A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion

Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P(2), driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on late-stage vesicles in the vicinity of the plasma membrane

A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on macropinosomes and phagosomes

Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P

Formal Safety Assessment of a Marine Seismic Survey Vessel Operation, Incorporating Risk Matrix and Fault Tree Analysis

In maritime safety research, risk is assessed usually within the framework of formal safety assessment (FSA), which provides a formal and systematic methodology to improve the safety of lives, assets, and the environment. A bespoke application of FSA to mitigate accidents in marine seismic surveying is put forward in this paper, with the aim of improving the safety of seismic vessel operations, within the context of developing an economically viable strategy. The work herein takes a close look at the hazards in North Sea offshore seismic surveying, in order to identify critical risk factors, leading to marine seismic survey accidents. The risk factors leading to undesirable events are analysed both qualitatively and quantitatively. A risk matrix is introduced to screen the identified undesirable events. Further to the screening, Fault Tree Analysis (FTA) is presented to investigate and analyse the most critical risks of seismic survey operation, taking into account the lack of historical data. The obtained results show that man overboard (MOB) event is a major risk factor in marine seismic survey operation; lack of training on safe work practice, slippery deck as a result of rain, snow or water splash, sea state affecting human judgement, and poor communication are identified as the critical risk contributors to the MOB event. Consequently, the risk control options are focused on the critical risk contributors for decision-making. Lastly, suggestions for the introduction and development of the FSA methodology are highlighted for safer marine and offshore operations in general

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner

A PI(3,5)P2 probe reveals PIKfyve is required for Rab7 acquisition and the delivery and fusion of early macropinosomes to phagosomes

Phosphoinositide signalling lipids (PIPs) are key regulators of membrane identity and vesicle trafficking. Of these, PI(3,5)P2 is one of the least understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is predominantly generated by the phosphoinositide 5-kinase PIKfyve, and is critical for phagosomal digestion and the killing of engulfed microbes. However, the regulation of PIKfyve activity, PI(3,5)P2 dynamics, and how they control phagosome maturation remains unclear. Using the model professional phagocyte Dictyostelium discoideum we have identified a highly selective PI(3,5)P2-binding protein that allows faithful observation of PI(3,5)P2 dynamics in live cells. Using this probe we demonstrate that PIKfyve recruitment and activity are separable, and that PIKfyve activation stimulates its own dissociation from membranes. We show that PI(3,5)P2 accumulates on phagosomes 2-3 minutes after engulfment drives fusion of a specific population of PI(3,5)P2 and Rab7-positive macropinosomes. We find PIKfyve is required for the fusion between macropinosomes and phagosomes, which enables phagosomes to efficiently accumulate Rab7 and other components of the lysosomal machinery. These findings uncover key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2 likely to have general relevance across endocytic pathways.</p

Quantitative grounding risk assessment and management

Grounding is a phenomenon in which the bottom part of a structural system, such as a ship, offshore platform, automobile, or aircraft, is accidentally damaged. Three types of grounding accidents are relevant, namely grounding, stranding, and squatting (as described in Chap. 1). As far as ship grounding is concerned, the first type usually occurs due to navigational errors associated with failures in the process of passage planning and piloting and nautical charts with out-of-date data. Stranding in the shipping industry happens when a ship is swept away by waves and tides as its engine power fails, where bottom structures are damaged on a rock near shore by vertical loading due to the difference between buoyancy and weight in ebb tide. Squatting may happen in ships operating in shallow waterways. In the aviation industry, grounding can occur upon landing when the landing gear system is malfunctional. This chapter describes the quantitative risk assessment and management of grounding accidents, with a focus on the first type of ship grounding. The methods are described in association with the shipping industry, but can be applied to other types of structural systems in grounding