Les lysosomes sont importants pour la survie et la fonction des cellules du système nerveux central et en particulier des neurones. Le mécanisme de la reformation des lysosomes est crucial pour maintenir une quantité adéquate de lysosomes fonctionnels dans les cellules. La spatacsine, qui joue un rôle dans le ce mécanisme est impliquée dans la paraplégie spastique de type SPG11 ; une maladie caractérisée par des troubles moteurs et cognitifs sévères. L’utilisation de modèles cellulaires de cette pathologie permet d’étudier les mécanismes physiopathologiques à l’origine d’altérations de la reformation des lysosomes. J’ai montré que la perte de fonction de la spatacsine est responsable de l’accumulation de lipides dans les lysosomes. Ces accumulations sont constituées de gangliosides et de cholestérol et sont présentes dans les autolysosomes perturbant leur recyclage en lysosomes, notamment en empêchant le recrutement de protéines impliquées dans le mécanisme. Les accumulations de gangliosides rendent les neurones à l’exposition au glutamate ce qui suggère que ces altérations pourraient avoir un rôle dans la neurodégénérescence. J’ai aussi montré que l’absence de spatacsine provoque une dérégulation de l’import de Ca2+ extracellulaire par le « store-operated calcium entry » ce qui conduit à altération de l’homéostasie calcique. L’inhibition de l’import de calcium par le SOCE permet de réduire les accumulations de lipides et de rétablir partiellement le recyclage des lysosomes. Ainsi, l’absence de spatacsine induit une altération de l’homéostasie calcique qui participe à l’accumulation de lipides dans le système lysosomal ce qui est délétère pour la survie des neurones. / Lysosomal dysfunctions are involved in a large number of neurodegenerative diseases highlightingthe crucial function of lysosomes in neuron survival and function. The mechanism of lysosomereformation from autolysosomes allow cells to maintain the ool of functional lysosomes.Disruptions of this rocess are involved in athologies affecting the central nervous system. Inparticular, spatacsin that lays a role in lysosome recycling is implicated in hereditary spasticparaplegia type SPG11, a severe disease characterized by motors and cognitive alterations. Thispathology is caused by loss of function mutations in SPG11, encoding spatacsin. The study ofSPG11 cellular models gives the opportunity to decipher the hysiopathological mechanismsunderlying lysosomal reformation disruptions.During my thesis, I showed that loss of spatacsin function induces lipid accumulation in lysosomesand articularly in autolysosomes both in fibroblasts and neurons from Spg11-/- mice. Gangliosidesand cholesterol are among lipids that accumulate in autolysosomes impairing lysosomal membranerecycling by disrupting the recruitment of keys roteins. Neurons with ganglioside accumulationsare more sensitive to glutamate induced neuronal death, suggesting that these accumulations areinvolved in neurodegeneration. These results could be of great importance since accumulations ofgangliosides in lysosomes arise in many diseases.I also showed that loss of spatacsin disrupts extracellular calcium import by the store-operatedcalcium entry (SOCE) leading to an increase in cytosolic calcium levels. Lysosomal calcium contentis also increased in Spg11-/- cells and calcium release from lysosome by TRPML1 is reduced.Inhibiting SOCE and stimulating lysosomal calcium release by TRPML1 reduced lipidsaccumulations in lysosomes and artially restored lysosome reformation.Our data suggest that absence of spatacsin is responsible for a disruption of calcium homeostasisthat contributes to lipid accumulation in autolysosomes, disturbing reformation of lysosomes fromautolysosomes. Inhibiting gangliosides synthesis could be used as a therapeutic strategy. However,understanding how loss of function of spatacsin alters these cellular athways will allow thedevelopment of targeted therapeutic approaches.

Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.

Store-operated calcium entry (SOCE) is the major Ca2+ entry pathway in most non-excitable cells maintaining prolonged elevation of cytosolic Ca2+ levels required for gene transcription. SOCE is activated by the loss of endoplasmic reticulum (ER) Ca2+ through stromal interaction molecules (STIM), ER-membrane associated Ca2+ sensors. In humans, STIM1 and STIM2 share 65% sequence similarity but differentially regulate SOCE. Biophysical studies on the luminal Ca2+-binding region suggests that STIM2 EF-SAM is more stable than STIM1. The NMR structure of Ca2+-loaded STIM2 EF-SAM determined in this work suggests a more stable SAM and a tighter EF-hand and SAM interaction in STIM2 may be account for its higher stability. Chimeric swapping of the EF-hand and SAM domains generates an unstable ES211. Introducing ES211 into cherryFP-STIM1 shows constitutive puncta which activate SOCE independent of ER depletion. The current work demonstrates that the instability of the EF-SAM plays an important role in regulating SOCE initiation.

No description available.

No description available.

Store-operated Ca2+ entry (SOCE) triggered by the depletion of endoplasmic reticulum (ER) luminal Ca2+ stores is a major Ca2+ entry pathway in non-excitable cells and is essential in physiological Ca2+ signaling and homeostasis. STIM proteins are sensors of ER luminal Ca2+, which translocate to ER-plasma membrane (PM) junctional regions to activate the family of Orai channels mediating Ca2+ entry. This study is focused on dissecting the mechanism of STIM interacting with Orai. A powerful modifier of SOCE, 2-aminoethoxydiphenyl borate (2-APB) is utilized. First, the action of 2-APB on the mammalian Orai homologues are characterized using the DT40 STIM knockout cells. 50 ìM 2-APB directly activates Orai3 but not Orai1 or Orai2. Second, while it stimulates the STIM2-mediated constitutive Ca2+ entry through Orai, 2-APB also induces the cytosolic STIM C-terminus fragments to translocate to the PM and activate Orai1. These data reveal 50 ìM 2-APB enhances STIM-Orai coupling. Further, this enhanced binding of STIM and Orai leads to a conformational change within the STIM-Orai complex, which is possibly the underlying mechanism for the 50 ìM 2-APB inhibitory effect on SOCE. Finally, six residues (344-349) at the N-terminus of the STIM-Orai activation region (SOAR) prove to be critical for this inhibitory action. These same six amino acid region also constitutes an ancillary Orai binding site within SOAR, in addition to the main polybasic region. The deletion of this ancillary site abolishes the ability of SOAR to bind to and activate Orai1, but can be compensated for by the STIM-Orai binding enhancing effect of 50 ìM 2-APB. The majority of STIM1 is located on the ER membrane, while a small proportion of STIM1 is on the PM. Using an extracellularly applied STIM1 antibody, the PM STIM1 can be aggregated to exert an influence on the ER STIM1. Although the PM STIM1 is not obligatory for STIM1-mediated Orai activation, it nevertheless may have a functional presence in the PM. Lastly, a regulatory link between voltage-gated Ca2+ channels (Cav channels) and the STIM proteins is established. After activation by store depletion, STIM strongly suppresses the Cav1.2 channels. There is a biochemical interaction between STIM1 and the Cav1.2 pore subunit á1C. This inhibitory effect is independent of Orai1 activation. Hence, STIM1 interacts with and reciprocally controls two major Ca2+ channels. / Biochemistry

Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

Optogenetics has emerged as a branch of biotechnology that combines genetic engineering with optics to observe intracellular changes as well as control cellular function. Despite recent progress, there still remains the need for an optogenetic tool that can specifically control Ca2+. Such a tool would greatly facilitate the study of highly Ca2+-dependent cellular processes that are regulated both spatially and temporally. Ca2+ signaling regulates many cellular processes in both healthy and diseased cells. The ability to modulate the shape, duration, and amplitude of Ca2+ signaling is important for elucidating mechanisms by which endogenous Ca2+ concentrations are maintained. In this thesis, we used optogenetic approaches to explore a number of strategies to control Ca2+ influx through store-operated Ca2+ entry (SOCE) mediated by Stim1 and Orai1. To better study Ca2+ signaling in live cells, protein-based biosensors can be developed to monitor intracellular Ca2+ changes. To aid in this, we developed a computational modeling tool called FPMOD to improve both new and existing biosensor designs. Although FPMOD was initially intended for evaluating biosensor designs, other research groups have since used it to construct models of other proteins to answer questions related to protein conformation. We next studied the modulation of SOCE by using drug-inducible fusion proteins to study the regulation of Stim1 puncta formation. Interestingly, recruiting a Ca2+-buffering protein to Stim1 led to puncta formation, a previously unknown means of inducing puncta. These results suggest Stim1 may additionally be regulated by cytoplasmic Ca2+ levels. Finally, we developed LOVS1K, an optogenetic tool to directly activate Orai1 channels and specifically control Ca2+ influx. Photo-sensitive LOVS1K was used to generate both local Ca2+ influx at the membrane and global cytoplasmic Ca2+ signals. As proof of concept, LOVS1K was further used to modulate engineered Ca2+-dependent proteins. Ca2+ is a remarkably versatile intracellular messenger. The combination of high spatiotemporal control of irradiation and the ability of LOVS1K to generate both local and global Ca2+ changes provides a promising platform to study cellular processes that are highly dependent on different Ca2+ signals. Together, biosensors and engineered Ca2+-modulating tools can be used to study the many different aspects of Ca2+ signaling and controllably manipulate endogenous Ca2+ signaling pathways.

No description available.

Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.