Deciphering the roles of cell shape and Fat and Dachsous planar polarity in arranging the Drosophila apical microtubule network through quantitative image analysis.

In epithelial cells, planar polarization of subapical microtubule networks is thought to be important for both breaking cellular symmetry and maintaining the resulting cellular polarity. Studies in the Drosophila pupal wing and other tissues have suggested two alternative mechanisms for specifying network polarity. On one hand, mechanical strain and/or cell shape have been implicated as key determinants; on the other hand, the Fat-Dachsous planar polarity pathway has been suggested to be the primary polarizing cue. Using quantitative image analysis in the pupal wing, we reassess these models. We found that cell shape was a strong predictor of microtubule organization in the developing wing epithelium. Conversely, Fat-Dachsous polarity cues do not play any direct role in the organization of the subapical microtubule network, despite being able to weakly recruit the microtubule minus-end capping protein Patronin to cell boundaries. We conclude that any effect of Fat-Dachsous on microtubule polarity is likely to be indirect, via their known ability to regulate cell shape.

Multifaceted control of E-cadherin dynamics by Adaptor Protein Complex 1 during epithelial morphogenesis.

Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargoes. We examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. We found that AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. We discovered a distinct pool of AP-1 that localizes at the subapical adherens junctions. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. We then provide evidence that E-cadherin hyperinternalization acts upstream of cell death in a potential tumor-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion.

Transcriptional and post-transcriptional regulation of checkpoint genes on the tumour side of the immunological synapse.

Cancer is a disease of the genome, therefore, its development has a clear Mendelian component, demonstrated by well-studied genes such as BRCA1 and BRCA2 in breast cancer risk. However, it is known that a single genetic variant is not enough for cancer to develop leading to the theory of multistage carcinogenesis. In many cases, it is a sequence of events, acquired somatic mutations, or simply polygenic components with strong epigenetic effects, such as in the case of brain tumours. The expression of many genes is the product of the complex interplay between several factors, including the organism's genotype (in most cases Mendelian-inherited), genetic instability, epigenetic factors (non-Mendelian-inherited) as well as the immune response of the host, to name just a few. In recent years the importance of the immune system has been elevated, especially in the light of the immune checkpoint genes discovery and the subsequent development of their inhibitors. As the expression of these genes normally suppresses self-immunoreactivity, their expression by tumour cells prevents the elimination of the tumour by the immune system. These discoveries led to the rapid growth of the field of immuno-oncology that offers new possibilities of long-lasting and effective treatment options. Here we discuss the recent advances in the understanding of the key mechanisms controlling the expression of immune checkpoint genes in tumour cells.

Interactions and Feedbacks in E-Cadherin Transcriptional Regulation.

Epithelial tissues rely on the adhesion between participating cells to retain their integrity. The transmembrane protein E-cadherin is the major protein that mediates homophilic adhesion between neighbouring cells and is, therefore, one of the critical components for epithelial integrity. E-cadherin downregulation has been described extensively as a prerequisite for epithelial-to-mesenchymal transition and is a hallmark in many types of cancer. Due to this clinical importance, research has been mostly focused on understanding the mechanisms leading to transcriptional repression of this adhesion molecule. However, in recent years it has become apparent that re-expression of E-cadherin is a major step in the progression of many cancers during metastasis. Here, we review the currently known molecular mechanisms of E-cadherin transcriptional activation and inhibition and highlight complex interactions between individual mechanisms. We then propose an additional mechanism, whereby the competition between adhesion complexes and heterochromatin protein-1 for binding to STAT92E fine-tunes the levels of E-cadherin expression in Drosophila but also regulates other genes promoting epithelial robustness. We base our hypothesis on both existing literature and our experimental evidence and suggest that such feedback between the cell surface and the nucleus presents a powerful paradigm for epithelial resilience.

STORM imaging reveals the spatial arrangement of transition zone components and IFT particles at the ciliary base in Tetrahymena.

The base of the cilium comprising the transition zone (TZ) and transition fibers (TF) acts as a selecting gate to regulate the intraflagellar transport (IFT)-dependent trafficking of proteins to and from cilia. Before entering the ciliary compartment, IFT complexes and transported cargoes accumulate at or near the base of the cilium. The spatial organization of IFT proteins at the cilia base is key for understanding cilia formation and function. Using stochastic optical reconstruction microscopy (STORM) and computational averaging, we show that seven TZ, nine IFT, three Bardet-Biedl syndrome (BBS), and one centrosomal protein, form 9-clustered rings at the cilium base of a ciliate Tetrahymena thermophila. In the axial dimension, analyzed TZ proteins localize to a narrow region of about 30 nm while IFT proteins dock approximately 80 nm proximal to TZ. Moreover, the IFT-A subcomplex is positioned peripheral to the IFT-B subcomplex and the investigated BBS proteins localize near the ciliary membrane. The positioning of the HA-tagged N- and C-termini of the selected proteins enabled the prediction of the spatial orientation of protein particles and likely cargo interaction sites. Based on the obtained data, we built a comprehensive 3D-model showing the arrangement of the investigated ciliary proteins.

Robustness of the microtubule network self-organization in epithelia.

Robustness of biological systems is crucial for their survival, however, for many systems its origin is an open question. Here, we analyze one subcellular level system, the microtubule cytoskeleton. Microtubules self-organize into a network, along which cellular components are delivered to their biologically relevant locations. While the dynamics of individual microtubules is sensitive to the organism's environment and genetics, a similar sensitivity of the overall network would result in pathologies. Our large-scale stochastic simulations show that the self-organization of microtubule networks is robust in a wide parameter range in individual cells. We confirm this robustness in vivo on the tissue-scale using genetic manipulations of Drosophila epithelial cells. Finally, our minimal mathematical model shows that the origin of robustness is the separation of time-scales in microtubule dynamics rates. Altogether, we demonstrate that the tissue-scale self-organization of a microtubule network depends only on cell geometry and the distribution of the microtubule minus-ends.

Fluorescence Recovery After Photobleaching to Study the Dynamics of Membrane-Bound Proteins In Vivo Using the Drosophila Embryo.

The epithelial-to-mesenchymal transition is a highly dynamic cell process and tools such as fluorescence recovery after photobleaching (FRAP), which allow the study of rapid protein dynamics, enable the following of this process in vivo. This technique uses a short intense pulse of photons to disrupt the fluorescence of a tagged protein in a region of a sample. The fluorescent signal intensity after this bleaching is then recorded and the signal recovery used to provide an indicator of the dynamics of the protein of interest. This technique can be applied to any fluorescently tagged protein, but membrane-bound proteins present an interesting challenge as they are spatially confined and subject to specialized cellular trafficking. Several methods of analysis can be applied which can disentangle these various processes and enable the extraction of information from the recovery curves. Here we describe this technique when applied to the quantification of the plasma membrane-bound E-cadherin protein in vivo using the epidermis of the late embryo of Drosophila melanogaster (Drosophila) as an example of this technique.

The Cross-Talk Between EGFR and E-Cadherin.

Epidermal growth factor receptor (EGFR) and adhesion protein E-cadherin are major regulators of proliferation and differentiation in epithelial cells. Consistently, defects in both EGFR and E-cadherin-mediated intercellular adhesion are linked to various malignancies. These defects in either are further exacerbated by the reciprocal interactions between the two transmembrane proteins. On the one hand, EGFR can destabilize E-cadherin adhesion by increasing E-cadherin endocytosis, modifying its interactions with cytoskeleton and decreasing its expression, thus promoting tumorigenesis. On the other hand, E-cadherin regulates EGFR localization and tunes its activity. As a result, loss and mutations of E-cadherin promote cancer cell invasion due to uncontrolled activation of EGFR, which displays enhanced surface motility and changes in endocytosis. In this minireview, we discuss the molecular and cellular mechanisms of the cross-talk between E-cadherin and EGFR, highlighting emerging evidence for the role of endocytosis in this feedback, as well as its relevance to tissue morphogenesis, homeostasis and cancer progression.

Arf6 determines tissue architecture by stabilizing intercellular adhesion.

Correct cell shape is indispensable for tissue architecture, with cell shape being determined by cortical actin and surface adhesion. The role of adhesion in remodelling tissue is to counteract the deformation of cells by force, resulting from actomyosin contractility, and to maintain tissue integrity. The dynamics of this adhesion are critical to the processes of cell shape formation and maintenance. Here, we show that the trafficking molecule Arf6 has a direct impact on cell elongation, by acting to stabilize E-cadherin-based adhesion complexes at the cell surface, in addition to its canonical role in endocytosis. We demonstrate that these functions of Arf6 are dependent on the molecule Flotillin1, which recruits Arf6 to the plasma membrane. Our data suggest that Arf6 and Flotillin1 operate in a pathway distinct from clathrin-mediated endocytosis. Altogether, we demonstrate that Arf6- and Flotillin1-dependent regulation of the dynamics of cell adhesion contribute to moulding tissue in vivo. This article is part of the discussion meeting issue 'Contemporary morphogenesis'.

Interplay between actomyosin and E-cadherin dynamics regulates cell shape in the Drosophila embryonic epidermis.

Precise regulation of cell shape is vital for building functional tissues. Here, we study the mechanisms that lead to the formation of highly elongated anisotropic epithelial cells in the Drosophila epidermis. We demonstrate that this cell shape is the result of two counteracting mechanisms at the cell surface that regulate the degree of elongation: actomyosin, which inhibits cell elongation downstream of RhoA (Rho1 in Drosophila) and intercellular adhesion, modulated via clathrin-mediated endocytosis of E-cadherin (encoded by shotgun in flies), which promotes cell elongation downstream of the GTPase Arf1 (Arf79F in Drosophila). We show that these two mechanisms do not act independently but are interconnected, with RhoA signalling reducing Arf1 recruitment to the plasma membrane. Additionally, cell adhesion itself regulates both mechanisms - p120-catenin, a regulator of intercellular adhesion, promotes the activity of both Arf1 and RhoA. Altogether, we uncover a complex network of interactions between cell-cell adhesion, the endocytic machinery and the actomyosin cortex, and demonstrate how this network regulates cell shape in an epithelial tissue in vivo.

Typhoid toxin exhausts the RPA response to DNA replication stress driving senescence and Salmonella infection.

Salmonella Typhi activates the host DNA damage response through the typhoid toxin, facilitating typhoid symptoms and chronic infections. Here we reveal a non-canonical DNA damage response, which we call RING (response induced by a genotoxin), characterized by accumulation of phosphorylated histone H2AX (γH2AX) at the nuclear periphery. RING is the result of persistent DNA damage mediated by toxin nuclease activity and is characterized by hyperphosphorylation of RPA, a sensor of single-stranded DNA (ssDNA) and DNA replication stress. The toxin overloads the RPA pathway with ssDNA substrate, causing RPA exhaustion and senescence. Senescence is also induced by canonical γΗ2ΑΧ foci revealing distinct mechanisms. Senescence is transmitted to non-intoxicated bystander cells by an unidentified senescence-associated secreted factor that enhances Salmonella infections. Thus, our work uncovers a mechanism by which genotoxic Salmonella exhausts the RPA response by inducing ssDNA formation, driving host cell senescence and facilitating infection.

Diverse integrin adhesion stoichiometries caused by varied actomyosin activity.

Cells in an organism are subjected to numerous sources of external and internal forces, and are able to sense and respond to these forces. Integrin-mediated adhesion links the extracellular matrix outside cells to the cytoskeleton inside, and participates in sensing, transmitting and responding to forces. While integrin adhesion rapidly adapts to changes in forces in isolated migrating cells, it is not known whether similar or more complex responses occur within intact, developing tissues. Here, we studied changes in integrin adhesion composition upon different contractility conditions in Drosophila embryonic muscles. We discovered that all integrin adhesion components tested were still present at muscle attachment sites (MASs) when either cytoplasmic or muscle myosin II was genetically removed, suggesting a primary role of a developmental programme in the initial assembly of integrin adhesions. Contractility does, however, increase the levels of integrin adhesion components, suggesting a mechanism to balance the strength of muscle attachment to the force of muscle contraction. Perturbing contractility in distinct ways, by genetic removal of either cytoplasmic or muscle myosin II or eliminating muscle innervation, each caused unique alterations to the stoichiometry at MASs. This suggests that different integrin-associated proteins are added to counteract different kinds of force increase.

Microtubule organization is determined by the shape of epithelial cells.

Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia.

The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo.

Dynamic control of gene expression is essential for the development of a totipotent zygote into an embryo with defined cell lineages. The accessibility of genes responsible for cell specification to transcriptional machinery is dependent on chromatin remodelling complexes such as the SWI\SNF (BAF) complex. However, the role of the BAF complex in early mouse development has remained unclear. Here, we demonstrate that BAF155, a major BAF complex subunit, regulates the assembly of the BAF complex in vivo and regulates lineage specification of the mouse blastocyst. We find that associations of BAF155 with other BAF complex subunits become enriched in extra-embryonic lineages just prior to implantation. This enrichment is attributed to decreased mobility of BAF155 in extra-embryonic compared with embryonic lineages. Downregulation of BAF155 leads to increased expression of the pluripotency marker Nanog and its ectopic expression in extra-embryonic lineages, whereas upregulation of BAF155 leads to the upregulation of differentiation markers. Finally, we show that the arginine methyltransferase CARM1 methylates BAF155, which differentially influences assembly of the BAF complex between the lineages and the expression of pluripotency markers. Together, our results indicate a novel role of BAF-dependent chromatin remodelling in mouse development via regulation of lineage specification.

Drosophila p120-catenin is crucial for endocytosis of the dynamic E-cadherin-Bazooka complex.

The intracellular functions of classical cadherins are mediated through the direct binding of two catenins: ß-catenin and p120-catenin (also known as CTNND1 in vertebrates, and p120ctn in Drosophila). Whereas ß-catenin is crucial for cadherin function, the role of p120-catenin is less clear and appears to vary between organisms. We show here that p120-catenin has a conserved role in regulating the endocytosis of cadherins, but that its ancestral role might have been to promote endocytosis, followed by the acquisition of a new inhibitory role in vertebrates. In Drosophila, p120-catenin facilitates endocytosis of the dynamic E-cadherin-Bazooka subcomplex, which is followed by its recycling. The absence of p120-catenin stabilises this subcomplex at the membrane, reducing the ability of cells to exchange neighbours in embryos and expanding cell-cell contacts in imaginal discs.

Dynamic microtubules produce an asymmetric E-cadherin-Bazooka complex to maintain segment boundaries.

Distributing junctional components around the cell periphery is key for epithelial tissue morphogenesis and homeostasis. We discovered that positioning of dynamic microtubules controls the asymmetric accumulation of E-cadherin. Microtubules are oriented preferentially along the dorso-ventral axis in Drosophila melanogaster embryonic epidermal cells, and thus more frequently contact E-cadherin at dorso-ventral cell-cell borders. This inhibits RhoGEF2, reducing membrane recruitment of Rho-kinase, and increasing a specific E-cadherin pool that is mobile when assayed by fluorescence recovery after photobleaching. This mobile E-cadherin is complexed with Bazooka/Par-3, which in turn is required for normal levels of mobile E-cadherin. Mobile E-cadherin-Bazooka prevents formation of multicellular rosette structures and cell motility across the segment border in Drosophila embryos. Altogether, the combined action of dynamic microtubules and Rho signaling determines the level and asymmetric distribution of a mobile E-cadherin-Bazooka complex, which regulates cell behavior during the generation of a patterned epithelium.

Dynorphin B induces lateral asymmetric changes in feline spinal cord reflexes.

The effects of dynorphin B (an agonist of κ-opioid receptors) and naloxone (an antagonist of opioid receptors) on the field potentials (FPs) evoked in the lumbar spinal cord of spinalized cats were examined following successive stimulation of pairs of identical peripheral nerves on both sides of the body. The FPs were recorded bilaterally using microelectrodes from symmetrical sites of the gray matter between the L6 and L7 segments of the spinal cord transected at level of Th11. Significant changes (up to 75%) were registered in the areas of the initial positive components of the FPs evoked by sequential stimulation of the nn. gastrocnemius-soleus, flexor digitorum longus, and tibialis at both hind limbs; a difference between the effects of various nerves was not observed. Two-Way ANOVA analysis showed that two factors, the injection type and recording side, as well as a combination of these factors, strongly influenced the amplitudes of the FPs. Statistically significant side- and injection-dependent differences were registered in the majority of the tests. Both the directions of the changes in the FPs and their relative amplitudes were not strongly connected with a definite side of the spinal cord in different animals. Therefore, it is possible to postulate that the κ-opioid receptors are distributed inhomogeneously over the neuronal populations transmitting the peripheral afferent signals from different hind limbs, thus indicating a possible presence of the lateral asymmetry effects.

Fluorescent proteins enhance UVC PDT of cancer cells.

Cancer cells, with and without fluorescent protein expression, were irradiated with various doses of UVC (100, 400, and 600 J/m(2)). Dual-color Lewis lung carcinoma cells (LLC) and U87 human glioma cells, expressing GFP in the nucleus and RFP in the cytoplasm and non-colored LLC and U87 cells were cultured in 96-well plates. Eight hours after seeding, the cells were irradiated with the various doses of UVC. The resulting cell number was determined after 24 hours. Compared to non-colored LLC cells, the number of dual-color LLC cells decreased significantly due to UVC irradiation with 100 J/m(2) (p=0.003). Although there was no significant difference in the number of dual-color and non-colored U87 cells after 100 J/m(2) UVC irradiation (p=0.852), the number of dual-color U87 cells decreased significantly with respect to non-colored cells due to UVC irradiation with 400 J/m(2) and 600 J/m(2) (p=0.011 and p=0.009, respectively). Thus, both dual-color LLC and dual-color U87 cells were more sensitive to UVC light than non-colored LLC and U87 cells. These results suggest that the expression of fluorescent proteins in cancer cells can enhance photodynamic therapy (PDT) using UVC and possibly with other wavelengths of light as well.