This thesis deals with the organisation and function of actin and septins in the model filamentous fungus, Neurospora crassa. Firstly, study demonstrates the utility of the Lifeact peptide probe for the investigation of actin dynamics in N. crassa. Lifeact fused to fluorescent proteins allowed live-cell imaging of actin patches, cables and rings without interfering with cellular functions. Actin cables and patches localised to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organisation was markedly different between the tip regions of mature hyphae and germ tubes. Only mature hyphae displayed a sub-apical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Proper organisation of actin cables required the class-V myosin, MYO-5, and the frequency of rapid transport of actin patches was reduced in its absence, suggesting that MYO-5 participates in actin patch translocation. Deletion of myo-5 caused gross morphological and polarity defects, demonstrating the importance of this motor for normal cell function. GFP-tagged MYO-5 localised as a crescent at germ tube tips and to the core of the Spitzenkörper in mature hyphae. Secondly, analysis of septin null mutants demonstrated that septins limit the emergence of germ tubes and are important for septation and conidiation in N. crassa. Septins showed different patterns of localisation at hyphal tips, with GFP-CDC-10 and CDC- 11-GFP organised as a collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP constituted as a cap at the tip apex and GFP-SPN-1 forming an extended collar. Septins formed a range of different higher-order structures in N. crassa – rings, loops, fibres, bar-like structures, and caps – which can co-exist within the same cell. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of SPN-1 revealed that this septin interacts with the core septin complex.

Septins are a family of GTP-binding proteins implicated in mammalian cell division. Since these proteins form heterologous complexes and filaments in interphase cells, it has been assumed that depletion of any or all septins in a given cell type will give rise to the same phenotype. I demonstrate that while all septins expressed in HeLa cells localize to the cleavage furrow and midbody during cytokinesis, and co-immunoprecipitate throughout cell division, they do not all have identical roles during this process. Specific depletion of SEPT2 or SEPT11 caused defects in the early stages of cytokinesis, ultimately resulting in binucleation. Similar results were observed upon simultaneous depletion of all septins. In sharp contrast, SEPT9 was dispensable for the early stages of cell division, but was critical for the final separation of daughter cells. I demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody. Immunofluorescence microscopy suggests that SEPT9 may act to compartmentalize the exocyst at the site of abscission, analogous to the role performed by septins in Saccharomyces cerevisiae. I provide evidence that the N-terminal region of SEPT9, which is absent from the shorter SEPT9 isoforms, plays an important role in abscission. I describe a long-anticipated link between a mammalian septin and the cell cycle machinery by showing that the N-terminal region of SEPT9 is phosphorylated at threonine 24 upon mitotic entry by cyclin-dependent kinase 1. This creates a binding site for the WW domain of the peptidyl-prolyl isomerase Pin1. I provide evidence that Pin1 induces a conformational change in the N-terminal region of SEPT9 that is important for the completion of cytokinesis. I propose that mitotic regulation of SEPT9 by Cdk1 and Pin1 regulates an interaction between SEPT9 and an unidentified protein that is critical for abscission.

Septins are a family of GTP-binding proteins implicated in mammalian cell division. Since these proteins form heterologous complexes and filaments in interphase cells, it has been assumed that depletion of any or all septins in a given cell type will give rise to the same phenotype. I demonstrate that while all septins expressed in HeLa cells localize to the cleavage furrow and midbody during cytokinesis, and co-immunoprecipitate throughout cell division, they do not all have identical roles during this process. Specific depletion of SEPT2 or SEPT11 caused defects in the early stages of cytokinesis, ultimately resulting in binucleation. Similar results were observed upon simultaneous depletion of all septins. In sharp contrast, SEPT9 was dispensable for the early stages of cell division, but was critical for the final separation of daughter cells. I demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody. Immunofluorescence microscopy suggests that SEPT9 may act to compartmentalize the exocyst at the site of abscission, analogous to the role performed by septins in Saccharomyces cerevisiae. I provide evidence that the N-terminal region of SEPT9, which is absent from the shorter SEPT9 isoforms, plays an important role in abscission. I describe a long-anticipated link between a mammalian septin and the cell cycle machinery by showing that the N-terminal region of SEPT9 is phosphorylated at threonine 24 upon mitotic entry by cyclin-dependent kinase 1. This creates a binding site for the WW domain of the peptidyl-prolyl isomerase Pin1. I provide evidence that Pin1 induces a conformational change in the N-terminal region of SEPT9 that is important for the completion of cytokinesis. I propose that mitotic regulation of SEPT9 by Cdk1 and Pin1 regulates an interaction between SEPT9 and an unidentified protein that is critical for abscission.

Aspergillus fumigatus is the major etiology of invasive aspergillosis, a leading cause of death in immunocompromised patients. Septins are conserved GTPases that could be mediating host-spore interactions in A. fumigatus. To test this hypothesis, we are using a combination of microscopy techniques and cell culture-based methods. Using Atomic Force Microscopy, we found that spores from the ∆aspB strain have a disorganized rodlet layer compared to the parent strain. Disorganized rodlet layer can lead to an increase in immune recognition of common pathogen associated molecular patterns (PAMPs), chitin and β-glucan. It is unknown whether the increase in immune recognition of ∆aspB spores is due to more exposure to the host or if they total quantity of PAMPs has increased. To test this, we used several staining methods to compare total and exposed levels of PAMPs in the conidia. Our work shows that there is an increase in exposed chitin and total levels of chitin and β-glucan. To evaluate how this plays a role in vitro, we first exposed macrophage-like cells (J744.1) to septin deletion strain’s spores and measured the TNF-a to determine spore immunogenicity using an ELISA. As expected, only the deletions that abolish all septin complexes had a significant increase in TNF-a. Then, we determine how effective macrophages were at killing the spores. We found that the deletion strains were more susceptible to killing by the macrophages. A murine study revealed that ∆aspB infection results in more lung inflammation than the wildtype. Taken together, these results suggest the septin cytoskeleton is involved in A. fumigatus spore cell wall organization and immune evasion.

Septinas fazem parte de uma família de proteínas de ligação ao nucleotídeo guanina e já foram encontradas em diversos eucariontes, mas nunca em plantas. Essas proteínas têm sido descritas como atuantes na citocinese, estruturação celular e exocitose, mas pouco se conhece do seu modo de ação. Além disso, as septinas mostraram-se capazes de se polimerizar em heterofilamentos altamente organizados, a partir da interação com outras septinas, mas as referências à existência e funcionalidade de homofilamentos de septinas permanecem escassas e controversas. Este trabalho visou caracterizar a função da septina da alga unicelular Chlamydomonas reinhardtii, um eucarionte modelo que divergiu há muito de um ancestral comum a plantas e metazoários, e que possui uma única septina, diferente dos demais eucariontes estudados até o momento. Para tal, foram realizados ensaios de duplo híbrido para detecção de possíveis proteínas parceiras à septina de C. reinhardtii, além de análise de expressão gênica em diferentes pontos do ciclo celular por PCR quantitativo (qPCR), silenciamento gênico por micro-RNA artificial de interferência e imunolocalização por microscopia confocal. Os ensaios de duplo híbrido retornaram duas possíveis proteínas parceiras de interação à septina de Chalmydomonas reinhardtii (CrSept) – S-Adenosyl-Homocysteine-Hydrolase (CrSAHH) e Subtilase-like-Serino-Protease (esporangina) – ambas relacionada à estrutura flagelar. A interação entre CrSept e CrSAHH não foi validada através das técnicas de pulldown e crosslinking, porém, os experimentos de imunolocalização da proteína in situ mostraram uma grande concentração de CrSept na base flagelar durante as fases G0, e G1, com mudança no perfil de localização para o citoplasma durante as fases S e M do ciclo celular, evidenciando uma participação da septina na estrutura flagelar e não excluindo a possibilidade de uma interação in vivo entre CrSept e CrSAHH. Análises do nível de expressão gênica de CrSept mostraram uma tendência de maior expressão do gene da septina durante o período claro, com redução na fase escura do ciclo celular, resultados que se assemelham aos observados pela análise qualitativa, por western blot, da expressão da proteína ao longo do ciclo celular. Os experimentos de silenciamento gênico, por fim, mostraram um possível fenótipo relacionado à redução de mRNA de CrSept, não observado no grupo controle. Estes resultados mostram que a CrSept possui caráter estrutural na alga verde C. reinhardtii, podendo atuar como suporte para outras proteínas durante a fase de crescimento celular. Além disso, localizações pontuais por toda a célula durante a fase de divisão celular sugerem que a CrSept desempenha um importante papel na manutenção da estrutura celular para a conclusão da divisão celular. / Septins belong to a family of proteins that bind guanine nucleotide and have been found in many eukaryotes, but never in plants. These proteins have been described in cytokinesis process, cell structure and exocytosis, but little is known about their way of action. Besides, septins are capable of polymerize in high organized heterofilaments when interacting with other septins, but references and functional studies of septins homofilaments remain controversial. This work aimed to characterize the function of the unique septin from the green alga Chlamydomonas reinhardtii, a model eukaryote organism that long diverged from a common ancestor to plants and Metazoans and that has a single septin. For that, yeast two-hybrid assays were conducted in search of possible partner proteins to C.reinhardtii, septin (CrSept); also, gene expression analyses by qPCR of different points of the cell cycle helped characterize the expression profile of the CrSept gene and gene silencing by artificial micro-RNA (amiRNA) and immunolocalization by confocal microscopy were used to enrich functional and localization studies. The yeast two-hybrid assays returned two possible partner proteins to CrSept - S-Adenosyl-Homocysteine-Hydrolase (CrSAHH) e Subtilase-like-Serino-Protease (sporangin) – both related to the flagella structure. The interaction among CrSept and CrSAHH could not be validated in vitro by pulldown or crosslink assays, however, the in situ immunostaining showed CrSept can mostly be found in punctual concentrated spots close to the base of the flagella during G0 and G1 phases of the cell cycle, which differs from the profile observed during phases S and M, where the protein can be observed as punctual spots through the whole cell. These results strengths CrSept role on the flagellar structure and do not exclude the possibility of an in vivo interaction between CrSept and CrSAHH. Gene expression analyses showed that septin is mostly expressed during the light part of the cycle, which is also observed at the protein quantitative analysis by western blot. Gene silencing experiments showed a possible phenotype in clones expressing amiRNA against CrSept, which was not observed on control group. Together, these results suggest CrSept has a structural role in C. reinhardtii and might work as a scaffold to other proteins during cell growth stage. Besides, punctual staining observed during mitosis suggests CrSept might help maintaining cell structure until cell division is completed.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.

Septins are a family of guanine nucleotide-binding proteins that function in eukaryotic cell division, where they form a high-order cortical structure at the site of division, which is essential in most eukaryotes. Expanded roles have evolved for septins in metazoans, where they also have essential functions in terminally-differentiated cell types, such as neurons and spermatozoa. Specific details of septin function are lacking in most roles described, due at least in part to the limited number of characterized binding partners. In this work, yeast two-hybrid screens and pull-downs from tissue homogenate were used to identify novel septin binding partners for subsequent characterization. The neuron-enriched septin, SEPT5, interacted directly with SUMO E3 ligases of the PIAS family. However, I was not able to demonstrate endogenous sumoylation of SEPT5 and SUMO isoforms did not concentrate with the septins during cytokinesis. SEPT5 also interacted with a novel septin, SEPT12, which I further characterized to be testis-specific and localized to the annulus in mature spermatozoa. Further, using SEPT12-specific reagents, I determined that the annulus forms via sequestration and subsequent segregation from the Golgi during spermiogenesis. SEPT9 pull-downs identified another novel testis-specific septin, SEPT14. Reagents specific to SEPT2 and SEPT9 also revealed a septin-rich structure in the seminiferous epithelium in close association with the ectoplasmic specialization. The specific role of septins in this structure awaits further characterization. Several other intriguing candidate septin-interaction partners were identified and the further study of their possible in vivo interaction with septins may provide substantial insight into the mechanisms of septin function in eukaryotes.

Septinas fazem parte de uma família de proteínas de ligação ao nucleotídeo guanina e já foram encontradas em diversos eucariontes, mas nunca em plantas. Essas proteínas têm sido descritas como atuantes na citocinese, estruturação celular e exocitose, mas pouco se conhece do seu modo de ação. Além disso, as septinas mostraram-se capazes de se polimerizar em heterofilamentos altamente organizados, a partir da interação com outras septinas, mas as referências à existência e funcionalidade de homofilamentos de septinas permanecem escassas e controversas. Este trabalho visou caracterizar a função da septina da alga unicelular Chlamydomonas reinhardtii, um eucarionte modelo que divergiu há muito de um ancestral comum a plantas e metazoários, e que possui uma única septina, diferente dos demais eucariontes estudados até o momento. Para tal, foram realizados ensaios de duplo híbrido para detecção de possíveis proteínas parceiras à septina de C. reinhardtii, além de análise de expressão gênica em diferentes pontos do ciclo celular por PCR quantitativo (qPCR), silenciamento gênico por micro-RNA artificial de interferência e imunolocalização por microscopia confocal. Os ensaios de duplo híbrido retornaram duas possíveis proteínas parceiras de interação à septina de Chalmydomonas reinhardtii (CrSept) – S-Adenosyl-Homocysteine-Hydrolase (CrSAHH) e Subtilase-like-Serino-Protease (esporangina) – ambas relacionada à estrutura flagelar. A interação entre CrSept e CrSAHH não foi validada através das técnicas de pulldown e crosslinking, porém, os experimentos de imunolocalização da proteína in situ mostraram uma grande concentração de CrSept na base flagelar durante as fases G0, e G1, com mudança no perfil de localização para o citoplasma durante as fases S e M do ciclo celular, evidenciando uma participação da septina na estrutura flagelar e não excluindo a possibilidade de uma interação in vivo entre CrSept e CrSAHH. Análises do nível de expressão gênica de CrSept mostraram uma tendência de maior expressão do gene da septina durante o período claro, com redução na fase escura do ciclo celular, resultados que se assemelham aos observados pela análise qualitativa, por western blot, da expressão da proteína ao longo do ciclo celular. Os experimentos de silenciamento gênico, por fim, mostraram um possível fenótipo relacionado à redução de mRNA de CrSept, não observado no grupo controle. Estes resultados mostram que a CrSept possui caráter estrutural na alga verde C. reinhardtii, podendo atuar como suporte para outras proteínas durante a fase de crescimento celular. Além disso, localizações pontuais por toda a célula durante a fase de divisão celular sugerem que a CrSept desempenha um importante papel na manutenção da estrutura celular para a conclusão da divisão celular. / Septins belong to a family of proteins that bind guanine nucleotide and have been found in many eukaryotes, but never in plants. These proteins have been described in cytokinesis process, cell structure and exocytosis, but little is known about their way of action. Besides, septins are capable of polymerize in high organized heterofilaments when interacting with other septins, but references and functional studies of septins homofilaments remain controversial. This work aimed to characterize the function of the unique septin from the green alga Chlamydomonas reinhardtii, a model eukaryote organism that long diverged from a common ancestor to plants and Metazoans and that has a single septin. For that, yeast two-hybrid assays were conducted in search of possible partner proteins to C.reinhardtii, septin (CrSept); also, gene expression analyses by qPCR of different points of the cell cycle helped characterize the expression profile of the CrSept gene and gene silencing by artificial micro-RNA (amiRNA) and immunolocalization by confocal microscopy were used to enrich functional and localization studies. The yeast two-hybrid assays returned two possible partner proteins to CrSept - S-Adenosyl-Homocysteine-Hydrolase (CrSAHH) e Subtilase-like-Serino-Protease (sporangin) – both related to the flagella structure. The interaction among CrSept and CrSAHH could not be validated in vitro by pulldown or crosslink assays, however, the in situ immunostaining showed CrSept can mostly be found in punctual concentrated spots close to the base of the flagella during G0 and G1 phases of the cell cycle, which differs from the profile observed during phases S and M, where the protein can be observed as punctual spots through the whole cell. These results strengths CrSept role on the flagellar structure and do not exclude the possibility of an in vivo interaction between CrSept and CrSAHH. Gene expression analyses showed that septin is mostly expressed during the light part of the cycle, which is also observed at the protein quantitative analysis by western blot. Gene silencing experiments showed a possible phenotype in clones expressing amiRNA against CrSept, which was not observed on control group. Together, these results suggest CrSept has a structural role in C. reinhardtii and might work as a scaffold to other proteins during cell growth stage. Besides, punctual staining observed during mitosis suggests CrSept might help maintaining cell structure until cell division is completed.

Septins are a family of GTP-binding proteins, and although highly conserved throughout many eukaryotes, their functions vary across species. In Aspergillus fumigatus, the etiological agent of invasive aspergillosis, septins participate in a variety of roles such as cell wall organization of conidia, septation, and response to anti-cell wall stress. Previous studies determined that the ∆aspB strain had a greater sensitivity to anti-cell wall drugs, especially the echinocandin caspofungin, yet mechanisms behind this augmented sensitivity are unknown. We performed cell viability staining post-caspofungin exposure and found that the ∆aspA, ∆aspB, and ∆aspC strains showed significant reduction in cell viability. Concomitant with the reduced viability, deletion strains are more susceptible to caspofungin on solid media. These results indicate that the septin cytoskeleton is important for A. fumigatus survival in the presence of caspofungin. Due to the potential of improved therapeutic outcome, we followed up using a neutropenic murine model of invasive aspergillosis. Deletion of the aspB gene resulted in improved survival when treated with caspofungin when compared to the akuBKU80 wild-type or untreated ∆aspB strains. Quantitative proteomics analyses were used to find proteins involved in the septin-dependent adaptation to caspofungin. We identified four candidates with roles in cell wall integrity. Deletion of these candidate genes resulted in increase in susceptibility to caspofungin and moderate reduction in viability post drug exposure. Taken together, these data suggest that septin AspB is essential in mediating the fungistatic response to caspofungin.

Septinas são GTPases capazes de se polimerizar. Essas proteínas, componentes do citoesqueleto, participam de diversos processos celulares nos quais elas se encontram associadas às membranas, como na citocinese, na ciliogênese e na exocitose. Para que possam exercer essas variadas funções, as septinas organizam-se como hetero-oligômeros (complexos) não-polares, os quais podem interagir entre si, formando estruturas maiores como filamentos, anéis e redes. Essas proteínas são altamente conservadas em eucariotos, todavia, o número de genes de septinas entre espécies é variável de uma, em Chlamydomonas reinhardtii, até mais de uma dezena de genes de septinas em humanos. Estudos anteriores do nosso grupo de pesquisa descreveram quatro septinas em Schistosoma mansoni, nomeadas SmSEPT5, SmSEPT10, SmSEPT7.1 e SmSEPT7.2. Ainda, recentemente, foi verificado que essas septinas são capazes de se ligar a membranas modelo, constituindo um modelo mais simples, quando comparadas às humanas, para estudar os mecanismos de associação às membranas. Neste trabalho, foram investigadas a influência da curvatura das membranas para a interação septinas-lipídios e a especificidade de septinas por diferentes fosfolipídios. Além das septinas de S. mansoni, o complexo de septinas de Ciona intestinalis foi também incluído, visando análises comparativas. Experimentos de microscopia confocal de fluorescência mostraram que tanto a SmSEPT10 isolada, quanto os complexos de septinas de S. mansoni e de C. intestinalis ligam-se, preferencialmente, a membranas com curvaturas de 2 μm-1(diâmetro de 0,96 μm). Essa tendência parece ser intrínseca de septinas, visto que complexos de outros organismos já haviam apresentado a mesma preferência. A capacidade de uma septina individual, no caso SmSEPT10, de distinguir curvaturas é um indicativo de que a polimerização não é necessária para esse mecanismo. A interação das septinas aos modelos de membrana só foi detectada na presença de dextrose, sugerindo que esse açúcar atue na estabilização dessas proteínas e abrindo novas frentes de estudo sobre a estabilidade das septinas. Os experimentos de microscopia, em conjunto com ensaios de PIP Strips, demonstraram que o complexo de septinas de C. intestinalis liga-se, preferencialmente, à fosfatidilserina, enquanto as septinas de S. mansoni apresentam uma preferência por fosfoinositóis. Finalmente, ensaios preliminares com construções mutantes do C-terminal da SmSEPT10 possibilitaram o desenvolvimento de uma hipótese para o mecanismo de associação dessa septina às membranas. / Septins are polymerizable GTPases. These cytoskeletal proteins are involved in several cellular processes in which they are associated to membranes, including cytokinesis, ciliogenesis and exocytosis. In order to perform these various functions, septins assemble into non-polar hetero-oligomers (complexes), which interact with each other forming higher-order structures such as filaments, ring and gauzes. These proteins are highly conserved in eukaryotes, yet the number of septin genes varies from one, in Chlamydomonas reinhardtii, to more than a dozen septin genes in humans. Previous studies from our research group described four septins in Schistosoma mansoni, named SmSEPT5, SmSEPT10, SmSEPT7.1 and SmSEPT7.2. Recently, it was verified that these proteins are capable of binding to model membranes, constituting a simpler model, when compared to human septins, to study the mechanism of membrane association. In this work, the influence of membrane curvature to the septin-lipid binding and the septin specificity to different phospholipids were investigated. In addition to S. mansoni septins, the septin complex from Ciona intestinalis was also included for comparative analyzes. Confocal fluorescence microscopy experiments showed that both SmSEPT10 and the septin complexes from S. mansoni and C. intestinalis bind, preferably, to membranes with 2 μm-1 curvatures (0,96 μm diameter). This tendency seems to be intrinsic to septins, as hetero-oligomers from other organisms had already presented the same binding preference. The capacity of an individual septin to distinguish curvatures is an indicative that the polymerization is not required for this mechanism. The interaction of these septins to the membrane models was only detected in the presence of dextrose, suggesting that this sugar acted in the protein stabilization, thus opening up new to study septin stability. These microscopy experiments, together with PIP Strips assays, demonstrated that the septin complex from C. intestinalis binds preferentially to phosphatidylserine, whereas septins from S. mansoni show a preference to phosphoinositides. Finally, preliminary assays with mutant constructions of SmSEPT10 C-terminal enabled the development of a hypothesis for the association mechanism of these proteins to membranes.