Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

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Cytokinesis is the physical partition of one cell into two. In Chapter 1, I provide a brief introduction to cytokinesis and some of the proteins whose functions I parse out throughout my studies. In Chapter 2, I present work I’ve contributed to elucidate the role of polarity proteins in cytokinesis, as well as a look at the differential requirement for canonically essential cytokinetic proteins in the 4-cell embryo. In Chapter 3, I address a long-standing controversy in the field regarding the relationship between the Rac GAP protein Cyk-4 and the small GTPase Rac, and in particular the inhibitory role of Rac during cell division. My major body of work highlights the necessity not to close the books on the GAP activity of Cyk-4 and its inhibition of Rac. I show that Rac is unable to rescue cytokinesis failure in downstream Rho effectors whose loss weakens the contractile ring, suggesting it is not a promiscuous suppressor of cytokinesis. Additionally, I found that levels of non-muscle myosin-II and the actin binding domain of Utrophin were unchanged with loss of Cyk-4. From this, I infer that Cyk-4 is unlikely to be an activator of the RhoGEF Ect-2. These results emphasize the need to probe further into the cross-talk between these GTPases. In chapter 4, I show inconclusive data addressing the role of cell fate signaling in protection against cytokinesis failure. Overall, this thesis represents my contributions to the field, revealing the complexity involved in assuring successful completion of cytokinesis.

Les mégacaryocytes (MK) sont des cellules géantes de la moelle osseuse, précurseurs des plaquettes sanguines. Leur différenciation se caractérise par un accroissement de la quantité de protéines et de membranes pour permettre l'élongation de prolongements cytoplasmiques, les proplaquettes (PPT), qui vont traverser l'endothélium médullaire pour libérer les plaquettes dans le sang. Il est admis que la tubuline joue un rôle essentiel dans l'élongation des PPT. Par contre, le rôle du cytosquelette d'actine reste encore peu clair et controversé. Dans ce contexte, le but de ma thèse a été d'étudier le rôle de l'actine et de ses régulateurs, les GTPases de la famille Rho et les phosphoinositides, dans la formation des PPT. Dans une première partie, j'ai montré que le cytosquelette d'actine était le principal régulateur de la formation du système de membranes de démarcation (DMS) qui constitue le réservoir de membranes nécessaires à la formation des PPT. Par la combinatoire de modèles murins et humains, j'ai montré que la GTPase Cdc42 et ses effecteurs Pak étaient les acteurs principaux de cette régulation. Mes résultats posent clairement les bases du processus de maturation interne des MK. Ainsi, en réponse à la thrombopoïétine, cytokine clé de la mégacaryopoïèse, le MK possède la propriété intrinsèque de se polariser de façon indépendante des contacts avec la niche hématopoïétique, pour former les territoires du DMS au niveau du futur site d'émission des PPT, et des noyaux. L'imagerie confocale en 3D et en FRET a démontré que la GTPase Cdc42 active était associée aux endomembranes. Logiquement, son inhibition pharmacologique induit une désorganisation majeure du DMS, abolissant totalement la formation des PPT. Pak2 intervient dans ce phénomène en aval de Cdc42 et son inhibition affecte également la production plaquettaire. Ces résultats originaux permettent de mieux comprendre la mécanistique physiologique de maturation des MK pour aboutir à une production plaquettaire normale et mettent en lumière d'éventuelles cibles moléculaires des dysfonctionnements observés dans les thrombopénies. En complément de ce travail, nous avons mis en place une collaboration avec l'équipe du Dr B. Nieswandt (University Clinic of Würzburg, Allemagne) qui vise à comparer l'impact de la délétion spécifique dans la lignée mégacaryocytaire des GTPases Rac1, Cdc42 et RhoA, seules ou en combinaison dans les lignées murines KO. Mes résultats valident le rôle central de Cdc42 et mettent en avant une régulation croisée inattendue entre RhoA et cette dernière. Dans une deuxième partie, j'ai abordé le rôle du phosphatidylinositol 5-phosphate (PtdIns5P), dont l'équipe a montré qu'il activait les GTPases Rac1 et Cdc42, et était responsable de l'invasion lymphomateuse en formant des podosomes invasifs. Les contacts avec la matrice extracellulaire (MEC) sont importants pour la différenciation terminale des MK qui forment des podosomes différents selon le type de matrice (fibrinogène ou collagène). J'ai montré que les MK formaient des podosomes linéaires le long des fibres de collagène alors qu'ils sont punctiformes sur fibrinogène. De façon intéressante, on constate un changement de GTPase selon la matrice. Les podosomes formés sur collagène sont riches en Rac1, alors que sur fibrinogène ils portent Cdc42. Par ailleurs, le PtdIns5P est alors un élément central de la structure des podosomes invasifs sur gel comprenant du fibrinogène. Ainsi, lorsque la matrice est proche de celle de l'environnement vasculaire (fibrinogène), on a apparition d'un phénotype invasif mettant en jeu le PtdIns5P et Cdc42, laissant supposer que la signalisation attenante pourrait réguler les contacts et la traversée endothéliale. L'ensemble des résultats obtenus au cours de ce travail de thèse contribue à une meilleure compréhension de la maturation des mégacaryocytes et de leur relation avec l'environnement médullaire et vasculaire pour aboutir à la production de plaquettes fonctionnelles. / Megakaryocytes (MK) are giant cells located in the bone marrow that produce blood platelets. Their differentiation is characterized by an increase in proteins and membranes that leads to the elongation of cytoplasmic protrusions called proplatelets (PPTs). PPTs will cross the medullar endothelium to release platelets in the blood flow. Tubulin plays a major role in PPT elongation. However, the involvement of the actin cytoskeleton remains unclear and controversial. In this context, the purpose of my thesis was to study the role of actin and its regulators, namely the Rho GTPases and phosphoinositides, in PPTs formation. In a first part, I demonstrated that the F-actin was the main regulator of the formation of the demarcation membranes system (DMS), which represents a membrane reservoir for PPTs. Using complementary mouse and human models, I demonstrated that the Cdc42 GTPase and its effector Pak2 were key regulators of this process. My data set the bases of the internal maturation process. I demonstrated that MKs display the intrinsic property of polarizing in response to thrombopoietin, which is the main cytokine regulating megakaryopoiesis, independently of contacts with the hematopoietic niche. Polarization results in the formation of the DMS territory at the future PPTs emission site, facing the nuclei territory. 3D confocal and FRET imaging demonstrated that active Cdc42 was associated with endomembranes. Interestingly, Cdc42 pharmacological inhibition resulted in total DMS disorganization, which completely abolished PPTs formation. In this phenomenon, Pak2 acted downstream of Cdc42 and its inhibition also abrogated platelets production. These data shed new light on MK physiological maturation that ends in normal platelet production and points to new potential molecular targets that might be dysregulated in thrombocytopenia. We then set up a collaboration with Dr B. Nieswandt's group (University Clinic of Würzburg, Germany) to evaluate the impact of megakaryocytic specific knockout of the Rac1, Cdc42 and RhoA GTPases, alone or in combination, on murine MKs differentiation. My results with these models validate the central role of Cdc42 and demonstrated an unexpected cross-regulation between Cdc42 and RhoA. In a second part, I studied the role of the phosphatidylinositol 5-phosphate (PtdIns5P), which the team demonstrated to activate Rac1 and Cdc42, and identified as a central regulator of lymphoma invasion through elaboration of invasive podosomes. Contacts with the extracellular matrix (ECM) within the bone marrow are instrumental into MKs terminal differentiation. I demonstrated that MKs form linear podosomes along collagen fibers while they form regular dotted structures on fibrinogen. Interestingly, we observed a difference in podosome-associated GTPases according to the matrix. Podosomes formed on collagen were enriched in Rac1, while Cdc42 localized in podosomes when cells were cultivated on fibrinogen. In this case, PtdIns5P that was vesicular in MKs progenitors concentrated in the actin core of those invasive podosomes. Taken together, these data suggest that when the matrix is similar to the vascular environment (fibrinogen), concentration of PtdIns5P and Cdc42 in podosomes might lead to the emergence of the invasive phenotype seen in vitro on fibrinogen-containing gels. An interesting hypothesis regarding the in vivo outcome of this observation is that the PtdIns5P/Cdc42 pathway might be involved in the regulation of MKs contacts with endothelial cells to drive endothelium crossing. Overall, these data shed new lights on MKs maturation and its relationship with medullar and vascular environments that results in production of functional platelets.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Neuropathy is a major complication that affects nearly half of all patients with diabetes, greatly decreasing their quality of life. Patients experience a wide range of symptoms including pain, numbness, weakness and other morbidities. While its pathogenesis has been the focus of extensive research, there are still few effective treatment options available for this disease. The discovery of novel molecular targets underlying this diabetic neuropathy may lead to the development of new, more effective therapeutics. DLC2, a Rho GTPase-activating protein with specific activity for RhoA, was shown to be involved in pain signaling. Mice deficient for this protein (DLC2-/-) have increased RhoA activity in their peripheral nerves, and have heightened pain responses compared to wild type (DLC2+/+) in acute pain tests, displaying increased sensitivity to noxious thermal and inflammatory stimuli. DLC2-/- mice also show elevated blood glucose levels, lower body weight and increased sensitivity to blood glucose compared to wild type. Because of the hyperalgesia to acute pain displayed by DLC2-/- mice compared to wild type, and since the RhoA pathway is known to be involved in the pathogeneses and maintenance of diabetes and its complications, these mice were used to investigate more clinically relevant, chronic pain in a model of diabetic neuropathy. Streptozotocin (STZ), given in multiple low doses over five days (MLDS treatment), was used to induce diabetes in DLC2+/+ and DLC2-/- mice, and their pain responses were tested 8 weeks later. Diabetic DLC2-/- mice (DLC2-/--STZ) were hyperalgesic to thermal stimuli from the hot plate test compared to diabetic DLC2 wild type mice (DLC2+/+-STZ) and vehicle-treated controls of both genotypes (DLC2-/--Veh and DLC2+/+-Veh. Similar responses were seen from the von Frey filament test, where the DLC2-/--STZ group exhibited mechanical allodynia compared to the DLC2+/+-STZ group and both control groups. Dorsal root ganglia (DRG) were dissected from these four groups of mice for qPCR screening and protein analysis. DLC2-/--STZ mice showed significantly higher gene expression of the voltage-gated sodium channel Nav 1.9 compared to DLC2+/+-STZ mice, while there was a strong trend of increased levels in the DLC2-/--STZ group compared to both non-diabetic groups. Western blot analysis of the DRG from these mice shows increased levels of COX-2 expression of DLC2-/--STZ mice compared to DLC2+/+-Veh, and elevated levels of phosphorylated ERK (pERK) in DLC2-/--Veh and both diabetic groups compared to DLC2+/+-Veh. Overall, diabetic DLC2-/- mice have more severe painful diabetic neuropathy, with thermal hyperalgesia and mechanical allodynia. Increased RhoA activity and pERK, which are known to be involved in regulation, transcription and trafficking of sodium channels, may lead to increased Nav1.9 mRNA levels and activation. Localized mainly to nociceptors of the DRG, Nav1.9 is known to play a role in sensitizing neurons through lowering the threshold for action potentials, possibly leading to the observed heightened pain response. Additionally, elevated COX-2 levels in DLC2-/--STZ mice may lead to further deficits through activation of inflammatory responses. Future studies will further investigate how these mechanisms are involved in the altered pain response from diabetes. / published_or_final_version / Anatomy / Master / Master of Philosophy

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Osteoclasts are multinucleated, bone resorbing cells that carry out their function using specialized actin-based structures called actin rings and podosomes. Rho GTPases function as molecular switches that regulate the actin cytoskeleton in osteoclasts and many other cell types. Filamin A (FLNa) and SBDS are two proteins that have the potential to interact with both F-actin and Rho GTPases, and thus regulate osteoclast formation, differentiation, or function. We found that in FLNa-null pre-osteoclasts, activation of RhoA, Rac1, and Cdc42 was perturbed, leading to defective pre-osteoclast migration prior to fusion. Ablation of SBDS resulted in the blockage of osteoclast differentiation downstream of RANK and defective RANKL-mediated upregulation of Rac2 that is required for pre-osteoclast migration. Therefore, both FLNa and SBDS are required to coordinate Rho GTPase activation during osteoclastogenesis, in addition to a role for SBDS in osteoclast differentiation downstream of RANK.

Osteoclasts are multinucleated, bone resorbing cells that carry out their function using specialized actin-based structures called actin rings and podosomes. Rho GTPases function as molecular switches that regulate the actin cytoskeleton in osteoclasts and many other cell types. Filamin A (FLNa) and SBDS are two proteins that have the potential to interact with both F-actin and Rho GTPases, and thus regulate osteoclast formation, differentiation, or function. We found that in FLNa-null pre-osteoclasts, activation of RhoA, Rac1, and Cdc42 was perturbed, leading to defective pre-osteoclast migration prior to fusion. Ablation of SBDS resulted in the blockage of osteoclast differentiation downstream of RANK and defective RANKL-mediated upregulation of Rac2 that is required for pre-osteoclast migration. Therefore, both FLNa and SBDS are required to coordinate Rho GTPase activation during osteoclastogenesis, in addition to a role for SBDS in osteoclast differentiation downstream of RANK.

A família das septinas caracteriza-se pela capacidade de ligar nucleotídeos de guanina e de se associarem formando filamentos. Diversas funções biológicas têm sido reportadas para esses filamentos e sua dissociação pode estar relacionada a patologias. A septina 12 humana expressa especificamente em testículos, foi identificada em filamentos que compõem o annulus do espermatozoide, cuja integridade está relacionada com a morfologia deste. Embora muitos estudos tenham sido reportados, vários aspectos das bases moleculares e fisiológicas de sua função e automontagem permanecem desconhecidos. Neste trabalho, procurou-se obter informações estruturais para o domínio de ligação ao nucleotídeo da SEPT12 (SEPT12G), do mutante SEPT12GT89M e do heterodímero SEPT7-SEPT12. A expressão destas proteínas foi realizada em células de E. coli da linhagem Rosetta(DE3) pelos vetores de expressão pET28a(+) e pETDuet-1. As etapas de purificação foram cromatografia de afinidade e exclusão molecular. A proteína SEPT12G foi submetida à avaliação do estado oligomérico, fluorescência intrínseca, ensaios de conteúdo de nucleotídeo, atividade GTPásica e transição térmica. O heterodímero SEPT7-SEPT12 foi submetido à avaliação do estado oligomérico e conteúdo de nucleotídeo. Ensaios de cristalização foram realizados para todas as proteínas. A coleta de dados realizada na linha I24 do Diamond Light Source (Didcot, Inglaterra) resultou em conjuntos de dados de alta resolução para a SET12G, SEPT12GT89M e baixa resolução para a SEPT7NGc. Os estudos biofísicos mostraram que a SEPT12G foi obtida em sua forma nativa ou, seja, capaz de ligar e hidrolisar o nucleotídeo GTP e que o heterodímero obtido apresentou ambas as proteínas. As estruturas cristalográficas foram resolvidas e permitiram realizar observações importantes para o grupo I das septinas humanas (SEPT3, SEPT9 e SEPT12). Para a SEPT12 pôde-se observar como a mudança que ocorre no motivo G4 pode alterar a estabilidade da interface G. No contexto do grupo I esta estrutura permitiu concluir que todas as proteínas deste subgrupo podem formar duas interfaces NC, dos tipos aberta e fechada. Além disso, reforçou a observação da orientação diferencial da hélice α5\', cuja função ainda não está esclarecida, mas sem dúvidas é um diferencial que caracteriza este grupo, possivelmente relacionado com a ancoragem da região polibásica na conformação aberta. A estrutura cristalográfica do mutante SEPT12T89M permitiu observar que a mutação levou a uma alteração na primeira esfera de coordenação do íon Mg2+, mudança que interrompe o mecanismo do switch universal e deixa a proteína catalíticamente inativa. Por fim, o estudo cristalográfica do complexo entre a SEPT12 e SEPT7 não foi possível, uma vez que todas as tentativas resultaram em cristais contendo apenas a SEPT7, o que pode ser consequência da precipitação da SEPT12 ou da condição de cristalização utilizada, que desestabiliza o heterodímero. / The septin family of proteins is characterized by their ability to bind guanine nucleotides and associate into filaments. Several biological functions have been reported for these filaments and their dissociation may be related to pathologies. Human septin is 12 specifically expressed in testes and has been identified in filaments that form the sperm annulus, whose integrity is related to its morphology. Although many studies have been reported, the molecular and physiological bases of septin filament function and self-assembly have yet to be completely elucidated. This study aims to obtain structural information for the nucleotide binding domain of SEPT12 (SEPT12G), the SEPT12GT89M mutant and the SEPT7-SEPT12 heterodimer. Expression of these proteins was performed in E. coli Rosetta(DE3) strain using the pET28a (+) and pETDuet-1 expression vectors. Purification was performed by affinity and size exclusion chromatography. The SEPT12G protein was submitted to an evaluation of its oligomeric state, intrinsic fluorescence, nucleotide content, GTPase activity and thermal transition. The oligomeric state and nucleotide content of SEPT7-SEPT12 was also evaluated. Crystallization assays were performed for all proteins. Data collection on line I24 of the Diamond Light Source (Didcot, England) resulted in high-resolution data sets for SET12G and SEPT12GT89M but only low resolution data for the SEPT7NGc. Biophysical studies showed that SEPT12G was obtained in its native form or, in other words, capable of binding and hydrolyzing GTP and that the purified heterodimer presented both proteins. The crystallographic structures were solved by molecular replacement allowing the identification of features characteristics of the group I septins (SEPT3, SEPT9 and SEPT12). The structure also confirmed that all the proteins of this group are able to form two different NC interfaces: open and closed. In addition, it reinforced the observation that the α5\' helix assumes a different orientation, whose function has not yet been clarified, but without doubt is a characteristic of this group which may be related to anchoring the polybasic regions whilst in the open conformation. The SEPT12T89M mutant crystal structure shows that the first shell coordination of the Mg2+ ion is altered, leading to an interruption of the universal switch mechanism and a consequent lack of catalytic activity. Finally, structural studies of the interaction between SEPT12 and SEPT7 were not possible, since all attempts resulted in crystals containing only SEPT7. This may be a consequence of SEPT12 precipitation or the crystallization condition used, destabilizing the heterodimeric interface.