Mapping the Interactions between a RUN Domain from DENND5/Rab6IP1 and Sorting Nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses

Thousands of Rab GTPases for the Cell Biologist

Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology

The management of acute venous thromboembolism in clinical practice. Results from the European PREFER in VTE Registry

Venous thromboembolism (VTE) is a significant cause of morbidity and mortality in Europe. Data from real-world registries are necessary, as clinical trials do not represent the full spectrum of VTE patients seen in clinical practice. We aimed to document the epidemiology, management and outcomes of VTE using data from a large, observational database. PREFER in VTE was an international, non-interventional disease registry conducted between January 2013 and July 2015 in primary and secondary care across seven European countries. Consecutive patients with acute VTE were documented and followed up over 12 months. PREFER in VTE included 3,455 patients with a mean age of 60.8 ± 17.0 years. Overall, 53.0 % were male. The majority of patients were assessed in the hospital setting as inpatients or outpatients (78.5 %). The diagnosis was deep-vein thrombosis (DVT) in 59.5 % and pulmonary embolism (PE) in 40.5 %. The most common comorbidities were the various types of cardiovascular disease (excluding hypertension; 45.5 %), hypertension (42.3 %) and dyslipidaemia (21.1 %). Following the index VTE, a large proportion of patients received initial therapy with heparin (73.2 %), almost half received a vitamin K antagonist (48.7 %) and nearly a quarter received a DOAC (24.5 %). Almost a quarter of all presentations were for recurrent VTE, with >80 % of previous episodes having occurred more than 12 months prior to baseline. In conclusion, PREFER in VTE has provided contemporary insights into VTE patients and their real-world management, including their baseline characteristics, risk factors, disease history, symptoms and signs, initial therapy and outcomes

Involvement of the Rho-mDia1 pathway in the regulation of Golgi complex architecture and dynamics

10.1091/mbc.E11-01-0007Molecular Biology of the Cell22162900-2911MBCE

C11ORF24 Is a Novel Type I Membrane Protein That Cycles between the Golgi Apparatus and the Plasma Membrane in Rab6-Positive Vesicles.

The Golgi apparatus is an intracellular compartment necessary for post-translational modification, sorting and transport of proteins. It plays a key role in mitotic entry through the Golgi mitotic checkpoint. In order to identify new proteins involved in the Golgi mitotic checkpoint, we combine the results of a knockdown screen for mitotic phenotypes and a localization screen. Using this approach, we identify a new Golgi protein C11ORF24 (NP_071733.1). We show that C11ORF24 has a signal peptide at the N-terminus and a transmembrane domain in the C-terminal region. C11ORF24 is localized on the Golgi apparatus and on the trans-Golgi network. A large part of the protein is present in the lumen of the Golgi apparatus whereas only a short tail extends into the cytosol. This cytosolic tail is well conserved in evolution. By FRAP experiments we show that the dynamics of C11ORF24 in the Golgi membrane are coherent with the presence of a transmembrane domain in the protein. C11ORF24 is not only present on the Golgi apparatus but also cycles to the plasma membrane via endosomes in a pH sensitive manner. Moreover, via video-microscopy studies we show that C11ORF24 is found on transport intermediates and is colocalized with the small GTPase RAB6, a GTPase involved in anterograde transport from the Golgi to the plasma membrane. Knocking down C11ORF24 does not lead to a mitotic phenotype or an intracellular transport defect in our hands. All together, these data suggest that C11ORF24 is present on the Golgi apparatus, transported to the plasma membrane and cycles back through the endosomes by way of RAB6 positive carriers

Recycling Endosome Tubule Morphogenesis from Sorting Endosomes Requires the Kinesin Motor KIF13A

Early endosomes consist of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. The tubular transport intermediates that constitute recycling endosomes function in cell polarity, migration, and cytokinesis. Endosomal tubulation and fission require both actin and intact microtubules, but although factors that stabilize recycling endosomal tubules have been identified, those required for tubule generation from vacuolar sorting endosomes (SEs) remain unknown. We show that the microtubule motor KIF13A associates with recycling endosome tubules and controls their morphogenesis. Interfering with KIF13A function impairs the formation of endosomal tubules from SEs with consequent defects in endosome homeostasis and cargo recycling. Moreover, KIF13A interacts and cooperates with RAB11 to generate endosomal tubules. Our data illustrate how a microtubule motor couples early endosome morphogenesis to its motility and function