615 research outputs found
Correction to Supporting Information for Sammon et al., Two-step release of kinase autoinhibition in discoidin domain receptor 1
No full textBIOCHEMISTRY Correction to Supporting Information for “Two-step release of kinase autoinhibition in discoidin domain receptor 1,” by Douglas Sammon, Erhard Hohenester, and Birgit Leitinger, which was first published August 24, 2020; 10.1073/pnas.2007271117 (Proc. Natl. Acad. Sci. U.S.A. 117, 22051–22060).
The authors note that Table S2 in the SI Appendix appeared incorrectly. The SI Appendix has been corrected online
Activation of Discoidin Domain Receptors by collagen VI and collagen I fibrils
No full textDiscoidin domain receptors (DDRs) are members of the Receptor Tyrosine Kinase (RTK) family. There are two DDRs, DDR1 and DDR2. In embryo development, DDR1 function is vital for mammary gland development, whereas DDR2 is important in long bone growth. The DDRs also have key roles in several cellular functions, such as cell migration, adhesion, proliferation and matrix remodeling. The DDRs are unique RTK members due to the fact that they are activated by an extracellular matrix component, collagen. It is well known that the DDRs are activated by fibrillar collagens, such as collagen I, when present as single triple helices. However, it is less clear whether collagen I fibrils or fibers, the form in which collagen I is present in tissues, can act as functional DDR ligands and induce DDR phosphorylation. In this thesis, I show that DDR1 activation is induced by collagen I fibrils generated in vitro from different sources, and furthermore by extracellular matrix secreted from fibroblastic cells. I also show that DDR2 is activated to a much lower extent in comparison to DDR1. Another collagen type that is present in many tissues is collagen VI. Collagen VI has not been characterized as a DDR ligand. Collagen VI is secreted in the form of tetramers to extracellular matrices, where it associates to form microfibrils. In this thesis, I show that collagen VI tetramers and microfibrils bind strongly to both DDRs. However, they can induce only DDR1 phosphorylation, not DDR2 phosphorylation. Our preliminary data further suggest that the triple-helical region in collagen VI is the functional region that promotes DDR1 activation. Furthermore, I show that collagen VI binds to DDR2 by occupying another binding site than the fibrillar binding site. Finally, preliminary experiments suggest that collagen VI microfibrils can inhibit collagen I-induced DDR2 autophosphorylation. These data suggest that collagen VI binding to DDR2 could serve as a limiting factor for DDR2 activation in pathological conditions, such as osteoarthritis.Open Acces
Understanding the requirements of DDR1 clustering in DDR1 activation
No full textDiscoidin domain receptors (DDRs), DDR1 and DDR2, are unique receptor tyrosine kinases that perform essential roles in both physiological and pathological conditions. They are constitutive dimers on the cell surface and respond to collagen binding, leading to the activation of the kinase and several downstream signalling pathways. Previous biochemical and cellular studies have shown that collagen-induced DDR1 clustering is essential for DDR1 activation, and that the transmembrane domain (TMD) plays a crucial role in mediating DDR1 dimerization and autophosphorylation. However, the detailed molecular mechanisms remain unclear. Here, a variety of studies were conducted to bring further insights into the DDR1 dimerization and activation mechanisms. The activation of DDR1 was enforced by a ligand-independent optical approach using Cryptochrome Circadian Regulator 2 (Cry2), and ligand-induced DDR1 activation was modulated by co-expression with transmembrane peptides in a second approach. Furthermore, the TMD interaction interfaces were analyzed by disulphide cross-linking studies via cysteine scanning mutagenesis, and the dimerization motifs within the DDR1 TMD were validated using a series of mutagenesis studies and a Venus fluorescent protein-based bimolecular fluorescence complementation (BiFC) assay. This project demonstrates that collagen-induced DDR1 activation can be boosted by co-expression with one of the transmembrane peptides (DDR1-TMD) generated in this project, demonstrating the ability of the TMD to regulate DDR1 activation. Although no ligand-specific TMD dimerization motifs were revealed by the presented experiments, a strong cross-linking within the N-terminal part of DDR1 TMD was shown to constitutively exist and contribute to the DDR1 intrinsic interactions. Overall, this project provides further understanding and approaches for future investigations in studying DDR1 dimerization and collagen-induced receptor activation.Open Acces
Identifying a Targeting Factor for Rab27a
No full textThe Rab family of small GTPases regulate intracellular membrane trafficking pathways and each Rab GTPase localises to a discrete membrane compartment. The mechanism(s) by which this targeting is achieved remains to be elucidated. Previous work identified that the C‐terminal hypervariable domain and effector binding were not necessary for Rab27a targeting in melanocytes. However, GEF activity of the non‐redundant Rab27a GEF Rab3GEP (R3G) was required but not sufficient for Rab27a targeting. This led to the hypothesis of the involvement of a Rab27a targeting factor.
A Rab27a mutant (Rab27aSF1/F4) had previously been characterised that maintained melanosomal targeting and GTP‐loading by R3G but bound to no known Rab27a effectors. Using this mutant, novel Rab27a interacting partners were identified and assessed for their possible role in Rab27a targeting. The current research identified ATP1a1 (the α1 subunit of Na+,K+‐ATPase) as a novel interacting partner for Rab27a in melanocytes, depletion of which resulted in perinuclear clustering of melanosomes, indicative of loss of Rab27a function. Depletion of ATP1a1 did not disrupt Rab27a membrane localisation but did reduce the levels of GTP‐bound Rab27a, comparable to what is seen following depletion of R3G. Crucially, depletion of ATP1a1 resulted in displacement of endogenous Rab27a from melanosomal membranes indicating a role in Rab27a targeting. Furthermore, ATP1a1 depletion disrupted the levels of other melanosomal proteins indicative of a disruption in melanogenesis. ATP1a1 is proposed to transiently associate with melanosomes and through its Na+,K+‐ATPase pump function regulate the lumenal pH, which indirectly regulates melanosome maturation. How the maturation of the lumenal environment is conveyed to Rab27a, and is subsequent recruitment to melanosomes remains to be clarified
Structure of the Discoidin Domain Receptor 1 Extracellular Region Bound to an Inhibitory Fab Fragment Reveals Features Important for Signaling
No full text06.09.12 KB. Accepted version, ok to add to spiral. Elsevier says it ok while mandate is not enforced
Mirror Landing - As Remembered by Birgit Hult
No full textNotes - This account, Memories Mirror Landing by Birgit Hult, was compiled by Birgit's daughter, Jean Elvira Male, it documents the Hult family's experiences in Mirror Landing from 1912 - 1916. The Hults, who were originally from Sweden, arrived in Mirror Landing with two young children, a third child was born during their stay in Mirror Landing. Upon arriving in the area, the Hults made friends with the Gauthier family. The wives became good friends and would swap piano lessons for English lessons. Details of the log home where the family lived and the surrounding landscape were recalled. A memory about a large forest fire that occurred near the family home and dances that were attended in the town are discussed. Jean recalls her mother's memories regarding the animosity towards the North West Mounted Police that was felt by the people of Mirror Landing. The Hult family retained a strong connection to the Swedish heritage and practised many Swedish traditions, such as flying the Swedish flag and eating hot cross buns stuffed with Swedish Marzipan soaked in warm milk. Photos and a postcard written in 1915 are included in this article (10 pages
Knowing Through Popular Music in the Western Pacific Island World
No full textPacific Indigenous scholars have long emphasized the role of relationality for Pacific Islanders’ epistemologies. In this article, the author rethinks music in terms of the procedural knowledge inherent in and specific to popular music-making by exploring the latter as knowledge practices in Micronesia. This approach opens new vistas on the relationality at the heart of Western Pacific music-making. The author calls the musical manifestation of that relational capacity sound ties, suggesting that if, following Epeli Hau‘ofa, Oceania is “humanity rising from the depths of brine”, then it is not least the sound ties of knowing in and through music that mould that very humanity of people who are at home with the sea into aquapelagic assemblages that are, after all, so much more than water and land
Analysis of DDR1 function at epithelial cell contacts
No full textDiscoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase (RTK) family, and binds to collagen in the extracellular matrix (ECM). It therefore plays an important role in relaying information from outside the cell to intracellular components. Accordingly, DDR1 contributes to many cellular processes including migration and differentiation amongst others. In malignant states, cell-matrix interactions are often deregulated, resulting in the pro-invasive phenotype characteristic of tumours. Increased DDR1 expression is a negative prognostic marker for many cancers, however the molecular mechanisms are not fully understood. Interestingly, novel ligand-independent roles of DDR1 have recently emerged that potentially implicate the receptor at epithelial cell contacts. In this thesis, I show that during new keratinocyte contact formation, DDR1 is recruited after E-cadherin. In contrast to previous literature, DDR1 does not form a complex with E-cadherin, and distinct separate clusters of DDR1 and E-cadherin are observed at mature cell contacts. DDR1 depletion decreases the junctional E-cadherin and actin levels during cell contact formation. This phenotype is independent of actin recruitment to clustered E-cadherin receptors. Actin thin bundles are also visibly disrupted during contact formation with DDR1 depletion, which is further linked to a reduction in Rho-ROCK signalling and actomyosin contractility. Not only are the levels of phosphorylated myosin light chain and myosin phosphatase reduced, but ROCK1 levels are also reduced by DDR1 knockdown, suggesting that DDR1 has a regulatory role upstream of ROCK1. Preliminary experiments demonstrate potential binding between DDR1 and some members of the catenin protein family, however the significance of these interactions requires further investigation. Data collected from keratinocytes and a series of lung cancer cell lines, suggest that E-cadherin-mediated cell contacts inhibit collagen-mediated DDR1 activation, possibly by preventing DDR1 ligand accessibility. Overall, my results suggest that DDR1 stabilizes epithelial cell contacts through regulation of actomyosin contractility.Open Acces
Novel insights into discoidin domain receptor 1 activation and regulation
No full textThe discoidin domain receptors, DDR1 and DDR2, are intriguing receptor tyrosine kinases (RTKs) that signal in response to collagen with unusually delayed autophosphorylation kinetics. Both DDRs play essential roles in development and regulate cell adhesion, migration, invasion, proliferation, and survival. The DDRs are attractive drug targets for atherosclerosis, arthritis, organ fibrosis, and various forms of cancer, but how collagen binding translates to DDR activation is not well understood. Previous studies suggested that DDR1 activation occurs in intracellular compartments following collagen-induced endocytosis. To define this mechanism further, I used biochemical assays and immunofluorescence. Contrary to the proposed activation mechanism, collagen did not induce DDR1 internalisation, and ligand-induced DDR1 phosphorylation was limited to the plasma membrane, supporting a mechanism whereby DDR1 is activated on the cell surface. Activation of the majority of RTKs occurs by ligand-induced dimerisation. However, the DDRs are constitutive, non-covalent dimers, which led to the hypothesis that activation of DDRs arises by collagen-induced lateral association (clustering) of DDR dimers on the cell membrane. To investigate this mechanism, different types of signalling-incompetent DDR1 mutants (‘receiver’) were co-expressed with functional DDR1 (‘donor’), and phosphorylation of receiver DDR1 by donor DDR1 was monitored by SDS-PAGE and Western blotting, or by immunofluorescence. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor activation, I demonstrate that the donor phosphorylates receiver DDR1 dimers in trans in a process that requires kinase activity of the donor but not the receiver. Intriguingly, DDR1 phosphorylation in trans is not dependent on ectodomain contacts, specific intracellular regions, the fibrillar nature of collagen, or a particular mode of activation, but requires intact transmembrane domain interactions. Moreover, collagen induces redistribution of DDR1 into a more compact structure, and mutant DDR1 that cannot bind collagen is recruited to collagen when co-expressed with a functional receptor. In summary, this thesis uncovered a mechanism of DDR1 activation whereby collagen induces clustering of DDR1 dimers on the cell surface, which leads to phosphorylation between dimers.Open Acces
EU-Behörde: Warum wir sie brauchen
No full textJan Cremers (UvT) contributed to the magazine Gute Arbeit (in German), published by BUND-Verlag. Together with co-author Birgit Krämer, he discusses the plans of the European Commission to introduce a European Labour Authority. They reflect on the possible contribution of such a body in checking respect for and compliance with labour legislation and conventional standards
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