Mitochondria are dynamic organelles that constantly undergo opposing fission and fusion events which impact many aspects of mitochondrial and cellular homeostasis including bioenergetic activity, calcium buffering and organelle transport. The large GTPase dynamin-related protein 1 (Drp1) acts as a mechanoenzyme to catalyze fission of mitochondria. Drp1 activity is regulated through a series of reversible posttranslational modifications. Phosphorylation of the conserved serine residue, S656, by cAMP dependent protein kinase A (PKA) acts as a master regulator of Drp1 activity. Two phosphatases oppose PKA by dephosporylating Drp1 S656, a mitochondrial isoform of protein phosphatase 2A and the calcium-calmodulin dependent phosphatase calcineurin (CaN). Here I report the characterization of a conserved CaN docking site on Drp1, an LxVP motif, just upstream of the Drp1 S656 site. Mutational modification of the Drp1 LxVP motif resulted in selective bidirectional modulation of formation of the CaN:Drp1 complex. Stability of the CaN:Drp1 LxVP motif mutant complexes was qualitatively described by affinity purification and quantitatively described by isothermal titration calorimetry. Stability of the CaN:Drp1 complex was found to directly correlate with Drp1 S656 dephosphorylation kinetics as demonstrated by studies conducted in vitro and in intact cells. Further, the CaN:Drp1 signaling axis was shown to shape basal mitochondrial morphology in a heterologous cell line system and in primary hippocampal neurons. Finally, disruption of the CaN:Drp1 signaling axis was found to protect neurons from oxygen-glucose deprivation, an in vitro model of ischemic injury. While these results suggest that the CaN:Drp1 signaling axis may be a potential target for neuroprotective therapeutic exploitation, the mechanism by which disruption of the CaN:Drp1 signaling axis specifically and mitochondrial elongation generally results in resistance to ischemic injury remains unknown. Additional studies reported here demonstrate that mitochondrial fragmentation remains a prominent feature of injured neurons regardless of the fidelity of the CaN:Drp1 signaling axis. Mitochondrial fragmentation at the time of injury was found to occur in a Drp1-independent manner. Chronic mitochondrial elongation was also found to leave unaltered the ability of neurons to detoxify reactive oxygen species, buffer intracellular calcium and supply ATP for homeostatic function.

Given the debilitating effect that mitochondrial dysfunction has on human health, it is important to understand mitochondrial dynamics that are vital for the maintenance of mitochondrial function, genome, morphology, and quality control. Mitochondrial dynamics result from a balance in mitochondrial fusion and fission. Although the mechanism and regulation of mitochondrial fission are largely elucidated, less is known about mitochondrial fusion. Mgm1 is a protein that mediates mitochondrial fusion in yeast. However, the molecular mechanism of Mgm1 function in mediating mitochondrial fusion is unclear. In this thesis, first, I show that Mgm1 contains a lipid-binding domain by demonstrating that purified Mgm1 has lipid-binding activity and by identifying mutations in conserved residues that abrogate these interactions. Second, I show that Mgm1 assembles into hexameric rings and undergoes nucleotide-dependent structural transitions that, I believe, initiate membrane fusion. Lastly, I demonstrate that Mgm1 exhibits membrane-remodeling activities that are crucial for the tethering and lipid-mixing steps in the membrane fusion event. Together, I propose a mechanistic model of Mgm1 function in mediating mitochondrial fusion that advances the fields of mitochondrial biology, cellular protein-membrane dynamics, and the etiology of neurodegenerative diseases.

La cryptogéine, une protéine sécrétée par l’oomycète Phytophthora cryptogea, provoque la mise en place de mécanismes de défense chez le tabac, mobilisant au cours des étapes précoces de la signalisation associée, des protéines localisées dans la membrane plasmique (MP). Une fraction membranaire résistante à la solubilisation par les détergents (DIM pour Detergent Insoluble Membrane), enrichie en stérols et en sphingolipides avait été purifiée à partir de la MP de tabac : cette fraction contenait plusieurs protéines impliquées dans la cascade de signalisation induite par la cryptogéine. Chez l’animal, l’association dynamique de protéines à des microdomaines riches en stérols et sphingolipides en réponse à un stress biotique joue un rôle essentiel dans la régulation de la signalisation cellulaire. L’objectif de ce travail était de tester l’hypothèse qu’un tel phénomène puisse se produire dans notre modèle d’étude. La comparaison du protéome de fractions DIMs, purifiées à partir de cellules traitées ou non pendant 5 minutes à la cryptogéine a été réalisée à l’aide d’un marquage isotopique (15N ou 14N) et d’une approche de protéomique quantitative. Le premier résultat est que l’abondance de la majorité des protéines n’est pas modifiée dans les DIMs en réponse à la cryptogéine. Une seule protéine est enrichie dans les DIMs, une isoforme de 14-3-3, tandis que quatre dynamines (DRPs pour Dynamin Related Proteins), impliquées dans le trafic vésiculaire, sont exclues des DIMs en réponse à la cryptogéine. L’étude d’une des dynamines identifiées, DRP1A, a été menée. Nous avons caractérisé les différents gènes codant DRP1A dans le génome du tabac, puis utilisé une approche ARN antisens pour altérer l’expression de cette protéine et nous avons étudié sa localisation subcellulaire à l’aide d’anticorps spécifiques et en observant en microscopie confocale cette protéine fusionnée à la GFP. Cette approche a permis de confirmer la présence de DRP1A dans la fraction DIMs et la diminution transitoire de son association à cette fraction en réponse à la cryptogéine, suite à une dissociation de la fraction membranaire. Ces travaux constituent la première mise en évidence d’une association/dissociation dynamique de protéines aux DIMs de plantes en réponse à un stimulus biologique / Cryptogein, a protein secreted by the oomycete Phytophthora cryptogea, induces defense mechanisms in tobacco. Several proteins involved in the associated signaling pathway were identified and localized on the plasma membrane (PM). A fraction resistant to solubilization by detergent named DIMs for Detergent Insoluble Membranes, enriched in sterols an sphigolipids had been isolated from tobacco PM. It was proved to contain proteins previously identified as actors of the signaling cascade triggered by cryptogein. In animal cells, the dynamic association of proteins to sterol and sphingolipid rich microdomains under the influence of a biological stimulus plays an essential role in the regulation of cellular signaling. The purpose of this work was to test the hypothesis that such a phenomenon might occur in our model. The comparison using isotopic labeling (15N or 14N) and quantitative proteomics, of the composition of DIMs extracted from tobacco cells treated or not by cryptogein, revealed that, although the association to DIMs of most proteins remained unchanged, five proteins had their relative abundance modified after 5 minutes of treatment. One of these was a signaling protein (a 14-3-3 protein) and the four others were related to cell trafficking (4 DRPs, Dynamin Related Proteins). We characterized the DRP1A gene family in tobacco, and set up an antisens RNA antisense to down-regulate the expression of this protein. We studied the intracellular localization of DRP1 using specific antibodies and a GFP fusion. The results confirmed the presence of DRP1 in DIMs and its depletion from this fraction upon cryptogein treatment, through a dissociation from the PM. This is the first evidence of a dynamic association/dissociation of proteins to microdomains in plants upon a biological stimulus

Mitochondria are critical for energy production and Ca2+ homeostasis and undergo fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by Drp1 (dynamin-related protein 1), a large GTPase tightly controlled by various posttranslational modifications, including phosphorylation. Bβ2 is a neuron-specific postnatally induced protein phosphatase 2A (PP2A) regulatory subunit that mediates PP2A translocation to the outer mitochondrial membrane (OMM) to promote mitochondrial fragmentation and sensitize neurons to various injuries. Opposing PP2A/Bβ2's effect on mitochondrial morphology and cell death is protein kinase A (PKA) anchored to the OMM via A kinase anchoring protein 1 (AKAP1). This dissertation describes how reversible phosphorylation of Drp1 at a conserved Serine residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase complex (PP2A/Bβ2) affects dendrite and synapse development in hippocampal neurons and synaptic plasticity and learning and memory in vivo. Inducing mitochondria fragmentation decreases dendritic arbor complexity, but increases spine and synapse number. Mitochondrial elongation induces opposite effects. L-carnitine increases mitochondria membrane potential and recapitulates the dendritic and synaptic effects of mitochondrial elongation. Epistasis experiments substantiate our hypothesis that PP2A/Bβ2 dephosphorylates and PKA/AKAP1 phosphorylates Drp1 to change mitochondrial shape and regulate mitochondria localization, dendrite outgrowth, and synapse development. Bβ2 null mice are viable and fertile, without obvious abnormalities. Bβ2 null mice demonstrate significantly larger cortical and hippocampal neuronal mitochondria than in wildtype. Bβ2 deletion decreases spine number on apical and basal cortical dendrites and hippocampal dendrites. Bβ2 null mice display significantly decreased input/output relationship in the hippocampus, consistent with a decrease in synapse number. In a combined context and cued fear-conditioning protocol, the hippocampal-dependent context recall trial revealed significant deficits in Bβ2 null and heterozygous mice. This deficit is also seen in hippocampal-dependent Barnes maze performance. These results are consistent with the reduced hippocampal long-term potentiation (LTP) found in Bβ2 null mice and demonstrate the importance of Bβ2 in hippocampal synaptic plasticity and memory. In conclusion, PP2A/Bβ2 and PKA/AKAP1 have important roles in mitochondria regulation and dendritic and synaptic development as seen in our results in vitro with rat hippocampal cultures and in vivo with Bβ2 null mice.

L’auto-incompatibilité est une barrière génétique qui permet à une plante de reconnaître et rejeter son propre pollen tout en acceptant le pollen d’individus moins apparentés d’un point de vue génétique. Chez les Brassicacées, l’auto-incompatibilité est contrôlée par un locus hautement polymorphe appelé le locus S, qui contient les déterminants mâle et femelle. Le stigmate exprime le déterminant femelle de l’auto-incompatibilité, S-LOCUS RECEPTOR KINASE (SRK). Chez Brassica oleracea, la localisation subcellulaire d’SRK est unique en son genre : le récepteur est localisé principalement au niveau des endosomes et dans une moindre mesure à la membrane plasmique.Nous avons étudié la fonction de la localisation endosomale de SRK chez Arabidopsis thaliana. Premièrement, nous avons réintroduit l’auto-incompatibilité chez Arabidopsis thaliana grâce à l’expression d’un allèle fonctionnel de SRK en provenance d’Arabidopsis lyrata (une espèce auto-incompatible). Deuxièmement, nous avons montré qu’un mutant perte de fonction de DYNAMIN-RELATED PROTEIN1A, une protéine requise pour l’endocytose, abolissait l’auto-incompatibilité. Nos résultats suggèrent que l’endocytose est requise pour l’auto-incompatibilité, et que SRK pourrait activer sa voie de signalisation depuis les endosomes. / Self-incompatibility is a genetic barrier by which a plant recognizes and rejects its own pollen while allowing pollen from more distantly related plants to germinate. In the Brassicacea family, it is controlled by a highly polymorphic locus called the S-locus, which contains the male and female determinants of self-incompatibility. The stigma expresses the female determinant of self-incompatibility, the plant receptor kinase (PRK) S-LOCUS RECEPTOR KINASE (SRK). In Brassica oleracea, SRK has a unique subcellular localization among PRK: the receptor is mostly localized in endosomes and to a lesser extent at the plasma membrane.We investigated the function of the endosomal localization of SRK in Arabidopsis thaliana. Firstly, we reintroduced self-incompatibility in Arabidopsis thaliana by expression of a functional SRK allele from Arabidopsis lyrata (a self-incompatible species). Secondly, we showed that a loss-of-function mutant of DYNAMIN-RELATED PROTEIN1A, a protein required for endocytosis, abolished self-incompatibility. Our results suggest that endocytosis is required for self-incompatibility, and that SRK may be signaling from endosomal compartments.

Proteins are essential parts of living organisms that initiate and control almost all cellular processes. Despite the widely accepted belief that all functional proteins fold into stable and well-defined three-dimensional (3D) structures mandatory for protein activity, the existence of biologically functional disordered proteins has been increasingly recognized during past two decades. Proteins with inherent structural disorder, commonly known as intrinsically disordered proteins (IDPs), play many roles in a biological context. However, in contrast to their folded counterparts, they are dynamically unstructured and typically fluctuate among many conformations even while performing biological functions. In fact, it is this dynamical structural heterogeneity that that allows for IDPs to interact with other biological macromolecules in unique ways. Moreover, while a majority of proteins in eukaryotic proteomes have been found to have intrinsically disordered regions (IDR), the mechanisms by which protein disorder fives rise to biological functionality is still not well understood. Through a series of simulation studies on specific systems, this thesis probes several aspects of the emerging structure-function paradygm of IDPs, namely the mechanics, intermolecular assembly, and structural transitions occurring in these proteins. The lack of well-defined 3D structure in IDPs gives rise to distinct mechanical properties, the subject of the first study in the thesis on the elasticity of a elastomeric gluten-mimetic polypeptide with an intrinsically disordered character. This disordered polypeptide was shown to exhibit distinctively variable elastic response to a wide range of tensions, which a classical worm-like chain model failed to accurately describe, thus requiring a molecular-level analysis. IDPs frequently are frequently involved in protein-protein interactions, the focus of the second study on the propensity of an IDR, the B domain in dynamin-related protein 1 (Dpr1), to self-assemble into dimer structures while remaining disordered in all solution conditions. Despite a hypothesized auto-inhibitory role for this domain in Dpr1 that was assumed to be triggered by an disordered-to-order transition, the B domains in solution showed no tendency to form ordered structures even in the presence of order promoting osmolytes. Instead, self-association in the presence of osmolyte was found to occur by favorable intermolecular intereactions between specific region on the surface of the B-domains. Other IDPs do undergo a disorder-to-order transition in response to environmental cues, in ways that are unique disordered proteins, the focus of the last study on intermolecular ordering transitions in silk-like proteins. Factors such as protein sequence and physical tension were investigated, and results suggested that tyrosine residues in the key silk sequence motifs promote templating of beta structure from disordered precursors and that elongational stresses preferentialy stabilize antiparallel beta-sheet order. Together, these three computational studies provide insight into the nature of the structure-function mechanisms of IDPs.

La cryptogéine, une protéine sécrétée par l'oomycète Phytophthora cryptogea, provoque la mise en place de mécanismes de défense chez le tabac, mobilisant au cours des étapes précoces de la signalisation associée, des protéines localisées dans la membrane plasmique (MP). Une fraction membranaire résistante à la solubilisation par les détergents (DIM pour Detergent Insoluble Membrane), enrichie en stérols et en sphingolipides avait été purifiée à partir de la MP de tabac : cette fraction contenait plusieurs protéines impliquées dans la cascade de signalisation induite par la cryptogéine. Chez l'animal, l'association dynamique de protéines à des microdomaines riches en stérols et sphingolipides en réponse à un stress biotique joue un rôle essentiel dans la régulation de la signalisation cellulaire. L'objectif de ce travail était de tester l'hypothèse qu'un tel phénomène puisse se produire dans notre modèle d'étude. La comparaison du protéome de fractions DIMs, purifiées à partir de cellules traitées ou non pendant 5 minutes à la cryptogéine a été réalisée à l'aide d'un marquage isotopique (15N ou 14N) et d'une approche de protéomique quantitative. Le premier résultat est que l'abondance de la majorité des protéines n'est pas modifiée dans les DIMs en réponse à la cryptogéine. Une seule protéine est enrichie dans les DIMs, une isoforme de 14-3-3, tandis que quatre dynamines (DRPs pour Dynamin Related Proteins), impliquées dans le trafic vésiculaire, sont exclues des DIMs en réponse à la cryptogéine. L'étude d'une des dynamines identifiées, DRP1A, a été menée. Nous avons caractérisé les différents gènes codant DRP1A dans le génome du tabac, puis utilisé une approche ARN antisens pour altérer l'expression de cette protéine et nous avons étudié sa localisation subcellulaire à l'aide d'anticorps spécifiques et en observant en microscopie confocale cette protéine fusionnée à la GFP. Cette approche a permis de confirmer la présence de DRP1A dans la fraction DIMs et la diminution transitoire de son association à cette fraction en réponse à la cryptogéine, suite à une dissociation de la fraction membranaire. Ces travaux constituent la première mise en évidence d'une association/dissociation dynamique de protéines aux DIMs de plantes en réponse à un stimulus biologique

Self-incompatibility is a genetic barrier by which a plant recognizes and rejects its own pollen while allowing pollen from more distantly related plants to germinate. In the Brassicacea family, it is controlled by a highly polymorphic locus called the S-locus, which contains the male and female determinants of self-incompatibility. The stigma expresses the female determinant of self-incompatibility, the plant receptor kinase (PRK) S-LOCUS RECEPTOR KINASE (SRK). In Brassica oleracea, SRK has a unique subcellular localization among PRK: the receptor is mostly localized in endosomes and to a lesser extent at the plasma membrane.We investigated the function of the endosomal localization of SRK in Arabidopsis thaliana. Firstly, we reintroduced self-incompatibility in Arabidopsis thaliana by expression of a functional SRK allele from Arabidopsis lyrata (a self-incompatible species). Secondly, we showed that a loss-of-function mutant of DYNAMIN-RELATED PROTEIN1A, a protein required for endocytosis, abolished self-incompatibility. Our results suggest that endocytosis is required for self-incompatibility, and that SRK may be signaling from endosomal compartments.

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