Browsing by Author "Tavares, Alexandra"
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- CCTα and CCTδ chaperonin subunits are essential and required for cilia assembly and maintenance in TetrahymenaPublication . Seixas, Cecília; Cruto, Teresa; Tavares, Alexandra; Gaertig, Jacek; Soares, HelenaBackground - The eukaryotic cytosolic chaperonin CCT is a hetero-oligomeric complex formed by two rings connected back-to-back, each composed of eight distinct subunits (CCTalpha to CCTzeta). CCT complex mediates the folding, of a wide range of newly synthesised proteins including tubulin (alpha, beta and gamma) and actin, as quantitatively major substrates. Methodology/Principal findings - We disrupted the genes encoding CCTalpha and CCTdelta subunits in the ciliate Tetrahymena. Cells lacking the zygotic expression of either CCTalpha or CCTdelta showed a loss of cell body microtubules, failed to assemble new cilia and died within 2 cell cycles. We also show that loss of CCT subunit activity leads to axoneme shortening and splaying of tips of axonemal microtubules. An epitope-tagged CCTalpha rescued the gene knockout phenotype and localized primarily to the tips of cilia. A mutation in CCTalpha, G346E, at a residue also present in the related protein implicated in the Bardet Biedel Syndrome, BBS6, also caused defects in cilia and impaired CCTalpha localization in cilia. Conclusions/Significance - Our results demonstrate that the CCT subunits are essential and required for ciliary assembly and maintenance of axoneme structure, especially at the tips of cilia.
- Characterization of a MOB1 homolog in the Apicomplexan parasite toxoplasma gondiiPublication . Delgado, Inês L. S.; Tavares, Alexandra; Francisco, Samuel; Santos, Dulce; Coelho, João; Basto, Afonso P.; Zúquete, Sara; Müller, Joachim; Hemphill, Andrew; Meissner, Markus; Soares, Helena; Leitão, Alexandre; Nolasco, S.Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites’ ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.
- Mob1, hippo pathway member, is critical for toxoplasma gondii replicationPublication . Tavares, Alexandra; Delgado, Inês; Francisco, Samuel; Coelho, João; Leitão, Alexandre; Soares, Helena; Nolasco, SofiaToxoplasma gondii is an obligate intracellular parasite of great veterinary and medical importance. It is able to evade the immune system of the host by converting from rapidly proliferating tachyzoites to latent bradyzoite cysts and this parasite number control is a key to the success of the infection. Pathways controlling cell division/proliferation like the Hippo pathway are likely candidates for regulating parasite replication. Human Mob1 participates in this pathway and our recent data suggests it is an excellent candidate for the control of parasite replication/number. Our research group has identified a single mob1 gene in T. gondii. A phylogenetic analysis of this gene showed it to be similar to other Apicomplexa but distant from protozoan parasites like the Trypanosomatida. We confirmed that this gene is expressed and our data show that its expression dramatically decreases (94%) during the parasite replication inside the host cell. We have constructed a transgenic parasite strain that overexpresses Mob1 and these parasites show a significant delay in the replication process. Using an in-house polyclonal antibody against this protein we observed a very clear polarized localization of the protein in the parasite posterior pole, where the basal complex, a structure involved in cytokinesis in T. gondii, is localized. To better understand the role of Toxoplasma Mob1 we have created, by using the by CRISPR/Cas9 approach, a strain where Mob1 loss of function can be induced. Our preliminary results, by immunofluorescence microscopy, show that after induction Toxoplasma parasites in parasitophorous vacuoles (PV) lose their intrinsic polarity and their normal rosette organization. Indeed, inside of the PV, it is difficult to identify the individual dividing parasites that seem to have originated a mass of abnormal cells where multiple nuclei are present. This result suggests that Toxoplasma cells have abnormal division and/or fail the cytokinesis. Altogether, the data support that Mob1 is involved in the control of T. gondii replication and is a promising candidate to target therapeutic agents against Toxoplasma parasites proliferation.
- Mob1: at the crossroad between morphogenesis, cytokinesis and cell proliferation control in unicellular organismsPublication . Tavares, Alexandra; Delgado, Inês; Nolasco, Sofia; Carmona, Bruno; Leitão, Alexandre; Soares, Helena
- Mob1: defining cell polarity for proper cell divisionPublication . Tavares, Alexandra; Gonçalves, João; Florindo, Cláudia; Tavares, Álvaro A.; Soares, HelenaMob1 is a component of both the mitotic exit network and Hippo pathway, being required for cytokinesis, control of cell proliferation and apoptosis. Cell division accuracy is crucial in maintaining cell ploidy and genomic stability and relies on the correct establishment of the cell division axis, which is under the control of the cell's environment and its intrinsic polarity. The ciliate Tetrahymena thermophila possesses a permanent anterior-posterior axis, left-right asymmetry and divides symmetrically. These unique features of Tetrahymena prompted us to investigate the role of Tetrahymena Mob1. Unexpectedly, we found that Mob1 accumulated in basal bodies at the posterior pole of the cell, and is the first molecular polarity marker so far described in Tetrahymena. In addition, Mob1 depletion caused the abnormal establishment of the cell division plane, providing clear evidence that Mob1 is important for its definition. Furthermore, cytokinesis was arrested and ciliogenesis delayed in Tetrahymena cells depleted of Mob1. This is the first evidence for an involvement of Mob1 in cilia biology. In conclusion, we show that Mob1 is an important cell polarity marker that is crucial for correct division plane placement, for cytokinesis completion and for normal cilia growth rates.
- MOB1APublication . Carmona, Bruno; Tavares, Alexandra; Nolasco, Sofia; Leitão, Alexandre; Soares, HelenaThe accuracy of cell division is fundamental for the maintenance of cell ploidy and genomic stability. During cell division, many events, like DNA replication, chromosome segregation, mitosis completion, and cytokinesis, must be tightly controlled. The deregulation of these events is closely associated with severe pathology. Among other factors, the accuracy of cell division relies on the correct placement of the division plane which is dependent on the polarity axis. Both in unicellular organisms and in metazoan, the cell spindle position is regulated to be perpendicular or planar to the division plane, allowing this way to equally segregate the chromosomes between the two daughter cells.
- The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturationPublication . Nolasco, Sofia; Bellido, Javier; Gonçalves, João; Zabala, Juan Carlos; Soares, Helena; Tavares, AlexandraAbstract - Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules. Methodology/Principal findings: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line. Conclusions/Significance: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.