Mechanisms and Mechanosensitivity: Exceptional Cadherins for Hearing and Balance

Légende manuscrite sur le document original : ''Madras. Quartier de Triplicane. '' -- Géolocalisation approximative centrée sur le quartier de Triplican

DYNAMIC REGULATION OF STORE-OPERATED CALCIUM ENTRY BY PROTEIN S-ACYLATION

Calcium plays a pivotal role in many physiological functions in cells. Cytosolic calcium levels are finely tuned by calcium ion channels, pumps, and intracellular organelles. Store-operated calcium entry (SOCE) is when depletion of endoplasmic reticulum (ER) calcium stores activates a calcium sensor known as stromal interaction molecule 1 (STIM1). Activation of STIM1 leads to a conformational change from a compact state to an extended state. This extended state of STIM1 allows it to bind to a calcium channel in the plasma membrane (PM) known as Orai1. The binding of Orai1 and STIM1 leads to opening of Orai1 channels and calcium entry into the cell. This is known as the calcium-release activated calcium (CRAC) current. Multiple subunits of Orai1 and STIM1 coalesce together at the ER:PM junctions to form oligomers known as “puncta”. Many cellular stimuli lead to store-depletion, most of which are mediated by inositol 1,4,5 trisphosphate (IP3)-mediated calcium release thorough the IP3 receptor calcium channels in the ER. Despite being discovered over two decades ago, the precise mechanism of how these two proteins in distinct subdomains in the cells colocalize to promote calcium entry remains elusive. A predominant model for how STIM1 activates Orai1 in the current field is known as a “diffusion-trap model”, which postulates that the extended conformation of STIM1 upon store-depletion traps Orai1 in the plasma membrane by passive diffusion. However, this model has several shortcomings. Upon store-depletion, Orai1 and STIM1 show a directed movement toward membrane subdomains enriched in cholesterol and sphingolipids (also known as lipid rafts). The non-random assembly of Orai1/STIM1 puncta in the membrane suggests that additional mechanisms other than random diffusion mediate puncta formation. S-acylation is the reversible addition of a lipid moiety to cytosolic cysteine residues mediated by a set of enzymes known as palmitoyl acyltransferases (PATs). These enzymes are also known as DHHC enzymes owing to the aspartate-histidine-histidine-cysteine residues in their catalytic site. S-acylation regulates many protein functions such as stability, activity, trafficking, and recruitment to membrane subdomains. In our previous research endeavors, we found that some signaling proteins involved in T cell receptor pathway undergo S-acylation upon T cell activation. Activation of T cells is also known to induce ER store-depletion through IP3R activity. Depilated mice carry a deletion of phenylalanine residue at 233 (ΔF233) in the DHHC21 enzyme. In these mice, many components of the T cell receptor complex cannot undergo S-acylation. Based on these observations, we hypothesized that Orai1 and STIM1 proteins undergo DHHC21-mediated S-acylation to promote SOCE. Here, we show that both Orai1 and STIM1 undergo S-acylation upon ER store-depletion. Using cysteine mutant versions of Orai1 and STIM1, we also show that these proteins that cannot undergo S-acylation and have deficits in puncta formation and SOCE. Using the cells obtained from depilated mice, we show DHHC21 mediates the S-acylation of STIM1. Our data show that S-acylation of Orai1 and STIM1 regulates CRAC channel formation and SOCE

Systems Modeling of Calcium Homeostasis and Mobilization in Platelets Mediated by Ip3 and Store-Operated Calcium Entry

Platelet aggregation is one of the body\u27s first responses to vascular damage to prevent blood loss; upon injury to the endothelium platelets react to the exposed extracellular matrix and undergo a host of intracellular biochemical changes enabling them to activate and form a plug at the site of injury. Internally, platelets respond to their environment by exhibiting a sharp rise in cytosolic calcium that triggers a series of chemical and morphological changes which are critical to platelet activation and subsequent clot propagation. This thesis develops a mechanistic, computational model of platelet calcium regulation using coupled sets of ordinary differential equations. This thesis extends previous work modeling calcium release mediated by inositol 1,4,5-trisphosphate (IP3) to engineer what is the first, to date, complete model of store-operated calcium entry (SOCE) integrated into a systems model for calcium signaling. SOCE is a ubiquitous extracellular calcium entry pathway which is activated by calcium store depletion, is seen in many cells types and is yet to be fully understood. Our model for SOCE regulation consists of diffusion-limited dimerization of the calcium sensor STIM1, followed by fast, cytosolic calcium-dependent association of STIM1 dimers with Orai1 channels in the plasma membrane resulting in graded store-operated channel activation. Appropriate model resting states were characterized using a dense Monte Carlo technique on an initial condition sampling space constrained by available data on species concentrations and protein copy numbers. From this set of resting configurations, following application of physiologic IP3 stimuli, we selected for resting states exhibiting calcium dynamics that are in agreement with experimental data. We also selected for states presenting significant SOCE current based on differences in cytosolic calcium between simulations run with and without extracellular calcium. Low resting levels of IP3 are required for system robustness and for simultaneous appropriate dynamic response to physiologic agonists. Platelets require a resting electrical potential across the membrane surrounding the calcium stores of greater than -70 mV in order to exhibit significant agonist-induced calcium release

The 2β Insert Perturbs Folding, Stability and Hydrophobic Exposure of Stromal Interaction Molecules

Stromal interaction molecule (STIM)1 and 2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels through oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil domains couple to PM Orai1 protein subunits to form Ca2+ released activated Ca2+ (CRAC) channels that facilitate store-operated Ca2+ entry (SOCE). One splice variant of STIM2, STIM2β, contains an extra 8-residue (2β insert) located within the coiled-coils and inhibits SOCE through an unresolved mechanism, adding another layer of complexity to Ca2+ regulation in mammals. I hypothesize that the 2β insert perturbs the coiled-coil conformation and dynamics commensurate with an ability to activate SOCE. My data show the 2β insertion induced an overall reduction in α-helicity, thermal stability, and promoted a conformation of greater exposed hydrophobicity which affected oligomerization. Previous studies show STIM2 more weakly couples to Orai1 compared to STIM1, and STIM2β is completely incapable of binding to Orai1. My functional studies show the 2β insertion in the STIM1 context also significantly inhibits SOCE. Therefore, my data suggests that the 2β insert inhibits STIM function and SOCE through a mechanism which perturbs α-helical levels and destabilizes the cytosolic domain coiled-coil structure, independent of the requirement for weaker STIM2 coupling to Orai1

Oxidative Stress-Induced STIM2 Cysteine Modifications Suppress Store-Operated Calcium Entry

Store-operated calcium entry (SOCE) through STIM-gated ORAI channels governs vital cellular functions. In this context, SOCE controls cellular redox signaling and is itself regulated by redox modifications. However, the molecular mechanisms underlying this calcium-redox interplay and the functional outcomes are not fully understood. Here, we examine the role of STIM2 in SOCE redox regulation. Redox proteomics identify cysteine 313 as the main redox sensor of STIM2 in vitro and in vivo. Oxidative stress suppresses SOCE and calcium currents in cells overexpressing STIM2 and ORAI1, an effect that is abolished by mutation of cysteine 313. FLIM and FRET microscopy, together with MD simulations, indicate that oxidative modifications of cysteine 313 alter STIM2 activation dynamics and thereby hinder STIM2-mediated gating of ORAI1. In summary, this study establishes STIM2-controlled redox regulation of SOCE as a mechanism that affects several calcium-regulated physiological processes, as well as stress-induced pathologies

Role of cytoskeletal remodeling in T cell receptor signaling and integrin activation at the immunological synapse

The efficiency of an immune response critically depends on the ability of T cells to respond to antigens. Upon encountering cognate antigenic peptides on the surface of antigen-presenting cells, T cells form a specialized interface, termed the immunological synapse (IS), which serves as the site of information transfer between the cells. This contact zone is characterized by the enrichment of signaling receptors, kinases and adaptor proteins, and is the site of extensive cytoskeletal remodeling. The versatile nature and spatio-temporal regulation of signaling cascades at the IS has long been recognized but the exact mechanisms that coordinate these processes remain poorly understood. In this work we have investigated the role of cytoskeletal remodeling in propagation of signaling events that lead to T cell activation. Using human T cell lines and primary T cells, we demonstrate that F-actin flow is largely driven by actin polymerization, rather than by myosin IIA contraction. While myosin IIA is able to exert forces on the cytoskeleton, it is dispensable for bulk network flow. Conversely, myosin IIA controls the extent of cell spreading and synaptic symmetry. We have also found that ongoing retrograde flow of F-actin sustains calcium mobilization at the level of release from endoplasmic reticulum stores. This defect is likely due to loss of PLCgamma1 activity at the IS, since the concentration of phosphorylated PLCgamma1 plummets upon F-actin immobilization. Furthermore, we have examined whether F-actin remodeling is required for integrin LFA-1 activation, which in turn strengthens conjugate formation and costimulation. Taking advantage of stimulatory planar lipid bilayers and cell-cell conjugates, we show that F-actin flow drives affinity maturation and spatial organization of LFA-1 at the IS. These observations are in line with a mechanotransduction model, in which F-actin-derived force induces integrin conformational change, thereby modulating binding affinity for ligand. The net inward movement of F-actin also recruits LFA-1 to the interface, thereby increasing its effective concentration. Taken together, these findings indicate that ongoing remodeling of actin cytoskeleton is required to sustain signaling and to choreograph spatio-temporal organization of receptors and their associated complexes at the IS during early phases of T cell activation

Modelling store operated calcium entry: creating a three dimensional spatio-temporal model to predict local calcium signals

Calcium is a signalling messenger that is crucial to cellular function, controlling a diverse range of processes such as apoptosis, cell proliferation and muscle contraction. Store operated calcium entry (SOCE) is a specific pathway coupling depletion of the calcium stores within the endoplasmic reticulum (ER) to calcium influx through Orai channels on the plasma membrane. SOCE occurs in small sub-cellular regions called 'ER-PM junctions' which are typically less than $300$nm in diameter. The small size of these domains prevent direct measurement of the calcium signals as current calcium imaging techniques cannot resolve the local signals within ER-PM junctions. The calcium signals associated with SOCE control many downstream cellular processes, such as gene expression and immune responses. There is substantial evidence demonstrating that the placement of the calcium signalling machinery, including Orai channels and SERCA pumps, is vital to the generation of spatially distinct calcium signals which then enhance the selectivity of the calcium signal. However, experimental techniques cannot investigate the local calcium dynamics occurring on a spatial scale of micrometres so mathematical modelling techniques can be used to close this gap in understanding how the local calcium dynamics affect the experimentally observed global calcium dynamics. In this thesis, we construct a three dimensional spatio-temporal model of calcium dynamics and investigate the relationship between the placement of core components of the calcium signalling machinery, e.g. Orai channels and SERCA pumps, and the spatial calcium profiles generated as well as the rates of ER refilling observed. The model includes a spatially extended ER-PM junction to examine the spatial signature of the calcium profiles generated and a spatially extended sub-PM ER to examine the impact of Orai channel and SERCA pump placement on ER refilling dynamics. The model is the first to include spatially extended versions of both the ER-PM junction and sub-PM ER. In this thesis, we first focus on the construction of the spatio-temporal model and the solution techniques used to solve the model. We implement a semi-analytical solution using Green's functions to calculate the analytical solution of the spatial component of the diffusion equation and use numerical time stepping methods in MATLAB to evolve the spatial calcium profile over time. We compare the predictions of the model to expected biological outcomes and then use the model to investigate how the placement of Orai channels, and in particular how clustering of Orai channels, creates spatially distinct calcium profiles. We then examine whether the spatial calcium profile affects ER refilling and what factors control ER refilling. We find that Orai channel clustering creates spatially distinct calcium profiles within the ER-PM junction but does not enhance ER refilling. ER refilling is more strongly controlled by the proximity of SERCA pumps to Orai channels. In fact, the placement of SERCA2b pumps weakly affects ER refilling but the major regulator of ER refilling is the placement of SERCA2a pumps within the ER-PM junction. However, ER refilling continues, albeit at reduced rates, regardless of Orai channel and SERCA pump placement which suggests that other factors, such as the geometry of the ER-PM junction, could be important regulators of ER refilling. This work is relevant to experimental biologists and mathematicians within the calcium signalling community as the calcium signals generated within the ER-PM junction are crucial for advancing the understanding of how calcium signals regulate cellular function. The local calcium dynamics are important regulators of whole cell calcium dynamics and so mathematical methods allowing rigorous investigation of the mechanisms controlling local calcium signalling will be invaluable to furthering our understanding of how SOCE regulates cell function

Integration Of Extracellular And Intracellular Signals Via The Calcium Sensing Receptor (CASR)

The Ca2+-sensing receptor (CaSR) regulates the calcium homeostasis in the human body via sensing fluctuations in the extracellular Ca2+ concentration. Naturally occurring mutations in the CaSR could result in Ca2+ regulation disorders. In the present study, we use several complementary approaches including imaging [Ca2+]i response in living cells at the cellular level and using molecular dynamic (MD) simulations at the atomic level to provide important insights into the behavior of the receptor in both normal and disease statuses. We demonstrated that the molecular connectivity between [Ca2+]o–binding sites is responsible for the functional positive homotropic cooperativity in the CaSR’s response to [Ca2+]o. Naturallyoccurring disease mutations near Site 1 disrupted the cooperativity. We further identified an L-Phe-binding pocket adjacent to Ca2+-binding Site 1, which is essential for functional positive heterotropic cooperativity by having a global impact on all five of the predicted Ca2+-binding sites in the ECD with regards to [Ca2+]o-evoked [Ca2+]i signaling. Furthermore, the CaSR’s ECDs have been expressed using both bacteria and mammalian systems and were characterized using the fluorescence titration spectroscopy, circular dichroism technique as well as the NMR spectroscopy. Our studies show calcium and Phe directly bind to the ECD domain directly and interactively. Moreover, we also demonstrated that intracellular trafficking of the CaSR is a complex process, which involves modulation by calmodulin and can possibly be affected by different CaSR isoforms when expressing in various cell lines. The studies on the isolated proteins will pave the way for future protein crystallization and related structural research

The μ-calpain-ezrin axis: a potential target for therapy in inflammatory disease

Neutrophils are the most abundant class of white blood cell in humans. They are among the first inflammatory cells to respond during infection or tissue damage, and their excessive recruitment to synovial joints in rheumatoid arthritis has been implicated in the progression of the disease. The process of neutrophil extravasation from blood vessels involves a rapid expansion of the available cell surface area. This ~200% increase is facilitated by the release of cell membrane microridges. As ezrin forms crucial cross-links between the plasma membrane and cortical F-actin, in neutrophils and other myeloid cells, it is thought to maintain the structure of these microridges, which act as a reservoir of plasma membrane for spreading. It has been suggested that elevating the cytosolic Ca2+ concentration, thereby activating the Ca2+-activated cysteine protease μ-calpain, would break the ezrin link and permit cell spreading. This thesis has investigated the relationship between, μ-calpain, ezrin and Ca2+ concentration. Through immunocytochemistry, confocal microscopy of live phagocytosis and chemotaxis experiments, observations in neutrophils have been complemented by investigations in a RAW 264.7 model cell line, amenable to transfection. By generating the novel genetically encoded Ca2+ indicator EPIC (Ezrin Peripheral Indicator of Ca2+), it has been possible to experimentally measure the cytosolic Ca2+ concentration in ezrin-rich microdomains beneath plasma membrane microridges, in transfected myeloid cells, during Ca2+ influx. It has thus been found that sub-membranous cytosolic Ca2+ in these microridges reaches highmicromolar concentrations, well within the range required to activate μ-calpain and result in ezrin cleavage. The work in this thesis contributes to the understanding of the molecular mechanisms which govern neutrophil morphological changes during events such as phagocytosis and extravasation. It is hoped that these findings will help contribute towards the body of research aimed at influencing the design of novel therapeutics to treat autoimmune conditions