Final Report Summary - COMITPOL (Understanding the coordination between mitosis and cell polarity in Drosophila)
The ability to physically separate different cellular compartments was a major evolutionary breakthrough of multicellular organisms, resulting on development of the epithelial tissue, surrounding their different organs. The roles of epithelial tissues require polarized intracellular distribution of adhesive structures and functionally distinct apical and basal domains. Examples of the relevance of this polarity can be seen in different human epithelia, which require transport of molecules in a polarized manner (absorption of nutrients in the gut, or filtration in the kidney) or protect the internal environment from external challenges, such as in the skin epidermis. Furthermore, defects in apico-basal architecture are a hallmark associated with poor cancer prognosis, being epithelia the source of most malignant tumors.
The molecular organization of epithelial polarity and the mechanisms that ensure correct chromosome segregation have been extensively studied, but how these processes are synchronized to maintain epithelial integrity during proliferation is now an emerging field. In proliferating tissues, epithelial cells round up during mitosis before producing daughter cells that physically separate by cytokinesis. This raises many challenges for a cell that has polarized domains during interphase, which has to adjust this organization to the dramatic cytoskeleton and shape alterations that occur during mitosis, and must maintain tissue architecture, while forming a new polarized interface between daughter cells. The overall goal of this project is to understand how epithelial cell polarity is coordinated with mitosis to maintain tissue architecture during cell division and to address the interplay between these processes in tumorigenesis. We progressed toward this goal by following the aims described below.
Aim 1) - Characterize how cortical polarity responds to mitotic cell division in epithelial cells
Epithelial polarity depends on three evolutionary conserved complexes, which establish mutually antagonistic interactions to maintain the specification of each distinct domain. The Crumbs and aPKC complexes function on the apical domain, whereas the Lgl complex occupies the lateral domain. We have used the Drosophila follicle epithelium to set up an in vivo model to address how the different polarized domains are remodeled during mitosis in epithelia. Using both time-lapse movies of intact ovaries and three-dimensional reconstructions of fixed samples, we found that whereas Adherens Junction (AJ) components are maintained during division, the apical polarity proteins, Crumbs, Par-6 and aPKC and the lateral polarity protein LGL depolarize during division. Furthermore, we found that cytokinesis is polarized in the follicle epithelium, and we therefore analyzed in detail the mechanism and purpose of this asymmetry. We show that it does not depend on intrinsic assymetric contractility of the cytokinetic cring, being instead determined by polarized AJs that contact with the contractile ring during constriction and mediate its apical positioning. Furthermore, we generated transgenic flies where we could misposition AJ components to induce ectopic midbody positioning, concluding that AJ-dependent midbody positioning transfers epithelial architecture to daughter cells during proliferation. This work has been published in EMBO reports (1), and we have written a feature in Cell Cycle summarizing this work and other three studies published in 2013 that addressed epithelial cytokinesis (2).
Aim 2) Analyse the role of distinct classes of mitotic regulators in the morphogenesis of epithelial tissue and in germline stem cell division.
The mechanisms that ensure proper chromosome segregation have been well studied by cell-culture experiments. However, the role of the different mitotic regulators is not well understood at the level of the organism where proliferation must be tightly regulated in a tissue specific fashion. We found that alterations in the fidelity of cell division caused by loss of polo function, bubR1, nuf2 and spc25 activity could produce defects in tissue organization, but these were attributed to failure in cell division and not due to an effect on these genes in the organization of epithelial polarity. More importantly, we found that aurora mosaic mutant clones affected the dynamics of the polarity protein Lgl during mitosis. Interestingly, our results suggest that AurA controls Lgl dynamics directly, instead of acting through the activation of the aPKC complex as previously described in Drosophila neuronal stem cells (Wirtz-Peitz F et al., Cell, 2008). Consistent with this, we found that in S2 cells, which do not display polarized localization of aPKC, Lgl displays a similar dynamic localization during mitosis, which is also phosphorylation-dependent and regulated by Aurora A. Thus, the mitotic kinase Aurora A couples the cell cycle to the redistribution of the cell polarity determinant Lgl regardless of the polarized state of the cell, suggesting that Lgl has general role during mitosis. Although it is not clear whether Lgl cortical exclusion is essential during mitosis, our results raise the hypothesis that a role in controlling faithful chromosome segregation may contribute for the well-established tumor suppressor function of Lgl.
Aim 3) Identify a genetic interaction between mitotic regulators and polarity complexes to explore a cooperative role in a multistage pathway to tumorigenesis
Altered expression of cell adhesion and polarity proteins has been associated with epithelial transformation and human cancer. Members of the Scribble (Scrib)-Lethal (2) giant larvae (Lgl) – Discs large (Dlg) complex, which defines the lateral domain of epithelial cells, were initially identified as tumor suppressors in Drosophila, and this role has been recently confirmed in mammalian carcinoma by several studies. Since misregulation of cell division and loss of epithelial organization are a hallmark of cancer, we tested the ability of more general regulators of mitotic cell division as well as different genes that control the fidelity of cell division at different levels (chromosome bi-orientation, kinetochore-microtubule attachment, spindle assembly checkpoint, centrosome biogenesis, chromosome condensation and cohesion) to enhance the loss of epithelial organization/overgrowth of hypomorphic alleles/RNAi for members of the Scribble complex. From this enhancer screen, we highlight the finding that simultaneous depletion of polarity and cytokinesis proteins enhanced the anomalous growth of tissue in the eye of the adult fly. These results indicate that unlike previously expected, tumorigenesis arising from cells in which polarity has been compromised can result from a second hit that promotes genetic instability without the apparent contribution of oncogenes. The major outcome of cytokinesis failure is tetraploidy, or the generation of cells that contain more than two sets of homologous chromosomes and increased number of centrosomes. Cytokinesis defects could therefore foster cell transformation via the increased chromosome instability resulting by the presence of supernumerary centrosomes or via the buffering effect of extra chromosomes that would allow the accumulation of other malignant genetic modifications. The results from this work favor the idea that the presence of extra chromosomes and not extra centrosomes is the major determinant of the tumorigenic ability of cells that have cytokinesis defects in a polarity defective context.
Conclusion
We consider that the research objectives for the project “COMITPOL” have been accomplished since the three aims of our research reflected into three key findings: 1) that the apical localization of Adherens junctions determines the asymmetry of epithelial cell cytokinesis, which is essential to maintain the architecture of epithelial tissue during proliferation 2) the discovery that that mitotic kinase Aurora A redirects the polarity protein Lgl to a new subcellular localization during mitosis, revealing a potential new role underlying Lgl´s tumor suppressor function 3) the identification of a cooperation between loss of polarity and defective cytokinesis in tumorigenesis, further validating the ability of Drosophila to model multistage transformation. Thus, since loss of epithelial architecture leads to a number of pathologies, understanding of how epithelial cells maintain their architecture while ensuring correct chromosome segregation paves the way to studies with direct interest to human disease. It is also important to note that the researcher Eurico Sá acquired new competences that are critical to its establishment has an independent researcher. Furthermore, the value of the results of the work funded by this project has been attested by the attribution of a grant to Eurico Sá as an independent researcher at the host country (Investigator FCT, Portuguese Foundation for Science and Technology). This grant starts at the end of the Marie Curie IEF, and therefore allows the extension of the line of research that was developed during this project.
The molecular organization of epithelial polarity and the mechanisms that ensure correct chromosome segregation have been extensively studied, but how these processes are synchronized to maintain epithelial integrity during proliferation is now an emerging field. In proliferating tissues, epithelial cells round up during mitosis before producing daughter cells that physically separate by cytokinesis. This raises many challenges for a cell that has polarized domains during interphase, which has to adjust this organization to the dramatic cytoskeleton and shape alterations that occur during mitosis, and must maintain tissue architecture, while forming a new polarized interface between daughter cells. The overall goal of this project is to understand how epithelial cell polarity is coordinated with mitosis to maintain tissue architecture during cell division and to address the interplay between these processes in tumorigenesis. We progressed toward this goal by following the aims described below.
Aim 1) - Characterize how cortical polarity responds to mitotic cell division in epithelial cells
Epithelial polarity depends on three evolutionary conserved complexes, which establish mutually antagonistic interactions to maintain the specification of each distinct domain. The Crumbs and aPKC complexes function on the apical domain, whereas the Lgl complex occupies the lateral domain. We have used the Drosophila follicle epithelium to set up an in vivo model to address how the different polarized domains are remodeled during mitosis in epithelia. Using both time-lapse movies of intact ovaries and three-dimensional reconstructions of fixed samples, we found that whereas Adherens Junction (AJ) components are maintained during division, the apical polarity proteins, Crumbs, Par-6 and aPKC and the lateral polarity protein LGL depolarize during division. Furthermore, we found that cytokinesis is polarized in the follicle epithelium, and we therefore analyzed in detail the mechanism and purpose of this asymmetry. We show that it does not depend on intrinsic assymetric contractility of the cytokinetic cring, being instead determined by polarized AJs that contact with the contractile ring during constriction and mediate its apical positioning. Furthermore, we generated transgenic flies where we could misposition AJ components to induce ectopic midbody positioning, concluding that AJ-dependent midbody positioning transfers epithelial architecture to daughter cells during proliferation. This work has been published in EMBO reports (1), and we have written a feature in Cell Cycle summarizing this work and other three studies published in 2013 that addressed epithelial cytokinesis (2).
Aim 2) Analyse the role of distinct classes of mitotic regulators in the morphogenesis of epithelial tissue and in germline stem cell division.
The mechanisms that ensure proper chromosome segregation have been well studied by cell-culture experiments. However, the role of the different mitotic regulators is not well understood at the level of the organism where proliferation must be tightly regulated in a tissue specific fashion. We found that alterations in the fidelity of cell division caused by loss of polo function, bubR1, nuf2 and spc25 activity could produce defects in tissue organization, but these were attributed to failure in cell division and not due to an effect on these genes in the organization of epithelial polarity. More importantly, we found that aurora mosaic mutant clones affected the dynamics of the polarity protein Lgl during mitosis. Interestingly, our results suggest that AurA controls Lgl dynamics directly, instead of acting through the activation of the aPKC complex as previously described in Drosophila neuronal stem cells (Wirtz-Peitz F et al., Cell, 2008). Consistent with this, we found that in S2 cells, which do not display polarized localization of aPKC, Lgl displays a similar dynamic localization during mitosis, which is also phosphorylation-dependent and regulated by Aurora A. Thus, the mitotic kinase Aurora A couples the cell cycle to the redistribution of the cell polarity determinant Lgl regardless of the polarized state of the cell, suggesting that Lgl has general role during mitosis. Although it is not clear whether Lgl cortical exclusion is essential during mitosis, our results raise the hypothesis that a role in controlling faithful chromosome segregation may contribute for the well-established tumor suppressor function of Lgl.
Aim 3) Identify a genetic interaction between mitotic regulators and polarity complexes to explore a cooperative role in a multistage pathway to tumorigenesis
Altered expression of cell adhesion and polarity proteins has been associated with epithelial transformation and human cancer. Members of the Scribble (Scrib)-Lethal (2) giant larvae (Lgl) – Discs large (Dlg) complex, which defines the lateral domain of epithelial cells, were initially identified as tumor suppressors in Drosophila, and this role has been recently confirmed in mammalian carcinoma by several studies. Since misregulation of cell division and loss of epithelial organization are a hallmark of cancer, we tested the ability of more general regulators of mitotic cell division as well as different genes that control the fidelity of cell division at different levels (chromosome bi-orientation, kinetochore-microtubule attachment, spindle assembly checkpoint, centrosome biogenesis, chromosome condensation and cohesion) to enhance the loss of epithelial organization/overgrowth of hypomorphic alleles/RNAi for members of the Scribble complex. From this enhancer screen, we highlight the finding that simultaneous depletion of polarity and cytokinesis proteins enhanced the anomalous growth of tissue in the eye of the adult fly. These results indicate that unlike previously expected, tumorigenesis arising from cells in which polarity has been compromised can result from a second hit that promotes genetic instability without the apparent contribution of oncogenes. The major outcome of cytokinesis failure is tetraploidy, or the generation of cells that contain more than two sets of homologous chromosomes and increased number of centrosomes. Cytokinesis defects could therefore foster cell transformation via the increased chromosome instability resulting by the presence of supernumerary centrosomes or via the buffering effect of extra chromosomes that would allow the accumulation of other malignant genetic modifications. The results from this work favor the idea that the presence of extra chromosomes and not extra centrosomes is the major determinant of the tumorigenic ability of cells that have cytokinesis defects in a polarity defective context.
Conclusion
We consider that the research objectives for the project “COMITPOL” have been accomplished since the three aims of our research reflected into three key findings: 1) that the apical localization of Adherens junctions determines the asymmetry of epithelial cell cytokinesis, which is essential to maintain the architecture of epithelial tissue during proliferation 2) the discovery that that mitotic kinase Aurora A redirects the polarity protein Lgl to a new subcellular localization during mitosis, revealing a potential new role underlying Lgl´s tumor suppressor function 3) the identification of a cooperation between loss of polarity and defective cytokinesis in tumorigenesis, further validating the ability of Drosophila to model multistage transformation. Thus, since loss of epithelial architecture leads to a number of pathologies, understanding of how epithelial cells maintain their architecture while ensuring correct chromosome segregation paves the way to studies with direct interest to human disease. It is also important to note that the researcher Eurico Sá acquired new competences that are critical to its establishment has an independent researcher. Furthermore, the value of the results of the work funded by this project has been attested by the attribution of a grant to Eurico Sá as an independent researcher at the host country (Investigator FCT, Portuguese Foundation for Science and Technology). This grant starts at the end of the Marie Curie IEF, and therefore allows the extension of the line of research that was developed during this project.