Microfluidic platform for bilayer experimatation from a research tooltowards drug screening

The aim of this thesis, which is the development of a microfluidic platform for bilayer experimentation with the potential for drug screening on ion channels, is introduced in this chapter. After a short presentation of the field of drug screening, an outline of this thesis is given, together with a brief summary of the different chapters

Electrophysiology

The outstanding evolution of recording techniques paved the way for better understanding of electrophysiological phenomena within the human organs, including the cardiovascular, ophthalmologic and neural systems. In the field of cardiac electrophysiology, the development of more and more sophisticated recording and mapping techniques made it possible to elucidate the mechanism of various cardiac arrhythmias. This has even led to the evolution of techniques to ablate and cure most complex cardiac arrhythmias. Nevertheless, there is still a long way ahead and this book can be considered a valuable addition to the current knowledge in subjects related to bioelectricity from plants to the human heart

Single-cell microfluidic impedance cytometry: From raw signals to cell phenotypes using data analytics

The biophysical analysis of single-cells by microfluidic impedance cytometry is emerging as a label-free and high-throughput means to stratify the heterogeneity of cellular systems based on their electrophysiology. Emerging applications range from fundamental life-science and drug assessment research to point-of-care diagnostics and precision medicine. Recently, novel chip designs and data analytic strategies are laying the foundation for multiparametric cell characterization and subpopulation distinction, which are essential to understand biological function, follow disease progression and monitor cell behaviour in microsystems. In this tutorial review, we present a comparative survey of the approaches to elucidate cellular and subcellular features from impedance cytometry data, covering the related subjects of device design, data analytics (i.e., signal processing, dielectric modelling, population clustering), and phenotyping applications. We give special emphasis to the exciting recent developments of the technique (timeframe 2017-2020) and provide our perspective on future challenges and directions. Its synergistic application with microfluidic separation, sensor science and machine learning can form an essential tool-kit for label-free quantification and isolation of subpopulations to stratify heterogeneous biosystems

Current technical approaches to brain energy metabolism

Neuroscience is a technology‐driven discipline and brain energy metabolism is no exception. Once satisfied with mapping metabolic pathways at organ level, we are now looking to learn what it is exactly that metabolic enzymes and transporters do and when, where do they reside, how are they regulated, and how do they relate to the specific functions of neurons, glial cells, and their subcellular domains and organelles, in different areas of the brain. Moreover, we aim to quantify the fluxes of metabolites within and between cells. Energy metabolism is not just a necessity for proper cell function and viability but plays specific roles in higher brain functions such as memory processing and behavior, whose mechanisms need to be understood at all hierarchical levels, from isolated proteins to whole subjects, in both health and disease. To this aim, the field takes advantage of diverse disciplines including anatomy, histology, physiology, biochemistry, bioenergetics, cellular biology, molecular biology, developmental biology, neurology, and mathematical modeling. This article presents a well‐referenced synopsis of the technical side of brain energy metabolism research. Detail and jargon are avoided whenever possible and emphasis is given to comparative strengths, limitations, and weaknesses, information that is often not available in regular articles.Fondecyt, Grant Number: 1160317; MINECO, Grant Numbers: SAF2016‐78114‐R and RTC‐2015‐3237‐1; CIBERFES, Grant Numer: CB16/10/00282; SP3‐People‐MC‐ITN program, Grant Number: 608381; EU BATCure, Grant Number: 666918; FEDER (European regional development fund); Investissement d'Avenir, Grant Number: ANR‐11‐INBS‐0011; French State in the context of the “Investments for the future” Program IdEx and the LabEx TRAIL, Grant Numbers: ANR‐10‐IDEX and ANR‐10‐LABX‐57; French–Swiss ANR‐FNS, Grant Numer: ANR‐15‐ CE37‐0012. University of Nottingham; BBSRC, Grant Numers: BB/L019396/1 and BB/K009192/1; MRC, Grant Number: MR/L020661/1; Deutsche Forschungsgemeinschaft, Grant Numers: DFG SPP 1757, SFB 894, and FOR 2289; European Commission, Grant Number: H2020‐FETPROACT 732344; Neurofibres, Grant Number: H2020‐MSCA‐ITN‐722053 EU‐GliaPhD; US National Institutes of Health, Grant Number: R01NS087611; Teva Pharmaceuticals; Agilent Technologies. IdEx, Grant Number: ANR‐10‐IDEX‐03‐02; French–Swiss ANR‐FNS, Grant number: 310030E‐164271; National Institutes of Neurologic Disease and Stroke at the National Institutes of Health, Grant Numer: R01 NS077773; University of Zurich and the Swiss National Science Foundation; Comisión Nacional de Investigación Científica y Tecnológica, Grant Numer: PB 01; Fondo Nacional de Desarrollo Científico y Tecnológico, Grant Numer: 1160317; Ministerio de Economía y Competitividad, Grant Numer: RTC‐2015‐3237‐1,SAF2016‐78114‐R; Agence Nationale de la Recherche, Grant Numers: ANR‐10‐IDEX, ANR‐10‐IDEX‐03‐02, ANR‐10‐LABX‐57, ANR‐11‐INBS‐0011, and ANR‐15‐ CE37‐0012; Biotechnology and Biological Sciences Research Council, Grant Numers: BB/L019396/1 and BB/K009192/1; Medical Research Council, Grant Numer: MR/L020661/1.Peer reviewe

Control of Membrane Excitability by Potassium and Chloride Leak Conductances

The permeability of the neuronal membrane to different ions determines both resting membrane potential (RMP) and input conductance. These parameters determine the cells response to synaptic input. In this thesis I have examined how the molecular properties of potassium and chloride ion channels can influence neuronal excitability in ways that have not previously been considered. For example, two‐pore domain potassium (K2P) channels open at rest to generate a persistent potassium ion efflux. In addition to its accepted role in setting the RMP, I have tested the hypothesis that this conductance is sufficient to repolarise the membrane during an action potential (AP) in the absence of voltage‐dependent potassium channels (Kv). We tested this prediction using heterologous expression of TASK3 or TREK1 K2P channels combined with conductance injection to simulate the presence of a voltage‐gated sodium conductance. These experiments demonstrated that K2P channels are sufficient to support APs during short and prolonged depolarising current pulses. The membranes permeability to chloride ions can also be affected by extrasynaptic GABAA receptors containing the delta subunit (δ‐GABAARs) that produce a tonic conductance due to their high apparent affinity for GABA. The anaesthetics Propofol and THIP are both believed to alter neuronal excitability by enhancing this persistent chloride flux. We have examined how this anaesthetic action is affected by the steady‐state ambient GABA concentrations that are believed to exist in vivo. Surprisingly, the anaesthetic enhancement of δ‐GABAARs is lost at low ambient GABA concentrations. Therefore, I would suggest that the anaesthetic potency of these drugs is affected by the resting ambient GABA concentration in a manner that has not previously been appreciated. In the current Thesis I have examined the molecular and pharmacological properties of two very different ion channel families that both generate a leak conductance, and I will present models that link the behaviour of these ion channels to their ability to modulate neuronal excitability

Na+ and Ca2+ Channels in the Lysosome: Opening the Gate to the Cell's Recycling Center.

Lysosomes primarily serve as the cell’s “garbage disposal and recycling center”, and are recently found to be involved in many important cellular functions. Lysosomes are also ion stores enriched with H+, Ca2+, and Na¬+. While it’s well known that the lysosomal ionic homeostasis is essential for its proper functions, the properties of ion transporters and channels residing on lysosomal membranes are barely understood, largely due to the lack of a reliable functional assay. Recently our lab has established a unique lysosomal patch-clamp method to directly record from native lysosomal membrane. Taking advantage of the technique, I discovered two novel lysosomal Na+-selective channels (Two-Pore-Channels TPC1 and TPC2), which are previously thought to be Ca2+ release channels, triggered by the second messenger NAADP. Using an integrative approach, I further demonstrated that TPCs are not activated by NAADP, but instead by PI(3,5)P2, a lysosome-specific phosphoinositide that regulates lysosomal ion homeostasis and membrane trafficking. TPCs represent the first intracellular Na+-selective channels, although their functions are not characterized. In addition, my colleagues and I found that PI(3,5)P2 also activates TRPML1, a principle lysosomal Ca2+ channel. Loss-of-function mutations in human TRPML1 cause type IV Mucolipidosis (ML4), a childhood neurodegenerative disease. My results showed that increasing TRPML1’s activity alleviated lysosomal trafficking defects in PI(3,5)P2-deficient cells, suggesting that PI(3,5)P2 controls Ca2+-dependent membrane trafficking by regulating TRPML1. To study the role of TRPML1 in membrane trafficking, I focused on the involvement of TRPML1 in Ca2+-dependent lysosomal exocytosis, a universal process important for many cellular functions, including cellular clearance, plasma membrane repair and phagocytosis. I found that gain-of-function mutations of TRPML1 caused a dramatic increase in lysosomal exocytosis. During particle uptake in macrophages, lysosomal exocytosis is required to provide membrane supplies to facilitate phagosome formation. By whole-cell recordings and newly developed whole-phagosome recordings, I found that upon particle binding, TRPML1-associated lysosomes are delivered to the newly-formed phagosomes via lysosomal exocytosis in a Ca2+-dependent manner. Overall, my thesis work has characterized two types of important channels (TPCs and TRPMLs) in the lysosome, identified their first endogenous activator PI(3,5)P2, and explored their functions in lysosomal biology.PHDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99812/1/xiangwa_1.pd

Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes