Investigation of the TOCA1-Cdc42 interaction

Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.JRW is supported by a Herchel Smith studentship. JLG is supported by a Wellcome Trust Research Career Development Fellowship (WT095829AIA), European Research Council Starting Grant (281971) and Gurdon Institute funding provided by the Wellcome Trust (092096) and CRUK (C6946/A14492). HMF is supported by a Wellcome Trust PhD Studentship (WT099740Z12Z). We would like to thank Dr A Walrant for help with the pyrene actin assays and liposome preparation. We are also grateful to Dr J.R. Peterson (Fox Chase Cancer Center) for human TOCA1 clones.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via http://dx.doi.org/10.1074/jbc.M116.72429

An improved database of coastal flooding in the United Kingdom from 1915 to 2016

Coastal flooding caused by extreme sea levels can produce devastating and wide-ranging consequences. The ‘SurgeWatch’ v1.0 database systematically documents and assesses the consequences of historical coastal flood events around the UK. The original database was inevitably biased due to the inconsistent spatial and temporal coverage of sea-level observations utilised. Therefore, we present an improved version integrating a variety of ‘soft’ data such as journal papers, newspapers, weather reports, and social media. SurgeWatch2.0 identifies 329 coastal flooding events from 1915 to 2016, a more than fivefold increase compared to the 59 events in v1.0. Moreover, each flood event is now ranked using a multi-level categorisation based on inundation, transport disruption, costs, and fatalities: from 1 (Nuisance) to 6 (Disaster). For the 53 most severe events ranked Category 3 and above, an accompanying event description based upon the Source-Pathway-Receptor-Consequence framework was produced. Thus, SurgeWatch v2.0 provides the most comprehensive and coherent historical record of UK coastal flooding. It is designed to be a resource for research, planning, management and education

A user-friendly database of coastal flooding in the United Kingdom from 1915–2014

Coastal flooding caused by extreme sea levels can be devastating, with long-lasting and diverse consequences. Historically, the UK has suffered major flooding events, and at present 2.5 million properties and £150 billion of assets are potentially exposed to coastal flooding. However, no formal system is in place to catalogue which storms and high sea level events progress to coastal flooding. Furthermore, information on the extent of flooding and associated damages is not systematically documented nationwide. Here we present a database and online tool called ‘SurgeWatch’, which provides a systematic UK-wide record of high sea level and coastal flood events over the last 100 years (1915-2014). Using records from the National Tide Gauge Network, with a dataset of exceedance probabilities and meteorological fields, SurgeWatch captures information of 96 storms during this period, the highest sea levels they produced, and the occurrence and severity of coastal flooding. The data are presented to be easily assessable and understandable to a range of users including, scientists, coastal engineers, managers and planners and concerned citizens

Endophilin drives the fast mode of vesicle retrieval in a ribbon synapse

Compensatory endocytosis of exocytosed membrane and recycling of synaptic vesicle components is essential for sustained synaptic transmission at nerve terminals. At the ribbon-type synapse of retinal bipolar cells, manipulations expected to inhibit the interactions of the clathrin adaptor protein complex (AP2) affect only the slow phase of endocytosis (τ = 10-15 s), leading to the conclusion that fast endocytosis (τ = 1-2 s) occurs by a mechanism that differs from the classical pathway of clathrin-coated vesicle retrieval from the plasma membrane. Here we investigate the role of endophilin in endocytosis at this ribbon synapse. Endophilin A1 is a synaptically enriched N-BAR domain-containing protein, suggested to function in clathrin-mediated endocytosis. Internal dialysis of the synaptic terminal with dominant-negative endophilin A1 lacking its linker and Src homology 3 (SH3) domain inhibited the fast mode of endocytosis, while slow endocytosis continued. Dialysis of a peptide that binds endophilin SH3 domain also decreased fast retrieval. Electron microscopy indicated that fast endocytosis occurred by retrieval of small vesicles in most instances. These results indicate that endophilin is involved in fast retrieval of synaptic vesicles occurring by a mechanism that can be distinguished from the classical pathway involving clathrin-AP2 interactions

EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 μM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in γ-synergin and use to predict that an uncharacterized EF-hand–containing protein will be a new gamma binding partner

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation.

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.Herchel Smith Fellowship, Funai Foundation scholarship, Austrian Science Fun

A direct role for SNX9 in the biogenesis of filopodia.

Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways

A longitudinal analysis of urological chronic pelvic pain syndrome flares in the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151272/1/bju14783.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151272/2/bju14783_am.pd