The Rho family small GTPases are key regulators of the cytoskeleton, through which they impact and control many vital cellular functions, including growth, vesicle trafficking, intercellular junctions, transepithelial transport, migration, and gene transcription. Activation of Rho GTPases is induced by Guanine Nucleotide Exchange Factors (GEFs). We have previously shown that Tumour Necrosis Factor-α (TNF), plasma membrane depolarization, and immunosuppressive drugs activate RhoA through a specific exchange factor, GEF-H1. However, the question of whether other stimuli, such as hyperosmolarity, that activate RhoA, act through GEF-H1 and whether GEF-H1 activates other RhoGTPases was not known. The overall objective of this research project has been to gain insights into the complex mechanism through which the Rho GTPases, Rac and RhoA, are regulated in tubular cells. Specifically, we wished to explore the role and pathway-specific regulation of GEF-H1 in hyperosmotic stress- and TNF-induced signalling in tubular cells. In order to accomplish our goals, we optimized and used affinity precipitation assays to detect GEF-H1 activation (RhoA(G17A) and Rac(G15A)). We found that 1) GEF-H1 is activated by hyperosmotic stress and mediates the hyperosmolarity-induced RhoA activation, as well as nuclear translocation of the Myocardin-Related Transcription Factor (MRTF); 2) TNF induces activation of both Rac and RhoA through GEF-H1, but via different mechanisms. Epidermal Growth Factor Receptor (EGFR)- and Extracellular signal Regulated Kinase (ERK)-dependent phosphorylation at the Thr678 site of GEF-H1 is a prerequisite for RhoA activation only, while both Rac and RhoA activation require GEF-H1 phosphorylation on Ser885. Interestingly, Rac is required for TNF-induced RhoA activation. Together these findings highlight a role for GEF-H1 as an osmosensitive molecule that regulates cellular reprogramming through MRTF. Importantly, we have also uncovered a novel mechanism explaining hierarchical activation of Rac and RhoA by TNF. Such a mechanism could be key in coordinating GEF function and fine-tuning Rac and RhoA activation.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [90]-109).

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease of unknown origin, which causes 80,000 deaths every year in the US and Europe combined. Unknown etiology and late diagnosis, combined with limited treatment options, contribute to a dismal survival rate of 3-5 years post diagnosis. Although molecular mechanisms underlying IPF pathogenesis and progression have been studied for over two decades, lack of in vivo models that recapitulate chronic, progressive, and irreversible nature of IPF have contributed to limited therapeutic success in clinical trials. Currently, only two drugs, Pirfenidone and Nintedanib, are approved for IPF treatment in the US, with their efficacy yet to be completely determined. Patients with IPF often observe lung infections, alveolar collapse, and respiratory failure, which are associated with focal edema and local hypoxia and contribute to development of hypoxemia associated with acute exacerbation of IPF (AE-IPF). In my thesis, I posit that hypoxic injury to the lung epithelium can initiate profibrotic signaling that can contribute to pathogenesis and progression of pulmonary fibrosis in vitro and in vivo. In my in silico studies, I analyzed human protein kinases to identify structural peculiarities that diversify their functions and highlight central hub kinases governing cell signaling. Using this approach, I identified Focal Adhesion Kinase 1 (FAK1) as a central hub kinase contributing to cytoskeletal remodeling. My proteomics and transcriptional studies defined in vitro effect of hypoxia in activation of lung epithelial cells. Using systems biology approaches, I identified interplay between transforming growth factor – β (TGF–β) signaling, hypoxia signaling, and FAK1 signaling. Further, my studies identified Galectin-1 as a novel mediator of hypoxia-induced pulmonary fibrosis. To mimic exacerbation of PF in patients, I developed a novel mouse model of exacerbated pulmonary fibrosis using subclinical bleomycin injury with chronic hypoxia. Further, to fill the existing requirement of an in vivo model of chronic PF, I characterized a triple transgenic mouse model that conditionally activates hypoxia signaling in the lung epithelial cells and causes progressive PF over a span of 12 weeks. Lastly, I performed RNA-Seq experiments on primary AEC2s isolated from our transgenic mouse model to identify a hypoxia-mediated profibrotic role of microRNA-96 in down-regulation of PTEN, a tumor suppressor and anti-fibrotic protein. In conclusion, my studies established in vitro and in vivo roles of hypoxia in profibrotic activation of lung epithelium and identifies FAK1 and Gal-1 as key drivers of hypoxia-mediated fibrosis, which should be further evaluated in animal and human studies to determine their therapeutic potential.

Orientadores: Antonio Carlos Boschiero, Everardo Magalhães Carneiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:06:06Z (GMT). No. of bitstreams: 1 Ferreira_SandraMara_M.pdf: 1558318 bytes, checksum: d69677a862aa6ba8e96725b7d5c76a87 (MD5) Previous issue date: 2011 / Resumo: Introdução e objetivos: A ARHGAP21 é uma Rho-GAP que promove a ativação de um fator intrínseco da Rho-GTPase Cdc42 responsável pela hidrólise de GTP à GDP e inativação da atividade da proteína. ARHGAP21 se associa à PKC? em cardiomiócitos e à porção C-terminal da FAK em glioblastomas, onde inibe a ativação de Rho-GTPases, e, com isso, o rearranjo do citoesqueleto de actina. A Cdc42, FAK e PKC? estão envolvidas na secreção de insulina estimula por glicose, diferenciação e proliferação de ilhotas pancreáticas. O objetivo deste trabalho foi investigar em células beta pancreáticas: 1) a expressão e localização da ARHGAP21; 2) o efeito da glicose na expressão das proteínas PKC?, FAK e Cdc42 e sua associação à ARHGAP21 e; 3) a função da ARHGAP21 no controle da secreção de insulina estimulada por glicose. Materiais e Métodos: A expressão e localização da ARHGAP21 em células MIN6 foram avaliadas por imunoflorescência. Células MIN6 foram tratadas na presença de 5,6 ou 22 mM de glicose ou, na ausência ou presença de insulina (0,2 U/ml) por 3 dias. Após a extração protéica a expressão das proteínas ARHGAP21, PKC, FAK e Cdc42 foi avaliada por Western blot. Células MIN6 foram incubadas em solução contendo 22 mM de glicose e coletadas em diferentes tempos (0, 5, 15, e 30 min) para avaliação da interação ARHGAP21/FAK e ARHGAP21/PKC? por imunoprecipitação e, para análise do grau de fosforilação tirosina 397 e 925 da FAK e em treonina 410 da PKC?. A expressão dessas proteínas também foi avaliada em ilhotas pancreáticas de camundongos Swiss neonatos previamente tratados por dois dias com 1 nM de anti-sense anti-ARHGAP21 ou mismatch. Essas ilhotas foram incubadas com 2,8 ou 16,7 mM de glicose e a secreção de insulina avaliada. Resultados: A ARHGAP21 localiza-se no citoplasma, mais acentuadamente na região da membrana plasmática. Glicose reduziu a expressão da ARHGAP21 e PKC? e não alterou a expressão da FAK e Cdc42, enquanto a insulina não alterou a expressão de nenhuma das proteínas estudadas. A glicose também promoveu a dissociação ARHGAP21/FAK, levando ao aumento na fosforilação da FAK seguido de aumento na associação ARHGAP21/PKC? e redução na fosforilação da PKC? em Thr 410. Animais Knockdown para ARHGAP21 apresentaram menor expressão de PKC? e maior expressão de Cdc42, além de um aumento na secreção de insulina por ilhotas pancreáticas tanto em condição sub- (2,8 mM) quanto supra-estimulatória (16,7 mM) de glicose. Conclusão: ARHGAP21 modula Cdc42 e FAK, proteínas responsáveis pelo rearranjo do citoesqueleto de actina e extrusão do grânulo de insulina, reduzindo sua expressão e atividade, o que inibe a secreção de insulina. Além disso, observamos que a glicose modula as interações ARHGAP21/FAK e ARHGAP21/PKC? / Abstract: Background/Aims: ARHGAP21 is a Rho-GAP that promotes activation of the Rho-GTPase Cdc42 intrinsic factor, which is responsible for the hydrolysis of GTP to GDP and those proteins inactivation. ARHGAP21 associates with PKC? in cardiomyocytes and to the C-terminal portion of FAK in glioblastoma, inhibiting Rho-GTPases and actin cytoskeleton rearrangement. Cdc42, FAK and PKC? promote glucose-stimulated insulin secretion, differentiation and proliferation in pancreatic islets. The aim this study was to assess in pancreatic beta cells: 1) ARHGAP21 expression and localization; 2) glucose effect in PKC?, FAK e Cdc42 expression and association to ARHGAP21; 3) The role of ARHGAP21 on glucose-stimulated insulin secretion. Materials and Methods: ARHGAP21 expression and localization in MIN6 cells were evaluated by immunofluorescence. MIN6 cells were treated with glucose (5.6 mM and 22 mM) or insulin (0.2 U/ml) for 3 days. After protein extraction, the ARHGAP21, PKC?, FAK e Cdc42 expressions were evaluated by Western blot. MIN6 cells were exposed for 0, 5, 15, e 30 min to 22 mM of glucose. ARHGAP21/FAK and ARHGAP21/PKC? interactions were evaluated for immunopreciption and phosphorylation of FAK in tyrosine 397 and 925 and phosphorylation of PKC? in threonine 410. The expression of these proteins also was evaluated in pancreatic islets of neonates Swiss mice previously treated for 2 days with 1 nM of ARHGAP21 antisense or mismatch oligonucleotides. These islets were incubated with 2.8 mM or 16.7 mM of glucose and insulin secretion was evaluated. Results: ARHGAP21 is localized in the cytoplasm, mainly the plasmatic membrane. Glucose reduced ARHGAP21 and PKC? expression but not altered FAK and Cdc42 expression, while insulin had no effect whatsoever on the expression of the studied proteins. Glucose also dissociated ARHGAP21/FAK, leading to increased FAK phosphorylation, which was followed by ARHGAP21/PKC? association and reduced Thr410 PKC? phosphorylation. ARHGAP21 Knockdown mice pancreatic islets had lower PKC? and higher Cdc42 expression, and also increased insulin secretion in both sub- (2.8 mM) and supra-stimulatory (16.7 mM) of glucose conditions. Conclusions: ARHGAP21 modulates Cdc42 and FAK, proteins responsible for rearrangement of actin cytoskeleton and extrusion of insulin vesicles, and reducing their expression leads to inhibited insulin secretion. Furthermore, we observed that glucose modulates ARHGAP21/FAK and ARHGAP21/ PKC? interactions / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

During embryonal development, axons of newly differentiated neurons need to properly interconnect and create a functional neuronal network. To achieve this, the cell requires a growth cone. The growth cone is a highly dynamic structure at the end of growing axons that serves both as the navigator and the propeller. Crosstalk between actin and microtubules is vital for proper axonal pathfinding. But the exact mechanism of this cooperation remains unknown. This diploma thesis investigates the possible role of a candidate scaffolding protein called anillin in this process. Anillin has been studied in two human cell lines. SH-SY5Y neuroblastoma cell line was used for overexpression and siRNA knock-down experiments. Anillin overexpression led to perturbed neurite morphology and growth cone dynamics in SH-SY5Y cells, whereas cells with lower anillin expression had fewer neurites. Next, neurons differentiated from human iPSC (induced pluripotent stem cells) expressing endogenous fluorescently tagged anillin were studied. Local dynamic high concentration spots of anillin have been observed at the base of cell protrusions of differentiating neurons. These anillin flares appeared during cell migration, early neurite initiation, and in newly created growth cones. These results suggest that anillin plays a...

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Using a forward genetic screen in Drosophila, we identified that loss of the Toll receptor adaptor dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway. This death signaling pathway can be activated without injury by loss of the N-terminal self-inhibitory domain, resulting in spontaneous neurodegeneration. To investigate the role of axon self-destruction in disease, we assessed the effects of Sarm1 loss on neurodegeneration in the SOD1-G93A model of amyotrophic lateral sclerosis (ALS), a lethal condition resulting in progressive motor neuron death and paralysis. Loss of Sarm1 potently protects motor axons and synapses from degeneration, but only extends animal survival by 10%. Thus, there appears to be at least two driving forces in place during ALS disease progression: (1) Sarm1 mediated axon death, and (2) cell body destruction via some unknown mechanism.

The fission yeast Schizosaccharomyces pombe has become a powerful model system for studying cytokinesis, a process of cytoplasmic division by which one cell divides into two identical daughter cells. Like mammalian cells, S. pombe divides through the use of an actomyosin contractile ring, which is composed of a set of highly conserved cytoskeletal proteins. Cytokinesis in S. pombe is primarily regulated by the SIN pathway, which is activated in late mitosis and is required for actomyosin contractile ring and septum assembly, and also plays a role in spindle checkpoint inactivation, and telophase nuclear positioning. The various functions of the SIN are carried out by the terminal kinase in the pathway called Sid2. The lack of information in the downstream targets of Sid2 has limited our understanding of the different functions of the SIN. We recently showed that, in addition to its other functions, the SIN promotes cytokinesis through inhibition the MOR signaling pathway, which normally drives cell separation and initiation of polarized growth following completion of cytokinesis (Ray et al, 2010). The molecular details of this inhibition and the physiological significance of inhibiting MOR during cytokinesis was unclear. The results presented in Chapter II describe our approach to identify Sid2 substrates, particularly focusing on Nak1 and Sog2 that function in the MOR signaling cascade. We identified and characterized Sid2 phosphorylation sites on the Nak1 and Sog2 proteins. Chapter III explores how post translational modification of MOR proteins by Sid2 regulates polarized growth during cytokinesis. This includes delineating the effect of Sid2 mediated phosphorylation of Nak1 and Sog2 on protein-protein interactions in the MOR pathway as well as on the regulation of their localization during late mitosis. Finally, results in Chapter IV demonstrate that failure to inhibit MOR signaling is lethal because cells initiate septum degradation/cell separation before completing cytokinesis thereby emphasizing the importance of cross-regulation between the two pathways to prevent initiation of the interphase polarity program during cytokinesis.

La cytokinèse est un processus minutieusement régulé par une structure corticale appelée l'anneau contractile d'actomyosine, sous le contrôle de la petite GTPase RhoA. La protéine d'échafaudage Anilline est un effecteur de RhoA et organise les différents éléments de l'anneau permettant sa fermeture. Via son N-terminus, l'Anilline interagit avec la Citron kinase Sticky, la myosine II et l'actine-F ce qui permet la stabilisation de l'anneau contractile et sa maturation en anneau du corps central ou «midbody». Via son domaine C-terminal, l'Anilline interagit avec la RhoA-GTP et les septines pour ancrer l'anneau à la membrane. De précédentes études du laboratoire ont montré que Sticky et les septines ont des actions opposées sur l'Anilline. Nous avons donc défini plusieurs ensembles d'interactions entre l'Anilline et les cytosquelettes d'actomyosine et de septines, qui possèdent des fonctions différentes voire opposées dans la cytokinèse: ce sont des réseaux cytosquelettiques. L'Anilline est ainsi impliquée dans la coordination de ces réseaux opposés et RhoA-dépendants. Nous devons encore déterminer si ces interactions de l'Anilline en N-ter et C-ter peuvent se produire en même temps ou si elles sont mutuellement exclusives pour coordonner les différents éléments cytosquelettiques pendant la fermeture de l'anneau contractile. Les cellules S2 de drosophile ont été utilisées pour déterminer comment l'Anilline coordonnait les réseaux cytosqulettiques de l'anneau contractile. Deux modèles ont été proposés : l'un où une seule molécule d'Anilline se lie simultanément aux réseaux en N-ter et C-ter; l'autre modèle suggère qu'une ou plusieurs populations d'Anilline coordonnent les réseaux de façon mutuellement exclusive. Pour distinguer entre ces deux modèles, des allèles de séparation de fonction de l'Anilline ont été testés : l'AnillinΔ1-5 qui n'interagit plus avec Sticky, ainsi que l'AnillinRBD* qui n'interagit plus avec Rho1-GTP et qui ne recrute plus les septines à l'anneau contractile. Des expériences de sauvetage suite à la déplétion de l'Anilline endogène ont été réalisées et les tentatives de division ont été captées par microscopie en temps réel. L'expression de chaque mutant individuellement menait à une fermeture de l’anneau décalée, ralentie et souvent incomplète. En revanche, la co-expression de l'AnillinΔ1-5 et AnillinRBD* en trans dans les mêmes cellules a restauré la cinétique normale de la fermeture de l'anneau. Ce résultat supporte le modèle des populations multiples d'Anilline. Cette étude avance significativement nos connaissances de l'organisation de l'anneau contractile qui gère la division de toutes cellules animales. / Cytokinesis is a process thoroughly regulated by a cortical structure called the actomyosine contractile ring, under the control of the RhoA GTPase. The scaffolding protein Anillin is a RhoA effector organizing the several elements of the ring, thus permitting its closure. The AnillinN-terminus interacts with the Citron kinase Sticky, Myosin II and F-actin to stabilize the contractile ring and drive its maturation to the midbody ring. The AnillinC-terminus interacts with the RhoA GTPase and the septins to anchor the ring to the membrane. Previous works revealed that Sticky retains Anillin while the septins shed Anillin from the ring. Therefore, Anillin is involved in opposed RhoA-dependent cytoskeletal sub-networks to generate or reduce the tension at the membrane, and their balance is necessary to improve the ring closure. This study aims to decipher the coordination between these opposed sub-networks. We proposed two models : either sub-networks on AnillinN-ter and AnillinC-ter are simultaneously organized by the same molecule of Anillin, or several pools of Anillin coordinate separately the opposed subnetworks. We generated and expressed several inducible Anillin mutants in drosophila S2 cells : AnillinΔ1-5 prevents the interaction with Sticky; AnillinRBD* does not interact with RhoA and perturbs the Anillo-septin assembly. The expression of each mutant individually delayed, slowed down and failed the ring closure. However, co-expression of single mutants in trans rescued the ring closure. Moreover, Sticky over-expression improved AnillinRBD* recruitment in the ring. These results support the multiple pools of Anillin model. This study improves our knowledge on the contractile ring organization, necessary to succeed cytokinesis in animal cells.

Cell migration and invasion play a role in many physiological and pathological processes and are therefore subject of intensive research efforts. Despite of the intensively investigated biochemical processes associated with the migration and invasion of cells, such as cancer cells, the contribution ofmechanobiological processes to themigratory capacity of cells as well as the role of physical polymeric phase transitions is not yet clearly understood. Unfortunately, these experiments are not very informative because they completely disregard the influence of the three-dimensional cell environment. Despite this data situation, it was possible to adequately demonstrate that there exists a direct mechanical interplay between cells and theirmicroenvironment in both directions, where both elements can bemechanically altered by one another. In line with these results, it has turned out that the mechanobiological molecular processes through which cells interact with each other and additionally sense their nearby microenvironment have an impact on cellular functions such as cellular motility. The mechanotransduction processes have become the major focus of biophysical research and thereby, diverse biophysical approaches have been developed and improved to analyze the mechanical properties of individual cells and extracellular matrix environments. Both, the cell mechanics and matrix environmentmechanics regulate the cellmigration types in confined microenvironments and hence it seems to be suitable to identify and subsequently present a common bidirectional interplay between cells and their matrix environment. Moreover, hallmarks of the mechanophenotype of invasive cells and extracellular matrices can be defined. This review will point out how on the one hand the intracellular cytoskeletal architecture and on the other hand the matrix architecture contribute to cellular stiffness or contractility and thereby determines the migratory phenotype and subsequently the emergence of a distinct migration mode. Finally, in this review it is discussed whether universal hallmarks of the migratory phenotype can be defined.

Anandamide (NAE 20:4) or arachidonlyethanolamine (AEA) is the most widely studied N-acylethanolamine (NAE) because it mediates several physiological functions in mammals. In vascular plants, 12-18C NAEs inhibit growth in an abscisic acid (ABA)-dependent and -independent manner. Anandamide, which is unique to bryophyte Physcomitrella patens, inhibited gametophyte growth and reduced chlorophyll content when applied exogenously. It is hypothesized that anandamide mediates its responses through morphological and cellular changes. Following growth inhibition by short-term anandamide-treatment, microscopic analyses revealed relocated chloroplasts and depolymerized F-actin in protonemal tips. Long-term treatment showed partially bleached gametophyte cells with degraded and browning chloroplasts. These anandamide-mediated responses have physiological implications as AEA may function as a signal for gametophytes to activate secondary dormancy as seen with ABA. Future studies will investigate the role of AEA in mediating stress responses and possible interaction with ABA.