Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.

Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.

During excitation-contraction coupling, Ca2+ transient induced by the depolarization of membrane potential is the trigger of mechanical contraction in cardiac myocytes, which is responsible for the pumping function of the heart. However, mechanisms underlying intracellular Ca2+ regulation and the coupling between Ca2+ transient and membrane potential are not completely understood. Abnormalities in intracellular Ca2+ regulation have been observed during heart failure and cardiac arrhythmias, such as intracellular Ca2+ alternans and T-tubule disorganization. In this project, intracellular Ca2+ dynamics in different types of cardiac myocytes were investigated by using computer modelling. For atrial myocytes, a biophysically detailed computer model was developed to describe the observations of Ca2+ alternans and Ca2+ wave propagation in cardiac myocytes lacking T-tubules. The model was validated by its ability to reproduce experimental observed Ca2+ wave propagation under normal condition and the influences on spatial Ca2+ distribution by modifying various aspects of Ca2+ cycling, such as Ca2+ influx, SR Ca2+ uptake and SR Ca2+ release in cardiac myocytes lacking T-tubules. Mechanisms underlying the genesis of Ca2+ alternans in this type of cell were investigated by the model. Furthermore, a spontaneous second Ca2+ release was observed in response to a single voltage stimulus pulse with enhanced Ca2+ influx as well as SR Ca2+ overload. For the ventricular myocytes, an existing canine model was used to study the genesis of APD and intracellular Ca2+ alternans under various conditions. The genesis of Ca2+ alternans was investigated by analyzing the relationship between systolic Ca2+ concentration and SR Ca2+ content. On the other side, the roles of SR Ca2+ regulation and action potential restitution in the genesis of intracellular Ca2+ and APD alternans were also examined under various conditions. In addition, it was shown that spatially discordant Ca2+ alternans was generated when the Ca2+-dependent inactivation of ICa,L was strong. It tended to be concordant for weak Ca2+-dependent inactivation of ICa,L. For the sinoatrial node cells, a mathematical model was developed to simulate stochastic opening of unitary L-type Ca2+ channel and single RyR channel, thereby reproducing experimental observed local Ca2+ release during diastolic depolarization phase of the action potential. Simulation results of ionic channel block and modifications of SR Ca2+ regulation suggested a limited role of intracellular Ca2+ in the automaticity of central SA node cells.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal