E-cadherin focuses protrusion formation at the front of migrating cells by impeding actin flow.

The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.

Germ cell migration-Evolutionary issues and current understanding.

In many organisms, primordial germ cells (PGCs) are specified at a different location than where the gonad forms, meaning that PGCs must migrate toward the gonad within the early developing embryo. Following species-specific paths, PGCs can be passively carried by surrounding tissues and also perform active migration. When PGCs actively migrate through and along a variety of embryonic structures in different organisms, they adopt an ancestral robust migration mode termed "amoeboid motility", which allows cells to migrate within diverse environments. In this review, we discuss the possible significance of the PGC migration process in facilitating the evolution of animal body shape. In addition, we summarize the latest findings relevant for the molecular and cellular mechanisms controlling the movement and the directed migration of PGCs in different species.

Tracking and line integration of diffuse cellular subdomains by mesh advection.

Active molecular transport ensures a purposeful spatiotemporal distribution of cellular proteins and is therefore key to a wide range of processes such as morphogenesis, homeostasis or migration. However, redistributions of molecules in bulk are seldom quantified because the regions involved are too diffuse to be segmented consistently. To bridge this gap, we propose a Laplace-corrected Runge-Kutta advection that is based on mesh triangulation. Our framework can follow the movement and deformation of multiple parts of a diffuse region at once and offers a seamless combination with spatiotemporal line integration in Lagrangian coordinates. This allows the flexibility to taylor specific measures to the question at hand, e.g. mechanical work, bringing long-established physics concepts into biology grounds. We exemplify our approach by quantifying how the isotropy of intracellular protein distributions changes during cargo transport.

Chemokine-Dependent pH Elevation at the Cell Front Sustains Polarity in Directionally Migrating Zebrafish Germ Cells.

Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent. Intracellular pH gradients were shown to occur in certain cells migrating in vitro, but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types, including metastatic cancer cells, is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells.

Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo.

Cell migration and polarization is controlled by signals in the environment. Migrating cells typically form filopodia that extend from the cell surface, but the precise function of these structures in cell polarization and guided migration is poorly understood. Using the in vivo model of zebrafish primordial germ cells for studying chemokine-directed single cell migration, we show that filopodia distribution and their dynamics are dictated by the gradient of the chemokine Cxcl12a. By specifically interfering with filopodia formation, we demonstrate for the first time that these protrusions play an important role in cell polarization by Cxcl12a, as manifested by elevation of intracellular pH and Rac1 activity at the cell front. The establishment of this polarity is at the basis of effective cell migration towards the target. Together, we show that filopodia allow the interpretation of the chemotactic gradient in vivo by directing single-cell polarization in response to the guidance cue.

Temporal control over the initiation of cell motility by a regulator of G-protein signaling.

The control over the acquisition of cell motility is central for a variety of biological processes in development, homeostasis, and disease. An attractive in vivo model for investigating the regulation of migration initiation is that of primordial germ cells (PGCs) in zebrafish embryos. In this study, we show that, following PGC specification, the cells can polarize but do not migrate before the time chemokine-encoded directional cues are established. We found that the regulator of G-protein signaling 14a protein, whose RNA is a newly identified germ plasm component, regulates the temporal relations between the appearance of the guidance molecules and the acquisition of cellular motility by regulating E-cadherin levels.

Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against T-cell acute lymphoblastic leukemia.

Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lend compelling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays. NVP-BEZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dependent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remarkably, NVP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which might correspond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide, cytarabine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation, where the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamycin. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferation and survival.

The emerging role of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in normal myelopoiesis and leukemogenesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway mediates diverse and important physiological cell functions which include proliferation, differentiation, survival, motility, autophagy, and metabolism. However, dysregulated PI3K/Akt/mTOR signaling has been documented in a wide range of neoplasias, including malignant hematological disorders. It is now emerging that this signaling network plays a key role during normal hematopoiesis, a tightly regulated process resulting in the formation of all blood lineages. Blood cell development encompasses a complex series of events which are mainly regulated by actions of cytokines, a family of extracellular ligands which stimulate many biological responses in a wide array of cell types. Hematopoiesis is strictly dependent on the correct function of the bone marrow microenvironment (BMM), as BMM cells secrete most of the cytokines. Several of these cytokines activate the PI3K/Akt/mTOR signaling network and regulate proliferation, survival, and differentiation events during hematopoiesis. Here, we review the evidence that links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of hematopoietic stem cells and the process of myelopoiesis, including lineage commitment. We then highlight the emerging role played by aberrant PI3K/Akt/mTOR signaling during leukemogenesis.

The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling network and the control of normal myelopoiesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in cell growth, proliferation, differentiation, and survival under physiological conditions. Aberrant regulation of the PI3K/Akt/mTOR signal transduction network has been observed in a wide range of neoplasias, including malignant hematological disorders. This observation suggests that this signaling cascade could also play a critical role during normal hematopoiesis, a highly regulated process which results in the formation of all blood lineages. The development of blood cells comprises a complex series of events which are mainly regulated through the actions of cytokines, a large family of extracellular ligands than can stimulate many biological responses in a wide array of cell types. Several of these cytokines are known to activate the PI3K/Akt/mTOR signal transduction network and thus regulate proliferation, survival, and differentiation events during hematopoiesis. Moreover, hematopoiesis is strictly dependent on the correct functions of the bone marrow microenvironment. Here, we review the evidence which links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of hematopoietic stem cells and the process of lineage commitment, which then gives rise to myeloid lineage-restricted cells. We then further highlight the key role played by the PI3K/Akt/mTOR network during erythropoiesis, megakaryocytopoiesis, and granulo-cytopoiesis/monocytopoiesis.

The emerging role of the phosphatidylinositol 3-kinase/ akt/mammalian target of rapamycin signaling network in cancer stem cell biology.

The cancer stem cell theory entails the existence of a hierarchically organized, rare population of cells which are responsible for tumor initiation, self-renewal/maintenance, and mutation accumulation. The cancer stem cell proposition could explain the high frequency of cancer relapse and resistance to currently available therapies. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway regulates a wide array of physiological cell functions which include differentiation, proliferation, survival, metabolism, autophagy, and motility. Dysregulated PI3K/Akt/mTOR signaling has been documented in many types of neoplasias. It is now emerging that this signaling network plays a key role in cancer stem cell biology. Interestingly, cancer stem cells displayed preferential sensitivity to pathway inhibition when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling pathways between neoplastic stem cells and healthy stem cells could be identified. In this review, we present the evidence which links the signals emanating from the PI3K/Akt/mTOR cascade with the functions of cancer stem cells, both in solid and hematological tumors. We then highlight how targeting PI3K/Akt/mTOR signaling with small molecules could improve cancer patient outcome.

Targeting the PI3K/AKT/mTOR signaling network in acute myelogenous leukemia.

BACKGROUND: The PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in cell growth, proliferation and survival not only under physiological conditions but also in a variety of tumor cells. Therefore, the PI3K/Akt/mTOR axis may be a critical target for cancer therapy. OBJECTIVE: This review discusses how PI3K/Akt/mTOR signaling network is constitutively active in acute myelogenous leukemia (AML), where it strongly influences proliferation, survival and drug-resistance of leukemic cells, and how effective targeting of this pathway with pharmacological inhibitors, used alone or in combination with existing drugs, may result in suppression of leukemic cell growth, including leukemic stem cells. METHODS: We searched the literature for articles dealing with activation of this pathway in AML and highlighting the efficacy of small molecules directed against the PI3K/Akt/mTOR signaling cascade. CONCLUSIONS: The limit of acceptable toxicity for standard chemotherapy has been reached in AML. Therefore, new therapeutic strategies are needed. Targeting the PI3K/Akt/mTOR signaling network with small molecule inhibitors, alone or in combinations with other drugs, may result in less toxic and more efficacious treatment of AML patients. Efforts to exploit selective inhibitors of the PI3K/Akt/mTOR pathway that show effectiveness and safety in the clinical setting are currently underway.

PKR activity is required for acute leukemic cell maintenance and growth: a role for PKR-mediated phosphatase activity to regulate GSK-3 phosphorylation.

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p-T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant-negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time-dependent augmentation of AKT S473 and GSK-3alpha S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK-3alpha was not dependent on PI3K activity. PKR inhibition augmented levels of p-S473 AKT and p-S21/9 GSK-3alpha/beta in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK-3alpha or beta phosphorylation in the presence of the AKT inhibitor, A443654. Pre-treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2alpha, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2alpha phosphorylation. The effects of PKR inhibition on AKT and GSK-3 phosphorylation were found to be, in part, PP2A-dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK-3.