12,342 research outputs found
The formation and function of ER-endosome membrane contact sites
No full textRecent advances in membrane contact site (MCS) biology have revealed key roles for MCSs in inter-organellar exchange, the importance of which is becoming increasingly apparent. Roles for MCSs in many essential physiological processes including lipid transfer, calcium exchange, receptor tyrosine kinase signalling, lipid droplet formation, autophagosome formation, organelle dynamics and neurite outgrowth have been reported. The ER forms an extensive and dynamic network of MCSs with a diverse range of functionally distinct organelles. MCSs between the ER and endocytic pathway are particularly abundant, suggesting important physiological roles. Here, our current knowledge of the formation and function of ER contact sites with endocytic organelles from studies in mammalian systems is reviewed. Their relatively poorly defined molecular composition and recently identified functions are discussed. In addition, likely, but yet to be established, roles for these contacts in lipid transfer and calcium signalling are considered. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim Levine and Anant K. Menon
Gedragsinzichten bieden meer beleidskansen dan er nu worden benut
No full textGedragsinzichten zijn onontbeerlijk om te komen tot effectief beleid, de mens is immers geen homo economicus. Dat geldt echter niet alleen voor uitvoeringsvraagstukken, maar ook voor het ontwerpen van beleid. Hier ligt er nog een groot onbenut potentieel.Policy Analysi
Embedding research (ER) led by nurses, midwives and allied health professionals (NMAHPs): the NMAHP-ER model.
No full textPrevious embedded researcher models have focused predominantly on an individual being a temporary team member and embedded for a project-limited short-term placement. To develop an innovative research capacity building model to address the challenges of developing, embedding and sustaining, research led by Nurses, Midwives, and Allied Health Professionals (NMAHPs) in complex clinical environments. This healthcare and academic research partnership model offers an opportunity to support the 'how' of enabling NMAHP research capacity building from within the researchers' clinical area of expertise. Collaboration between three healthcare and academic organisations and the iterative process of cocreation, development and refinement took place over 6 months during 2021. The collaboration relied on virtual meetings, emails, telephone calls and document review. A codesigned NMAHP embedded research (ER) model is ready for trialling with the individual being an existing clinician working collaboratively within the healthcare setting and with academia to develop the skills to become the ER. This model supports NMAHP-led research activity in clinical organisations in a visible and manageable way. As a shared, long-term vision, the model will contribute to research capacity and capability of the wider healthcare workforce. It will lead, facilitate and support research in and across clinical organisations in collaboration with higher education institutions. [Abstract copyright: © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
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No full textN.B.: When citing this work, cite the original article. This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Radiation Protection Dosimetry following peer review. The definitive publisher-authenticated version
Valt er geld te verdienen aan het voorspellen van Voetbaluitslagen?
No full textIn dit verslag is onderzocht of er geld te verdienen valt aan het spelen van online voetbaltoto. Er zijn twee modellen onderzocht; één op basis van onderlinge resultaten en de ander op basis van achtergrondvariabelen. Vervolgens zijn deze getest op fictieve en historische data en met elkaar vergeleken. Als conclusie is er gevonden dat er zeker een kans bestaat dat de modellen winstgevend kunnen zijn, maar dat er te weinig data is om dit met zekerheid te zeggen.StatisticsApplied mathematicsElectrical Engineering, Mathematics and Computer Scienc
The secretion inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and the trans-Golgi network
No full textThe small-molecule inhibitor Exo2 {4-hydroxy-3-methoxy-(5,6,7,8-tetrahydrol[1]benzothieno[2,3-d]pyrimidin-4-yl)hydraz-one benzaldehyde} has been reported to disrupt the Golgi apparatus completely and to stimulate Golgi–ER (endoplasmic reticulum) fusion in mammalian cells, akin to the well-characterized fungal toxin BFA (brefeldin A). It has also been reported that Exo2 does not affect the integrity of the TGN (trans-Golgi network), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2, and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in HeLa and in BS-C-1 cells. However, Exo2, unlike BFA, does not induce tubulation and merging of the TGN and endosomal compartments. Furthermore, and in contrast with its effects on cholera toxin, Exo2 significantly perturbs the delivery of Shiga toxin to the ER. Together, these results suggest that the likely target(s) of Exo2 operate at the level of the TGN, the Golgi and a subset of early endosomes, and thus Exo2 provides a more selective tool than BFA for examining membrane trafficking in mammalian cells
The role of the lectin VIP36 in the early secretory pathway
No full textLectins are of emerging importance for quality control and intracellular transport of glycoproteins in mammalian cells. One of the most prominent lectins involved in intracellular transport is ERGIC-53, which belongs to the family of L-type lectins. ERGIC-53 mediates the ER export of several glycoproteins like cathepsin Z, α1-antitrypsin (α1-AT) or blood coagulation factors. VIP36 belongs to the same family as ERGIC-53, but its cellular function remains poorly understood. VIP36 is a type I membrane protein. It cycles within the early secretory pathway and binds high mannose glycans. In order to gain insight into the function of VIP36 we decided to search for a luminal interaction partner for VIP36.
We used a YFP-protein fragmentation complementation (YFP-PCA) based FACS screen of a human adult liver library to unravel an interaction partner for VIP36. Complementation of YFP is irreversible. Therefore, the YFP-PCA is well suited to detect weak interactions, like those between mammalian lectins and glycoproteins. YFP2-VIP36 was used as the bait in our screen. The human liver library was tagged with YFP1. Our screen identified α1-AT as an interaction partner for VIP36. VIP36 recognized high mannose containing α1-AT, which is consistent with the previously obtained results about the glycan affinity of VIP36. This interaction was increased upon inhibition of complex glycosylation by kifunensine. The complex formed by α1-AT and VIP36 was localized to the Golgi and the ER. α1-AT was previously identified as a cargo for ERGIC-53. Knockdown of ERGIC-53 slowed down α1-AT transport, consistent with a role for ERGIC-53 in ER export of α1-AT. In contrast, knockdown of VIP36 accelerated transport of endogenous α1-AT in HepG2 cells. This effect was specific for α1-AT, as the non-glycosylated protein albumin showed no acceleration in transport. In addition, VIP36 knockdown did not affect general protein secretion. This finding makes it unlikely that VIP36 acts as an anterograde cargo receptor for α1-AT. Further studies on the dynamics of the complex formed by VIP36 and α1-AT revealed that VIP36 recycles α1-AT back to the ER, which argues for a role of VIP36 in post-ER quality control. This notion is further supported by the finding that the chaperone BiP co-immunoprecipitated with the complex of VIP36 and α1-AT. This chaperone was previously described as an interaction partner for VIP36. This argues for a complex consisting of VIP36 and BiP acting together in post-ER quality control to detect misfolded α1-antitrypsin in the Golgi and retrieve it back to the ER.
Apart from searching for an interaction partner, I also determined the effect of depletion of VIP36 on the morphology of the secretory pathway. The rationale behind this is the observation that cargo receptors contribute to the structural integrity of organelles of the secretory pathway. Knockdown of VIP36 had no effect on ER exit sites or on the ERGIC. However, VIP36 knockdown resulted in fragmentation of the Golgi apparatus. The fragmented Golgi was not the consequence of disturbed bidirectional protein transport and not due to effects on microtubules. Knockdown of VIP36 reduced COPI staining on the Golgi. VIP36 is likely to provide COPI binding sites on the Golgi via its cytosolic tail and thereby contribute to Golgi structural integrity. Our results underscore the importance of cargo receptors, not only for intracellular transport within the secretory pathway, but also to maintain the integrity of the secretory pathway itself.
In conclusion, my thesis provides a deeper insight into the function of VIP36 in the early secretory pathway
Constitutive ER stress and Wolfram syndrome
No full textBackground: Wolfram syndrome (WS) is a disorder characterized by the association of insulin-dependent diabetes mellitus (DM), diabetes insipidus, deafness, and optic nerve atrophy. WS is caused by WFS1 mutations encoding WFS1 protein expressed in endoplasmic reticulum (ER). During ER protein synthesis, misfolded and unfolded proteins accumulate, known as "ER stress". This is attenuated by the unfolded protein response (UPR), which recovers and maintains ER functions. Because WFS1 is a UPR component, mutant WFS1 might cause unresolvable ER stress conditions and cell apoptosis, the major causes underlying WS symptoms. We encountered an 11-month-old Japanese female WS patient with insulin-dependent DM, congenital cataract and severe bilateral hearing loss. Objective: Analyze the WFS1 and functional consequence of the patient WFS1 in vitro. Results: The patient WFS1 contained a heterozygous 4 amino acid in-frame deletion (p.N325_I328del). Her mutant WFS1 increased GRP78 and ATF6α promoter activities in the absence of thapsigargin, indicating constitutive ER stress and nuclear factor of activated T-cell reporter activity, reflecting elevated cytosolic Ca2+ signals. Mutant transfection into cells reduced mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase 2b (SERCA2b) compared with wild type. Because SERCA2b is required for ER and cytoplasmic Ca2+ homeostasis, decreased SERCA2b expression might affect ER Ca2+ efflux, causing cell apoptosis. Conclusion: A novel heterozygous mutation of WFS1 induced constitutive ER stress through ATF6α activation and ER Ca2+ efflux, resulting in cell apoptosis. These results provide new insights into the roles of WFS1 in UPR and mechanism of monogenic DM
A non-catalytic disulphide bond regulating redox flux in the ER oxidative folding pathway
No full textTFG Promotes Organization of Transitional ER and Efficient Collagen Secretion
No full textCollagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.</p
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