Microtubules, polarity and vertebrate neural tube morphogenesis

Microtubules (MTs) are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of MTs, their effects on cell shape and polarity, and their role in large-scale morphogenesis remain poorly understood. Here, these relationships were examined with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate; and cavitation of the teleost neural rod. The latter process has been compared with 'secondary' neurulation that generates the caudal spinal cord in mammals. MTs align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration, which indirectly impacts on cell shape. Whether MTs play other functional roles in mammalian neurulation remains unclear. In the zebrafish, MTs are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric MT apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of MT functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish 'cause' from 'effect'. Future developments will likely rely on novel ways to selectively impair MT function in order to investigate the roles they play

The role of Mob proteins in protozoan cell cycle regulation

Tese de doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e BiomédicasABSTRACT- Proper cell division and control of cell proliferation are critical aspects in cell biology, with implications during embryonic development and in the maintenance of organisms’ homeostasis. Mob1 is a core protein of the Mitotic Exit Network and of the Hippo pathway, fundamental signaling cascades for the correct metaphase to anaphase transition and for the proper balance between cell proliferation and death. In this work we took advantage of two protozoan organisms to investigate the role of Mob1, the most ancient protein of the Hippo pathway. In the ciliate Tetrahymena thermophila we demonstrated that Mob1 has a polarized subcellular distribution, concentrating in the basal bodies of the cell posterior pole. During cell division, the protein also localizes in the region where the division plane is formed and its absence in this specific place leads to the mispositioning of division axis and cytokinesis impairment. These results revealed that Mob1 directly links proper cell polarity to correct cell division. Our studies of Mob1 in the apicomplexan parasite Toxoplasma gondii, also a permanent polarized unicellular organism, contributed to a better understanding of how parasites may regulate cell proliferation inside the host cell, a critical aspect for the course of infection. In T. gondii, Mob1 also localizes preferentially in the posterior pole of the cell, where the basal complex, which is essential for cytokinesis, is localized. Interestingly, in agreement with a role for Mob1 in proliferation control in T. gondii, we observed that mob1 mRNA levels are dramatically diminished when parasites are actively replicating inside the cell and that Mob1 overexpression leads to a delay in the parasite replication rate. Altogether, the work presented clearly positions Mob1 as an ancestral molecule playing a critical role in the cross-road of cell polarity establishment, correct cell division and proliferation control.RESUMO - O papel das proteínas Mob1 na regulação da divisão celular em protozoários - A divisão celular e o controlo da proliferação são aspectos fundamentais em biologia celular com implicações no desenvolvimento embrionário e na manutenção da homeostasia nos organismos. A proteína Mob1 é uma componente de duas vias de sinalização celular, a Mitotic Exit Network e a via de sinalização Hippo, cascatas de fosforilação essenciais para a correcta transição entre a metáfase a e a anáfase e para o balanço entre a proliferação/morte celular. Neste trabalho, utilizámos dois protozoários modelo para investigar a função da proteína Mob1, a mais ancestral das proteínas nas vias de sinalização referidas. No ciliado Tetrahymena thermophila, demonstrámos que a proteína Mob1 apresenta uma localização polarizada, estando principalmente concentrada nos corpos basais do polo posterior das células. Aquando da divisão celular, a Mob1 também é observada na região da célula onde se forma o eixo de divisão. Esta localização é essencial visto a ausência de Mob1 no local conduzir ao deslocamento do eixo e impedir a citocinese. O nosso estudo no parasita apicomplexa Toxoplasma gondii, um organismo também permanentemente polarizado, contribuiu para compreender melhor, o possível mecanismo de regulação da proliferação dos parasitas dentro da célula hospedeira, um aspecto essencial no desenvolvimento da infecção. Em T. gondii, a proteína Mob1 também se concentra no polo posterior da célula onde se localiza o complexo basal, uma estrutura envolvida na citocinese. Claramente suportando a nossa hipótese que a Mob1 desempenha um papel no controlo da proliferação, observámos que os níveis de RNA mensageiro do gene mob1 são drasticamente diminuídos quando os parasitas estão no período de replicação activa dentro das células hospedeiras. Adicionalmente, a acumulação da proteína no citoplasma dos parasitas provoca um atraso significativo na sua taxa de replicação. Em conjunto, o trabalho apresentado posiciona a proteína Mob1 como uma molécula ancestral envolvida na conexão entre o estabelecimento da polaridade, a correcta divisão e o controlo da proliferação celular.Instituto de Emprego e Formação Profissional - IEF

Investigating the antitumoral activity and mechanism of action of a xanthone derivative

Chapter I refers to the introduction, and has the role of providing an initial overview of the issues addressed directly or indirectly in the rest of the study. Initially, an overview of the cell cycle, its regulation and control is given. In the sequence, a greater emphasis is given to mitosis, which is described in detail from its cascade of events to the molecular machinery of the mitotic spindle. Still at this stage, the dynamics that occur between kinetochore-microtubules, correlated errors and their due corrections are described. Next, we provide an overview of the Spindle Assembly Checkpoint (SAC), dissecting the functions triggered by this mechanism, as well as the proteins involved in this important cellular control mechanism. Following, an introduction was given about drugs that use the targeting of mitosis for cancer therapy, namely through microtubules, providing an overview of current approaches, their limitations and future directions. Finally, a correlation was made between xanthones and cancer, demonstrating how this class of compounds (as well as their derivatives) is already used as a starting point in the development of new anticancer drugs. Chapter II concerns what motivated the project, as well as its specific objectives. Chapter III refers to the materials and methods used throughout the study, so that it was possible to dissect the mechanism of action of the compound. Chapter IV will demonstrate the results about the compound's mechanism of action, through: in vitro characterization of the compound's antimitotic activity, identification of the underlying mechanism of action and evaluation of the combined treatment of PX2 with paclitaxel in promoting cell death of tumoral cells. Chapter V provides a discussion, correlating previous studies and the present study. Chapter VI provides general conclusions about the mechanism of action of PX2 and the prospects for future research. Chapter VII contains a list of all references cited in the course of the thesis.This work was supported by CESPU - Cooperativa de Ensino Superior Politécnico e Universitário Crl [grant number ComeTarget_CESPU_2017 and ComeTax-PFT-IINFACTS-2019]. This research was partially supported by FCT/MCTES - Foundation for Science and Technology from the Minister of Science, Technology and Higher Education and European Regional Development Fund (ERDF) under the projects, co-financed by COMPETE 2020, Portugal 2020, PTDC/SAU-PUB/28736/2017 (POCI-01-0145-FEDER-028736) and within the scope of UIDB/04423/2020, UID/QUI/5000612019, and UIDP/04423/2020 (Group of Natural Products and Medicinal Chemistry). ACH thanks FCT for her PhD grant (SFRH/BD/140844/2018). DRPL thanks FCT for her PhD grant (SFRH/BD/140844/2018). JXS thanks for the FCT PhD Programmes, specifically by the BiotechHealth Programme (PD/00016/2012), and for the grants (SFRH/BD/98105/2013 and SFRH/BD/116167/2016). To Departamento de Química da Universidade de Aveiro (Portuguese NMR network) for the NMR analysis

Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in \u3cem\u3eToxoplasma gondii\u3c/em\u3e

The Toxoplasma genome encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage-dependent manner. Replication in intermediate hosts occurs by endodyogeny, whereas a hybrid of schizogony and endopolygeny occurs in the gut of the definitive feline host. Here, we characterize the consequence of the loss of a cell cycle-regulated ovarian tumor (OTU family) deubiquitinase, OTUD3A of Toxoplasma gondii (TgOTUD3A; TGGT1_258780), in T. gondii tachyzoites. Rather than the mutation being detrimental, mutant parasites exhibited a fitness advantage, outcompeting the wild type. This phenotype was due to roughly one-third of TgOTUD3A-knockout (TgOTUD3A-KO) tachyzoites exhibiting deviations from endodyogeny by employing replication strategies that produced 3, 4, or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a dysregulation of centrosome duplication, causing a transient loss of stoichiometry between the inner and outer cores that resulted in a failure to terminate S phase at the attainment of 2N ploidy and/or the decoupling of mitosis and cytokinesis. The resulting dysregulation manifested as deviations in the normal transitions from S phase to mitosis (S/M) (endopolygeny-like) or M phase to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis, resulting in the generation of normal progeny. Our findings suggest that decisions regarding the utilization of specific cell cycle architectures are controlled by a ubiquitin-mediated mechanism that is dependent on the absolute threshold levels of an as-yet-unknown target(s). Analysis of the TgOTUD3A-KO mutant provides new insights into mechanisms underlying the plasticity of apicomplexan cell cycle architecture

Characterization of Human Cdc14B\u27s function in centrosome cycle control

Centrosomes are the only non-membranous organelles in most vertebrate cells and their major function is to nucleate microtubules, hence often recognized as the microtubule-organizing center (MTOC). Much like chromosome centrosome duplicates only once during the S phase of each cell cycle. The fidelity and timing of this duplication event will ensure equal division of duplicated chromosomes into the daughter cells. As a consequence, numerical and/or structural centrosome abnormalities will cause chromosome missegregation and lead to the generation of multiple mitosis and ultimately chromosomal instability, which typify many cancers. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B’s involvement in centrosome cycle control has never been explored. In this study, we identify Cdc14B as a negative regulator in centrosome cycle control: depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells; ectopic expression of Cdc14B leads to stepwise loss of centrioles and attenuates centriole amplification in HU/APH arrested S phase cells and cells treated with proteasome inhibitor Z-L3VS. This inhibitory function requires centriole-associated Cdc14B catalytic activity. In addition, our data suggests counterbalancing effects between Cdc14B phosphatase and kinases such as Plk4, Cdk2/Cyclin-E/A in centrosome duplication control potentially through modulating phosphorylation status of their common downstream effectors, HsSas-6 and B23 respectively. Taken together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle

Impact of Marine Drugs on Cytoskeleton-Mediated Reproductive Events

Marine organisms represent an important source of novel bioactive compounds, often showing unique modes of action. Such drugs may be useful tools to study complex processes such as reproduction; which is characterized by many crucial steps that start at gamete maturation and activation and virtually end at the first developmental stages. During these processes cytoskeletal elements such as microfilaments and microtubules play a key-role. In this review we describe: (i) the involvement of such structures in both cellular and in vitro processes; (ii) the toxins that target the cytoskeletal elements and dynamics; (iii) the main steps of reproduction and the marine drugs that interfere with these cytoskeleton-mediated processes. We show that marine drugs, acting on microfilaments and microtubules, exert a wide range of impacts on reproductive events including sperm maturation and motility, oocyte maturation, fertilization, and early embryo development

Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications stages, are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a “binary fission” mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal vs. external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota