The endocytic pathways of a secretory granule membrane protein in HEK293 cells : PAM and EGF traverse a dynamic multivesicular body network together

Peptidylglycine alpha-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4 h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes. (C) 2017 Elsevier GmbH. All rights reserved.Peer reviewe

Polarized THG Microscopy Identifies Compositionally Different Lipid Droplets in Mammalian Cells

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ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P-2 exchange

Low-density lipoprotein (LDL)-cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin-inducible rapid degradation of oxysterol-binding protein-related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL-derived cholesterol from late endosomes to focal adhesion kinase (FAK)-/integrin-positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P-2-containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P-2 generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P-2 in NPC1-containing late endosomes in a FAK-dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL-cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P-2 exchange between late and recycling endosomes.Peer reviewe

Oxidative stress and antioxidant defense responses in Acartia copepods in relation to environmental factors

On a daily basis, planktonic organisms migrate vertically and thus experience widely varying conditions in their physico-chemical environment. In the Gulf of Finland, these changes are larger than values predicted by climate change scenarios predicted for the next century (up to 0.5 units in pH and 5 degrees C in temperature). In this work, we are interested in how temporal variations in physico-chemical characteristics of the water column on a daily and weekly scale influence oxidative stress level and antioxidant responses in the planktonic copepod of the genus Acartia. Responses were determined from samples collected during a two-week field survey in the western Gulf of Finland, Baltic Sea. Our results showed that GST (Glutathione-S-transferase) enzyme activity increased in the surface waters between Weeks I and II, indicating antioxidant defense mechanism activation. This is most likely due to elevating temperature, pH, and dissolved oxygen observed between these two weeks. During Week II also GSSG (oxidized glutathione) was detected, indicating that copepods responded to stressor(s) in the environment. Our results suggest that Acartia copepods seem fairly tolerant to weekly fluctuations in environmental conditions in coastal and estuarine areas, in terms of antioxidant defense and oxidative stress. This could be directly connected to a very efficient glutathione cycling system acting as antioxidant defense system for neutralizing ROS and avoiding elevated levels of LPX

Sääntelytaakan arviointi ja vähentäminen

Tutkimuksessa on pyritty tuottamaan kattava, realistinen ja moniulotteinen yleiskuva sääntelytaakasta Suomessa. Erityisenä tavoitteena on ollut tunnistaa yrityksiin, kansalaistoimintaan ja yksittäisten ihmisten viranomaisasiointiin kohdistuvan turhan taakan keskeisimmät lähteet. Turhaa sääntelytaakkaa syntyy mm. seuraavista tekijöistä: epäselvä ja nopeasti muuttuva sääntely, ristiriitaiset ja päällekkäiset vaatimukset, kohtuuttoman raskaat menettelytavat asioinnissa viranomaisten kanssa ja sääntelyn epäyhtenäinen toimeenpano. Edes summittaisen ja uskottavan, numeerisen kokonaisarvion antaminen sääntelytaakasta ilman laajoja varaumia on mahdotonta. Tulosten mukaan 1) sääntelytaakka on epämääräinen käsite, mutta ilmiönä moniulotteinen, 2) sääntelytaakan mittaaminen on vaikeaa, 3) sääntelyn hallinta ja ongelmat toimeenpanossa muodostavat keskeisen osan sääntelytaakkaa, 4) kohdetahojen kokemukset sääntelytaakasta ovat monimuotoisia. Raportissa esitetään 15 kehittämisehdotusta koskien sääntelytaakan kohdentumista, sääntelyn toimeenpanoa ja lainvalmisteluprosessej

Expression of ODC Antizyme Inhibitor 2 (AZIN2) in Human Secretory Cells and Tissues

Ornithine decarboxylase (ODC) antizyme inhibitor 2 (AZIN2), originally called ODCp, is a regulator of polyamine synthesis that we originally identified and cloned. High expression of ODCp mRNA was found in brain and testis. We reported that AZIN2 is involved in regulation of cellular vesicle transport and/or secretion, but the ultimate physiological role(s) of AZIN2 is still poorly understood. In this study we used a peptide antibody (K3) to human AZIN2 and by immunohistochemistry mapped its expression in various normal tissues. We found high expression in the nervous system, in type 2 pneumocytes in the lung, in megakaryocytes, in gastric parietal cells co-localized with H, K-ATPase beta subunit, in selected enteroendocrine cells, in acinar cells of sweat glands, in podocytes, in macula densa cells and epithelium of collecting ducts in the kidney. The high expression of AZIN2 in various cells with secretory or vesicle transport activity indicates that the polyamine metabolism regulated by AZIN2 is more significantly involved in these events than previously appreciated.Peer reviewe

Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion

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Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.Peer reviewe

Multiparametric platform for profiling lipid trafficking in human leukocytes

Summary Systematic insight into cellular dysfunction can improve understanding of disease etiology, risk assessment, and patient stratification. We present a multiparametric high-content imaging platform enabling quantification of low-density lipoprotein (LDL) uptake and lipid storage in cytoplasmic droplets of primary leukocyte subpopulations. We validate this platform with samples from 65 individuals with variable blood LDL-cholesterol (LDL-c) levels, including familial hypercholesterolemia (FH) and non-FH subjects. We integrate lipid storage data into another readout parameter, lipid mobilization, measuring the efficiency with which cells deplete lipid reservoirs. Lipid mobilization correlates positively with LDL uptake and negatively with hypercholesterolemia and age, improving differentiation of individuals with normal and elevated LDL-c. Moreover, combination of cell-based readouts with a polygenic risk score for LDL-c explains hypercholesterolemia better than the genetic risk score alone. This platform provides functional insights into cellular lipid trafficking and has broad possible applications in dissecting the cellular basis of metabolic disorders.Peer reviewe

Carbon guide : An outlook on carbon in soil and on carbon cultivation

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