytokines, including tumor necrosis factor-alpha (TNFalpha) 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 TNFalpha 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 TNFalpha, 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 TNFalpha and from surface recycling transferrin. Thereafter, these cytokines are independently secreted, with TNFalpha 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.

A trans-Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo.

The transmembrane precursor of tumor necrosis factor-α (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 microRNA-vector 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.

Automated organelle-based colocalization in whole-cell imaging.

The use of fluorescence microscopy to investigate protein colocalization is an invaluable tool for understanding subcellular structures and their associated proteins. However, current techniques are largely limited to two‐dimensional (2D) imaging and often require manual segmentation. Here, we present OBCOL, a methodology to automatically segment and quantify protein colocalization not within an image as a whole but on all individual punctuate organelles within a 3D multichannel image. A wide variety of colocalization statistics may then be calculated on the objects found, and features reported for each such as position, degree of overlap between channels, and number of component objects. OBCOL was validated on imaging of two fluorescent markers (Dextran, EGF) in 3D microscopy imaging. OBCOLs application was then exemplified by investigating the colocalization of three fluorescently tagged proteins (VAMP3, Rab11, and transferrin) on recycling endosomes in mammalian cells. The methodology showed for the first time the diversity of endosomes labeled with one or more of these proteins and quantitatively demonstrated the degree of overlap among these proteins in individual recycling endosomes. The consistent segregation of these markers provides novel evidence for the subcompartmentalization of recycling endosomes. OBCOL is a flexible methodology for 3D multifluorophore image analysis. This study clearly demonstrated its value for investigating subcellular structures and their constituent proteins.

Developmental Vitamin D (DVD) Deficiency Reduces Nurr1 and TH Expression in Post-mitotic Dopamine Neurons in Rat Mesencephalon

Developmental vitamin D (DVD) deficiency has been proposed as an important risk factor for schizophrenia. Our previous study using Sprague Dawley rats found that DVD deficiency disrupted the ontogeny of mesencephalic dopamine neurons by decreasing the mRNA level of a crucial differentiation factor of dopamine cells, the nuclear receptor related 1 protein (Nurr1). However, it remains unknown whether this reflects a reduction in dopamine cell number or in Nurr1 expression. It is also unclear if any particular subset of developing dopamine neurons in the mesencephalon is selectively affected. In this study, we employed state-of-the-art spinning disk confocal microscopy optimized for the imaging of tissue sections and 3D segmentation to assess post-mitotic dopamine cells on a single-cell basis in the rat mesencephalon at embryonic day 15. Our results showed that DVD deficiency did not alter the number, morphology, or positioning of post-mitotic dopamine cells. However, the ratio of Nurr1+TH+ cells in the substantia nigra pars compacta (SNc) compared with the ventral tegmental area (VTA) was increased in DVD-deficient embryos. In addition, the expression of Nurr1 in immature dopamine cells and mature dopamine neurons in the VTA was decreased in DVD-deficient group. Tyrosine hydroxylase was selectively reduced in SNc of DVD-deficient mesencephalon. We conclude that DVD deficiency induced early alterations in mesencephalic dopamine development may in part explain the abnormal dopamine-related behaviors found in this model. Our findings may have broader implications for how certain environmental risk factors for schizophrenia may shape the ontogeny of dopaminergic systems and by inference increase the risk of schizophrenia.

Vitamin D regulation of GDNF/Ret signaling in dopaminergic neurons

1,25(OH)2D3 (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. FASEB J. 32, 819–828 (2018). www.fasebj.org

Maternal Vitamin D Prevents Abnormal Dopaminergic Development and Function in a Mouse Model of Prenatal Immune Activation

Dysfunction in dopamine (DA) systems is a prominent feature in schizophrenia patients and may result from the abnormal development of mesencephalic (mes)DA systems. Maternal immune activation (MIA) and developmental vitamin D (DVD)-deficiency both induce schizophrenia-relevant dopaminergic abnormalities in adult offspring. In this study, we investigated whether maternal administration of the vitamin D hormone (1,25OHD, VITD) could prevent MIA-induced abnormalities in DA-related behaviors and mesDA development. We administrated the viral mimetic polyriboinosinic-polyribocytidylic (poly (I:C)) simultaneously with 1,25OHD and/or their vehicles, to pregnant mouse dams at gestational day 9. Maternal treatment with VITD prevented MIA-induced hypersensitivity to acute DA stimulation induced by amphetamine, whereas it failed to block prepulse inhibition deficiency in MIA-exposed offspring. MIA and VITD both reduced fetal mesDA progenitor (Lmx1a + Sox2+) cells, while VITD treatment increased the number of mature (Nurr1 + TH+) mesDA neurons. Single-cell quantification of protein expression showed that VITD treatment increased the expression of Lmx1a, Nurr1 and TH in individual mesDA cells and restored normal mesDA positioning. Our data demonstrate that VITD prevents abnormal dopaminergic phenotypes in MIA offspring possibly via its early neuroprotective actions on fetal mesDA neurons. Maternal supplementation with the dietary form of vitamin D, cholecalciferol may become a valuable strategy for the prevention of MIA-induced neurodevelopmental abnormalities.