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Bella J.,Wellcome Trust Center for Cell Matrix Research
Journal of Structural Biology | Year: 2010

Collagen fibre diffraction patterns are typically interpreted assuming a monotonous, average triple helical conformation for the collagen molecule. Two different helical symmetries have been proposed: seven residues in two turns versus 10 residues in three turns. Collagen model peptides show predominantly the 7-fold symmetry but provide evidence for local changes in the helical twist, which are related to some extent to the local sequence of the peptides but also to the lattice interactions in the crystal. Thus, it is difficult to determine precisely to what degree the amino acid sequence dictates the fine details of collagen conformation. A new method is presented here in which an internal triple helical twist is defined. This method takes into consideration all three chains simultaneously, and facilitates investigating the sequence dependence of helical twist variation, the conformational consequences of collagen interruptions, and the effects on collagen conformation introduced upon receptor or ligand-binding. Analysis of the crystal structures of model peptides suggests that collagen varies gradually and continuously its helical twist according to the local distribution of imino acid residues, with the 7-fold and 10-fold symmetries representing the limits of this variation for the cases of imino acid saturation or absence, respectively. © 2010 Elsevier Inc.

Peffers M.J.,University of Liverpool | Thornton D.J.,Wellcome Trust Center for Cell Matrix Research | Clegg P.D.,University of Liverpool
Journal of Orthopaedic Research | Year: 2016

Osteoarthritis is characterized by a loss of extracellular matrix that leads to cartilage degradation and joint space narrowing. Specific proteases, including the aggrecanases ADAMTS-4 and matrix metalloproteinase 3, are important in initiating and promoting cartilage degradation in osteoarthritis. This study investigated protease-specific and disease-specific cleavage patterns of particular extracellular matrix proteins by comparing new peptide fragments, neopeptides, in specific exogenous protease-driven digestion of a crude cartilage proteoglycan extract and an in-vitro model of early osteoarthritis. Additionally, equine cartilage explants were treated with interleukin-1 and the media collected. Proteolytic cleavage products following trypsin digestion were then identified using tandem mass spectrometry. Complete sequences of proteolytically cleaved neopeptides were determined for the major cartilage proteoglycans aggrecan, biglycan, decorin, fibromodulin plus cartilage oligomeric matrix protein. The generation of neopeptides varied with enzyme specificity; however, some peptides were common to all samples. Previous known and novel cleavage sites were identifies. The identification of novel peptide fragments provides a platform for the development of antibodies that could assist in the identification of biomarkers for osteoarthritis (OA), as well as the identification of basic biochemical processes underlying OA. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Koper A.,Wellcome Trust Center for Cell Matrix Research | Schenck A.,Radboud University Nijmegen | Prokop A.,Wellcome Trust Center for Cell Matrix Research
PLoS ONE | Year: 2012

Synapse formation and maintenance crucially underlie brain function in health and disease. Both processes are believed to depend on cell adhesion molecules (CAMs). Many different classes of CAMs localise to synapses, including cadherins, protocadherins, neuroligins, neurexins, integrins, and immunoglobulin adhesion proteins, and further contributions come from the extracellular matrix and its receptors. Most of these factors have been scrutinised by loss-of-function analyses in animal models. However, which adhesion factors establish the essential physical links across synaptic clefts and allow the assembly of synaptic machineries at the contact site in vivo is still unclear. To investigate these key questions, we have used the neuromuscular junction (NMJ) of Drosophila embryos as a genetically amenable model synapse. Our ultrastructural analyses of NMJs lacking different classes of CAMs revealed that loss of all neurexins, all classical cadherins or all glutamate receptors, as well as combinations between these or with a Laminin deficiency, failed to reveal structural phenotypes. These results are compatible with a view that these CAMs might have no structural role at this model synapse. However, we consider it far more likely that they operate in a redundant or well buffered context. We propose a model based on a multi-adaptor principle to explain this phenomenon. Furthermore, we report a new CAM-independent adhesion mechanism that involves the basement membranes (BM) covering neuromuscular terminals. Thus, motorneuronal terminals show strong partial detachment of the junction when BM-to-cell surface attachment is impaired by removing Laminin A, or when BMs lose their structural integrity upon loss of type IV collagens. We conclude that BMs are essential to tie embryonic motorneuronal terminals to the muscle surface, lending CAM-independent structural support to their adhesion. Therefore, future developmental studies of these synaptic junctions in Drosophila need to consider the important contribution made by BM-dependent mechanisms, in addition to CAM-dependent adhesion. © 2012 Koper et al.

Goncalves-Pimentel C.,Wellcome Trust Center for Cell Matrix Research | Goncalves-Pimentel C.,University of Coimbra | Gombos R.,Hungarian Academy of Sciences | Mihaly J.,Hungarian Academy of Sciences | And 2 more authors.
PLoS ONE | Year: 2011

F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. Here, we have focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, we used embryonic Drosophila primary neurons as one consistent cellular model to study filopodia regulation. Our data for loss-of-function of capping proteins, enabled, different Arp2/3 complex components, the formin DAAM and profilin reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of our Drosophila model system. Using combinatorial genetics, we demonstrate that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. Our genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps. © 2011 Gonçalves-Pimentel et al.

Prokop A.,Wellcome Trust Center for Cell Matrix Research | Sanchez-Soriano N.,Wellcome Trust Center for Cell Matrix Research | Goncalves-Pimentel C.,Federal University of Pernambuco | Molnar I.,Hungarian Academy of Sciences | And 2 more authors.
Communicative and Integrative Biology | Year: 2011

Formins are an important and evolutionarily well conserved class of actin binding proteins with essential biological functions. Although their molecular roles in actin regulation have been clearly demonstrated in vitro, their functions at the cellular or organism levels are still poorly understood. To illustrate this problem, but also to demonstrate potential ways forward, we focus here on the DAAM group of formins. In vertebrates, DAAM group members have been demonstrated to be important regulators of cellular and tissue morphogenesis but, as for all formins, the molecular mechanisms underlying these morphogenetic functions remain to be uncovered. The genome of the fruitfly Drosophila encodes a single DAAM gene that is evolutionarily highly conserved. Recent work on dDAAM has already provided a unique combination of observations and experimental opportunities unrivalled by any other Drosophila formin. These comprise in vitro actin polymerisation assays, subcellular studies in culture and in vivo, and a range of developmental phenotypes revealing a role in tracheal morphogenesis, axonal growth and muscle organization. At all these levels, future work on dDAAM will capitalize on the power of fly genetics, raising unique opportunities to advance our understanding of dDAAM at the systems level, with obvious implications for other formins. © 2011 Landes Bioscience.

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