A study of SNARE-mediated autophagosome clearance using fluorescence lifetime microscopy

Cell survival requires the turnover of toxic cellular material and recycling of biomolecules in low nutrient conditions. An efficient degradation system is therefore essential for disease prevention and its dysfunction has been linked to both neurodegeneration and oncogenesis. Bulk degradation is accomplished through the collection of cytoplasmic material in a unique sequestration vesicle, which forms de novo and subsequently deposits cargo in the lysosome for degradation. This process, known as autophagy, therefore requires membrane fusion between the autophagosomal vesicle and the lysosome. SNARE proteins mediate membrane fusion events and therefore their careful regulation ensures the proper organisation of the membrane trafficking network. The SNARE proteins governing autophagosome clearance have been identified as syntaxin 17, SNAP29 and VAMP8 and SNARE assembly appears to be positively regulated by VPS33A. This well established model of SNARE-mediated autophagosome clearance has not, however, been demonstrated within the spatiotemporal framework of the cell and little is known about how VPS33A modulates SNARE function. The research presented in this thesis therefore aims to determine the applicability of the proposed SNARE model within the cellular environment and to investigate the regulatory mechanisms controlling syntaxin 17 function. To accomplish this, carefully validated fluorescence colocalisation and time-resolved fluorescence lifetime imaging techniques were primarily employed. The limitations of these techniques were also considered for data interpretation and a novel prototype SPAD array technology, designed for high-speed time-correlated single photon counting, was trialled for widefield FLIM-FRET. FLIM-FRET revealed that VAMP8 has been incorrectly assigned as the dominant autophagosomal R-SNARE and VPS33A studies evidence a multi-modal regulation of Stx17 that diverges from other studied syntaxin family modulation mechanisms. A new model of SNAREmediated autophagosome clearance is therefore proposed, where syntaxin 17 engages with SNAP29 and VAMP7 to drive membrane fusion with the endolysosome in a manner governed by VPS33A and dependent on the phosphorylation status of syntaxin 17

Biomolecule Trafficking and Network Tomography-based Simulations

International audienceDuring the past two decades many groundbreaking technologies, including Green Fluorescent Protein (GFP)-tagging and super-resolution microscopy, emerged and allowed the visualization of protein dynamics and molecular interactions at different levels of spatial and temporal resolution. In the meantime, the automated quantification of microscopy images depicting moving biomolecules has become of major importance in cell biology since it offers a better understanding of fundamental mechanisms including membrane transport, cell signaling, cell division and motility. Consequently, dedicated image analysis methods have been developed to process challenging temporal series of 2D-3D images and to estimate individual trajectories of biomolecules. Nevertheless, the current tracking methods cannot provide global information about biomolecule trafficking. This motivated the development of simulation techniques able to generate realistic fluorescence microscopy image sequences depicting trafficking of small moving particles in interaction, with variable velocities within the cell. In this chapter, we describe a simulation approach based on the concept of Network Tomography (NT) which is generally used in network communications and transport to infer the main routes of communication between origins and destinations. The trafficking model, scaled down for microscopy, is combined with real 2D-3D image sequences to generate artificial videos depicting fluorescently tagged moving proteins within cells. Simulation in bioimaging is timely since it has become essential to build ground truth datasets for image processing algorithm evaluation such as biomolecule detectors and trackers, as well as to generate training datasets for deep learning algorithms

In vivo Analysis and Modeling Reveals that Transient Interactions of Myosin XI, its Cargo, and Filamentous Actin Overcome Diffusion Limitations to Sustain Polarized Cell Growth

Tip growth is a ubiquitous process throughout the plant kingdom in which a single cell elongates in one direction in a self-similar manner. To sustain tip growth in plants, the cell must regulate the extensibility of the wall to promote growth and avoid turgor-induced rupture. This process is heavily dependent on the cytoskeleton, which is thought to coordinate the delivery and recycling of vesicles containing cell wall materials at the cell tip. Although significant work has been done to elucidate the various molecular players in this process, there remains a need for a more mechanistic understanding of the cytoskeletonÂ’s role in tip growth. For this reason, specific emphasis should be placed on understanding the dynamics of the cytoskeleton, its associated motors, and their cargo. Since the advent of fluorescence fusion technology, various quantitative fluorescence dynamics techniques have emerged. Among the most prominent of these techniques is fluorescence recovery after photobleaching (FRAP). Despite its prominence, it is unclear how to interpret fluorescence recoveries in confined cellular geometries such as tip-growing cells. Here we developed a digital confocal microscope simulation of FRAP in tip-growing cells. With this simulation, we determined that fluorescence recoveries are significantly influenced by cell boundaries. With this FRAP simulation, we then measured the diffusion of VAMP72-labeled vesicles in the moss Physcomitrella patens. Using finite element modeling of polarized cell growth, and the measured VAMP72-labeled vesicle diffusion coefficient, we were able to show that diffusion alone cannot support the required transport of wall materials to the cell tip. This indicates that an actin-based active transport system is necessary for vesicle clustering at the cell tip to support growth. This provides one essential function of the actin cytoskeleton in polarized cell growth. After establishing the requirement for actin-based transport, we then sought to characterize the in vivo binding interactions of myosin XI, vesicles, and filamentous actin. Particle tracking evidence from P. patens protoplasts suggests that myosin XI and VAMP72-labeled vesicles exhibit fast transient interactions. Hidden Markov modeling of particle tracking indicates that myosin XI and VAMP72- labeled vesicles move along actin filaments in short-lived linear trajectories. These fast transient interactions may be necessary to achieve the rapid dynamics of the apical actin, important for growth. This work advances the fieldÂ’s understanding of fluorescence dynamics, elucidates a necessary function of the actin cytoskeleton, and provides insight into how the components of the cytoskeleton interact in vivo

Proceedings of Abstracts, School of Physics, Engineering and Computer Science Research Conference 2022

© 2022 The Author(s). This is an open-access work distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For further details please see https://creativecommons.org/licenses/by/4.0/. Plenary by Prof. Timothy Foat, ‘Indoor dispersion at Dstl and its recent application to COVID-19 transmission’ is © Crown copyright (2022), Dstl. This material is licensed under the terms of the Open Government Licence except where otherwise stated. To view this licence, visit http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3 or write to the Information Policy Team, The National Archives, Kew, London TW9 4DU, or email: [email protected] present proceedings record the abstracts submitted and accepted for presentation at SPECS 2022, the second edition of the School of Physics, Engineering and Computer Science Research Conference that took place online, the 12th April 2022

Single particle trajectories reveal active endoplasmic reticulum luminal flow

The endoplasmic reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell[1, 2]. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicrometre distances. Understanding the ER structure–function relationship is critical in light of mutations in ER morphology-regulating proteins that give rise to neurodegenerative disorders[3, 4]. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, while fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.Wellcome Trust - 3-3249/Z/16/Z and 089703/Z/09/Z [Kaminski] UK Demential Research Institute [Avezov] Wellcome Trust - 200848/Z/16/Z, WT: UNS18966 [Ron] FRM Team Research Grant [Holcman] Engineering and Physical Sciences Research Council (EPSRC) - EP/L015889/1 and EP/H018301/1 [Kaminski] Medical Research Council (MRC) - MR/K015850/1 and MR/K02292X/1 [Kaminski

Towards Better Understanding of Failure Modes in Lithium-Ion Batteries: Design for Safety

In this digital age, energy storage technologies become more sophisticated and more widely used as we shift from traditional fossil fuel energy sources to renewable solutions. Specifically, consumer electronics devices and hybrid/electric vehicles demand better energy storage. Lithium-ion batteries have become a popular choice for meeting increased energy storage and power density needs. Like any energy solution, take for example the flammability of gasoline for automobiles, there are safety concerns surrounding the implications of failure. Although lithium-ion battery technology has existed for some time, the public interest in safety has become of higher concern with media stories reporting catastrophic cellular phone- and electric vehicle failures. Lithium-ion battery failure can be dangerously volatile. Because of this, battery electrochemical and thermal response is important to understand in order to improve safety when designing products that use lithium-ion chemistry. The implications of past and present understanding of multi-physics relationships inside a lithium-ion cell allow for the study of variables impacting cell response when designing new battery packs. Specifically, state-of-the-art design tools and models incorporate battery condition monitoring, charge balancing, safety checks, and thermal management by estimation of the state of charge, state of health, and internal electrochemical parameters. The parameters are well understood for healthy batteries and more recently for aging batteries, but not for physically damaged cells. Combining multi-physics and multi-scale modeling, a framework for isolating individual parameters to understand the impact of physical damage is developed in this work. The individual parameter isolated is the porosity of the separator, a critical component of the cell. This provides a powerful design tool for researchers and OEM engineers alike. This work is a partnership between a battery OEM (Johnson Controls, Inc.), a Computer Aided Engineering tool maker (ANSYS, Inc.), and a university laboratory (Advanced Manufacturing and Design Lab, University of Wisconsin-Milwaukee). This work aims at bridging the gap between industry and academia by using a computer aided engineering (CAE) platform to focus battery design for safety

Homeostasis and volume regulation in the Plasmodium falciparum infected red blood cell

The thesis reports on the application of advanced microanalytical techniques to answer a fundamental open question on the homeostasis of Plasmodium falciparum infected red blood cells, namely how infected cells retain their integrity for the duration of the parasite asexual reproduction cycle. The volume and shape changes of infected cells were measured and characterized at femtolitre resolution throughout the intraerythrocytic cycle using confocal microscopy. Fluorescence lifetime imaging and electron probe X-ray microanalysis were applied for the quantification of intracellular haemoglobin and electrolyte concentrations. The cytomechanical properties of uninfected and infected red cells were studied using a novel optical stretcher device, which enabled individual cells to be trapped and manipulated optomechanically in microfluidic channels. Combined, these methods offered a unique insight into the homeostatic and rheological behaviour of malaria-infected red cells. The results were analysed by comparison with predictions from a detailed physiological model of the homeostasis and volume regulation of infected cells, providing broad support to the view that excess haemoglobin consumptions by the parasite was necessary for the integrity of infected cells (the colloidosmotic hypothesis). The dissertation is introduced with an overview of malaria, red blood cells homeostasis and the changes induced by Plasmodium falciparum infection. In the following, this description is extended to an in-depth theoretical analysis of the infected red blood cell homeostasis, from which the need to characterise certain parameters arises. The subsequent chapters address sequentially the assessment of the haemoglobin and electrolyte concentration, cell shape and volume changes and ultimately alterations in cell elasticity. The experimental part is complemented with a comparison of the resulting data to the predictions from the theoretical analysis and an outlook on future work.This work has been made possible through the award of the EPRSC and the RSC analytical science studentship to the author