The cytoskeleton is an intrinsic aspect of all cells, and is essential for many cellular events including cell motility, endocytosis, cell division and wound healing. Remodeling of the cytoskeleton in response to these cellular activities leads to significant alterations in the morphology of the cell. One such alteration is the formation of an actomyosin contractile array required for cytokinesis, wound healing and embryonic development. / Cellular structure and shape depends upon tensional prestress brought about by the organization of cytoskeletal components. Using the Xenopus laevis oocyte wound healing model, it is first described how diminished cellular tension affects the balance of the Rho family of GTPases, and subsequently prevents the formation of actomyosin contractile arrays. This suggests that cellular tension in the cell is not created at the level of the cytoskeletal elements but rather via the upstream signaling molecules: RhoA and Cdc42. / The role of N-WASP (Neural-Wiscott Aldrich Syndrome Protein), a mediator of Arp2/3 based actin polymerization, is next examined for its putative role in cellular wound healing. Xenopus laevis oocytes injected with mutant N-WASP constructs reveals in vivo evidence that functional N-WASP is required for appropriate contractile array formation and wound closure. / Lastly, it is revealed that the cellular structures involved with single cell wound healing in other model systems are also important for the initial repair of severed muscle cells. Actin, non-muscle myosin-II, microtubules, sarcomeric myosin and Cdc42 are all recruited and reorganized at the edge of damaged C2C12 myotubes. This data promotes the possibility that an actomyosin array may be established in injured muscle cells as well.

Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed ). Includes bibliographical references.

In response to external stimuli, many intracellular signaling proteins undergo dynamic changes in localization to the plasma membrane. Using the Saccharomyces cerevisiaemating pathway as a model, I investigated the molecular interactions that govern plasma membrane localization of signaling proteins, and how the plasma membrane compartmentalization of a signaling complex influences the overall signaling behavior of the pathway. Signaling proteins often consist of multiple interaction domains that collectively dictate their localization and function. Ste20 is a p21-activated kinase (PAK) that functions downstream of the Rho-type GTPase Cdc42 to activate several mitogen-activated protein (MAP) kinase pathways in budding yeast, including the mating pathway. I identified a short domain in Ste20 that directly binds to membrane lipids via electrostatic interaction. A mutation in this domain abolishes both the localization and function of Ste20. Thus, the previously known Cdc42 binding is necessary but not sufficient; instead, direct membrane binding by Ste20 is also critical. By replacing this domain with heterologous membranebinding domains, I demonstrated that phospholipid specificity is not essential in vivo. Functionally important short membrane-binding domains were also found in the Cdc42 effectors Gic1 and Gic2, indicating that generic membrane binding can work in concert with the CRIB domain to regulate activation of Cdc42 targets. These results underscore the importance of cooperation between protein-protein and protein-membrane interaction in achieving proper localization of signaling proteins at the cell cortex. At the system level, MAP kinase cascades can be graded or switch-like. The budding yeast mating pathway exhibits a graded response to increasing levels of pheromone. Previously the scaffold protein Ste5 was hypothesized to contribute to this graded response. To test this idea, I activated the pathway in a variety of ways and measured the response at the single cell level. I found that the graded response is not perturbed by the deletion of negative regulators of the pathway whereas the response became switch-like when the pathway was activated by a crosstalk stimulus that bypasses the upstream components. Interestingly, activation of the pathway in the cytoplasm using the graded expression of MAPKKK resulted in an ultrasensitive response. In contrast, activation of the pathway at the plasma membrane using the graded expression of membranetargeted active pathway components remained graded. In these settings, the scaffold protein Ste5 increased ultrasensitivity when limited to the cytosol; however, if Ste5 was allowed to function at the plasma membrane, signaling was graded. The results suggest that, in the mating pathway, the inherently ultrasensitive MAPK cascade is converted to a graded system by the scaffoldmediated assembly of signaling complexes at the plasma membrane. Therefore, the plasma membrane localization of Ste5 helps shape the input-output properties of the mating MAPK pathway in a manner that is suitable for the biology of mating. Taken together, this thesis underscores the importance of plasma membrane localization during mating pathway signaling in yeast. The examples described here provide further appreciation of how multiple interaction domains can function together to achieve specific targeting of the signaling proteins, as well as advances in understanding the role of scaffold proteins in modulating signaling behavior to promote graded signaling at the plasma membrane.

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Les petites protéines G de la famille Rho sont des régulateurs importants de la fonction cellulaire. Elles lient les signaux extracellulaires à l'activation de diverses voies de signalisation telles que celles menant à la phagocytose, la mitogénèse, l'adhésion cellulaire, l'expression génique, Toutefois leur fonction principale est l'assemblage et l'organisation du cytosquelette d'actine. Ces GTPases fonctionnent comme des interrupteurs moléculaires, actifs lorsque liés au GTP et inactifs sous la forme liée au GDP. <p><p>Le but de notre thèse est d'investiguer, dans les cellules thyroïdiennes de chien en culture primaire, l'implication des protéines de la famille Rho et de l'organisation du cytosquelette d'actine dans les actions diverses que la TSH exerce, via l'AMPc, sur la morphologie, la prolifération, la différenciation et la fonction des thyrocytes de chien en culture primaire.<p><p>Trois cascades conduisant à la mitogénèse coexistent dans la cellule thyroïdienne de chien: la voie de l'AMPc stimulée par la TSH ou la forskoline (activateur direct de l'adénylate cyclase), la voie des facteurs de croissance (tels que l'EGF, l'HGF) activant leur récepteur à activité tyrosine kinase et la cascade dépendante de la protéine kinase C activée par les esters de phorbol (TPA). Contrairement aux voies indépendantes de l'AMPc qui répriment l'expression des caractéristiques de différenciation, la cascade de l'AMPc stimule à la fois la prolifération, l'expression des gènes de l'état différencié et la fonction (iodation, formation d'H2O2, sécrétion hormonale).<p><p>Dans la cellule thyroïdienne de chien, les agents activant les cascades dépendantes et indépendantes de l'AMPc ont des effets différents sur l'organisation du cytosquelette d'actine. La TSH/AMPc et le TPA induisent une destruction des microfilaments d'actine et un "ruffling" membranaire, tandis que les autres agents (insuline, EGF, HGF, sérum) ne modifient pas le réseau de fibres d'actine (fibres de stress) présent dans les cellules quiescentes.<p><p>Parmi les protéines de la famille Rho, RhoA, Rac1 et Cdc42 sont les premières à avoir été identifiées et sont actuellement les mieux caractérisées. Nous montrons que la TSH, via l'AMPc, induit une diminution de la concentration de la forme active des protéines Rac1, Cdc42 et RhoA. En revanche, les autres agents mitogènes, tels que l'EGF et le TPA, qui activent des voies indépendantes de l'AMPc, n'affectent pas les taux de Rac1 et Cdc42 activés, mais augmentent le taux de RhoA-GTP. L'activation ou l'inactivation des protéines RhoA, Rac1 et Cdc42 est donc un nouvel élément distinguant les voies dépendantes et indépendantes de l'AMPc.<p><p>Grâce à deux toxines bactériennes, la toxine B qui inactive les protéines Rho et la toxine CNF1 qui au contraire les active, nous montrons que, dans les thyrocytes, celles-ci jouent un rôle critique dans l'organisation du cytosquelette, dans la transition G1-S, dans l'expression des gènes de différenciation Tg, ThOXs, NIS et TPO, mais pas dans la génération d'H2O2. <p><p>En effet, l'activité d'un ou plusieurs membres de cette famille est nécessaire à l'entrée des thyrocytes en phase S et à la phosphorylation de la protéine pRb, étape pré-requise à la transition G1-S. L'activation de ces protéines n'induit cependant pas, à elle seule, la prolifération. Nous mettons également en évidence l'existence d'un nouveau mécanisme par lequel ces protéines contrôleraient l'activité des complexes cycline D3-CDK4 indépendamment de leur assemblage. Par l'utilisation de la dihydrocytochalasine B, qui comme la toxine B via l'inactivation des Rhos, désorganise le cytosquelette, nous démontrons que l'intégrité de celui-ci n'est pas requise pour la progression des thyrocytes en phases G1 et S. L'inactivation des protéines Rho est par contre nécessaire à l'induction, par l'AMPc, de l'expression des gènes de différenciation incluant Tg, ThOXs, NIS et TPO, puisque ce processus est inhibé par la toxine CNF1. De plus, l'inactivation des Rhos par la toxine B, ainsi que le désassemblage des fibres de stress et du cytosquelette induit par la dihydrocytochalasine B, suffisent à imiter l'induction dépendante de l'AMPc de Tg et ThOXs, mais pas de NIS et TPO. La toxine B et la dihydrocytochalasine B imitent aussi l’effet de la voie TSH/AMPc sur l’accumulation de p27kip1. Enfin, nous montrons que l'augmentation de la production d'H2O2, nécessaire à la synthèse des hormones thyroïdiennes, ne requiert pas l'activité de la protéine Rac (ni des autres protéines de la famille Rho) alors que celle-ci joue un rôle déterminant dans la génération d'H2O2 dans le leucocyte. <p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

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Indiana University-Purdue University Indianapolis (IUPUI) / Recent preclinical reports have provided evidence that endothelial colony forming cells (ECFCs), a subset of endothelial progenitor cells, significantly improve vessel formation, largely due to their robust vasculogenic potential. While it has been known that the Rho family GTPase Cdc42 is involved in this ECFC-driven vessel formation process, the effect of extracellular matrix (ECM) stiffness on its activity during vessel formation is largely unknown. Using a fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor, we examined the spatio-temporal activity of Cdc42 of ECFCs in three-dimensional (3D) collagen matrices with varying stiffness. The result revealed that ECFCs exhibited an increase in Cdc42 activity in a soft (150 Pa) matrix, while they were much less responsive in a rigid (1 kPa) matrix. In both soft and rigid matrices, Cdc42 was highly activated near vacuoles. However, its activity is higher in a soft matrix than that in a rigid matrix. The observed Cdc42 activity was closely associated with vacuole formation. Soft matrices induced higher Cdc42 activity and faster vacuole formation than rigid matrices. However, vacuole area is not dependent on the stiffness of matrices. Time courses of Cdc42 activity and vacuole formation data revealed that Cdc42 activity proceeds vacuole formation. Collectively, these results suggest that matrix stiffness is critical in regulating Cdc42 activity in ECFCs and its activation is an important step in early vacuole formation.