Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos.

The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

TFEB controls vascular development by regulating the proliferation of endothelial cells.

Transcription factor TFEB is thought to control cellular functions-including in the vascular bed-primarily via regulation of lysosomal biogenesis and autophagic flux. Here, we report that TFEB also orchestrates a non-canonical program that controls the cell cycle/VEGFR2 pathway in the developing vasculature. In endothelial cells, TFEB depletion halts proliferation at the G1-S transition by inhibiting the CDK4/Rb pathway. TFEB-deficient cells attempt to compensate for this limitation by increasing VEGFR2 levels at the plasma membrane via microRNA-mediated mechanisms and controlled membrane trafficking. TFEB stimulates expression of the miR-15a/16-1 cluster, which limits VEGFR2 transcript stability and negatively modulates expression of MYO1C, a regulator of VEGFR2 trafficking to the cell surface. Altered levels of miR-15a/16-1 and MYO1C in TFEB-depleted cells cause increased expression of plasma membrane VEGFR2, but in a manner associated with low signaling strength. An endothelium-specific Tfeb-knockout mouse model displays defects in fetal and newborn mouse vasculature caused by reduced endothelial proliferation and by anomalous function of the VEGFR2 pathway. These previously unrecognized functions of TFEB expand its role beyond regulation of the autophagic pathway in the vascular system.

A pilot study of next generation sequencing-liquid biopsy on cell-free DNA as a novel non-invasive diagnostic tool for Klippel-Trenaunay syndrome.

OBJECTIVES: Somatic mosaicism of PIK3CA gene is currently recognized as the molecular driver of Klippel-Trenaunay syndrome. However, given the limitation of the current technologies, PIK3CA somatic mutations are detected only in a limited proportion of Klippel-Trenaunay syndrome cases and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next generation sequencing liquid biopsy using cell-free DNA has emerged as an innovative non-invasive approach for early detection and monitoring of cancer. This approach, overcoming the space-time profile constraint of tissue biopsies, opens a new scenario also for others diseases caused by somatic mutations. METHODS: In the present study, we performed a comprehensive analysis of seven patients (four females and three males) with Klippel-Trenaunay syndrome. Blood samples from both peripheral and efferent vein from malformation were collected and cell-free DNA was extracted from plasma. Tissue biopsies from vascular lesions were also collected when available. Cell-free DNA libraries were performed using Oncomine™ Pan-Cancer Cell-Free Assay. Ion Proton for sequencing and Ion Reporter Software for analysis were used (Life Technologies, Carlsbad, CA, USA). RESULTS: Cell-free circulating DNA analysis revealed pathogenic mutations in PIK3CA gene in all patients. The mutational load was higher in plasma obtained from the efferent vein at lesional site (0.81%) than in the peripheral vein (0.64%) leading to conclude for a causative role of the identified variants. Tissue analysis, available for one amputated patient, confirmed the presence of the mutation at the malformation site at a high molecular frequency (14-25%), confirming its causative role. CONCLUSIONS: Our data prove for the first time that the cell-free DNA-next generation sequencing-liquid biopsy, which is currently used exclusively in an oncologic setting, is indeed the most effective tool for Klippel-Trenaunay syndrome diagnosis and tailored personalized treatment.

Irreversible Activation of Rho-activated Kinases Resulted from Evolution of Proteolytic Sites within Disordered Regions in Coiled-coil Domain.

Activation of Rho-associated protein kinase 1 (ROCK1) and myotonic dystrophy kinase-related CDC42-binding kinase alpha (MRCKα) by caspases during apoptosis in vertebrates represents a prototypical example of co-option of kinases by proteases. How caspases acquired the ability to control these proteins during evolution of vertebrates is still unknown. Here, we report a phylogenetic and molecular study on the acquisition of caspase-cleavage sites in the family of Rho-activated kinases (RaKs). We demonstrate that the acquisition of such sites has more frequently occurred in identifiable intrinsically disordered regions (IDRs) within or flanking the coiled-coil domain. Thanks to computational identification of IDRs in protein sequences of different organisms, we predicted and validated the independent evolution of two caspase-cleavage sites in ROCK of arthropods and the loss of one of the MRCKα caspase-cleavage sites in ray-finned fishes. In conclusion, we shed light on the propensity of RaKs to evolve novel proteolytic sites, causing kinase activation and uniform subcellular distribution.

Death for life: a path from apoptotic signaling to tissue-scale effects of apoptotic epithelial extrusion.

Apoptosis plays a crucial role in clearing old or critically compromised cells, and actively maintains epithelial homeostasis and epithelial morphogenesis during embryo development. But how is the apoptotic signaling pathway able to orchestrate such complex and dynamic multi-cellular morphological events at the tissue scale? In this review we collected the most updated knowledge regarding how apoptosis controls different cytoskeletal components. We describe how apoptosis can control epithelial homeostasis though epithelial extrusion, a highly orchestrated process based on high- order actomyosin structures and on the coordination between the apoptotic and the neighboring cells. Finally, we describe how the synergy among forces generated by multiple apoptotic cells can shape epithelia in embryo development.

PDK1: At the crossroad of cancer signaling pathways.

Rational target therapy of cancer would benefit from the identification of new targets that can be easily inhibited by small molecules. An increasing amount of evidence hints at 3-phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) as an intriguing and underexplored target for cancer therapy. Several reports show that PDK1 expression is dysregulated in multiple cancer types. Furthermore PDK1 is implicated in signaling pathways frequently altered in cancer, such as PI3K/Akt, Ras/MAPK and Myc. PDK1 targeting has been proven to be effective in experimental models harboring alterations of these pathways. In this paper we review PDK1 main biochemical mechanisms, its alterations in cancer and interactions with relevant cancer pathways. A potential role of PDK1 in tumor microenvironment is also discussed.

PDK1 regulates focal adhesion disassembly by modulating endocytosis of αvß3 integrin.

Non-amoeboid cell migration is characterised by dynamic competition among multiple protrusions to establish new adhesion sites at the cell's leading edge. However, the mechanisms that regulate the decision to disassemble or to grow nascent adhesions are not fully understood. Here we show that, in endothelial cells, 3-phosphoinositide-dependent protein kinase 1 (PDK1) promotes focal adhesion (FA) turnover by controlling endocytosis of integrin αvß3 in a PI3K-dependent manner. We demonstrate that PDK1 binds and phosphorylates integrin αvß3. Downregulation of PDK1 increases FA size and slows down their disassembly. This process requires both PDK1 kinase activity and PI3K activation but does not involve Akt. Moreover, PDK1 silencing stabilises FA in membrane protrusions decreasing migration of endothelial cells on vitronectin. These results indicate that modulation of integrin endocytosis by PDK1 hampers endothelial cell adhesion and migration on extracellular matrix, thus unveiling a novel role for this kinase.

PDK1: A signaling hub for cell migration and tumor invasion.

The ability of cells to migrate is essential for different physiological processes including embryonic development, angiogenesis, tissue repair and immune response. In the context of cancer such abilities acquire dramatic implications, as they are exploited by tumor cells to invade neighboring or distant healthy tissues. 3-Phosphoinositide dependent protein kinase-1 (PDK1 or PDPK1) is an ancient serine-threonine kinase belonging to AGC kinase family. An increasing amount of data points at a pivotal role for PDK1 in the regulation of cell migration. PDK1 is a transducer of PI3K signaling and activates multiple downstream effectors, thereby representing an essential hub coordinating signals coming from extracellular cues to the cytoskeletal machinery, the final executor of cell movement. Akt, PAK1, ß3 integrin, ROCK1, MRCKα and PLCγ1 are, according to the literature, the signaling transducers through which PDK1 regulates cell migration. In addition, PDK1 contributes to tumor cell invasion by regulating invadopodia formation and both amoeboid and collective cancer cell invasion. This and other pieces of evidence, such as its reported overexpression across several tumor types, corroborate a PDK1 role tumor aggressiveness. Altogether, these findings indicate the possibility to rationally target PDK1 in human tumors in order to counteract cancer cell dissemination in the organism.

Neuroligin 1 induces blood vessel maturation by cooperating with the α6 integrin.

The synaptic protein Neuroligin 1 (NLGN1), a cell adhesion molecule, is critical for the formation and consolidation of synaptic connectivity and is involved in vascular development. The mechanism through which NLGN1 acts, especially in vascular cells, is unknown. Here, we aimed at deepening our knowledge on the cellular activities and molecular pathways exploited by endothelial NLGN1 both in vitro and in vivo. We analyzed the phenotypic consequences of NLGN1 expression modulation in endothelial cells through in vitro angiogenesis assays and the mouse postnatal retinal angiogenesis model. We demonstrate that NLGN1, whereas not affecting endothelial cell proliferation or migration, modulates cell adhesion to the vessel stabilizing protein laminin through cooperation with the α6 integrin, a specific laminin receptor. Finally, we show that in vivo, NLGN1 and α6 integrin preferentially colocalize in the mature retinal vessels, whereas NLGN1 deletion causes an aberrant VE-cadherin, laminin and α6 integrin distribution in vessels, along with significant structural defects in the vascular tree.

Modeling human tumor angiogenesis in a three-dimensional culture system.

The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis.

Unraveling the influence of endothelial cell density on VEGF-A signaling.

Vascular endothelial growth factor-A (VEGF) is the master determinant for the activation of the angiogenic program leading to the formation of new blood vessels to sustain solid tumor growth and metastasis. VEGF specific binding to VEGF receptor-2 (VEGFR-2) triggers different signaling pathways, including phospholipase C-γ (PLC-γ) and Akt cascades, crucial for endothelial proliferation, permeability, and survival. By combining biologic experiments, theoretical insights, and mathematical modeling, we found that: (1) cell density influences VEGFR-2 protein level, as receptor number is 2-fold higher in long-confluent than in sparse cells; (2) cell density affects VEGFR-2 activation by reducing its affinity for VEGF in long-confluent cells; (3) despite reduced ligand-receptor affinity, high VEGF concentrations provide long-confluent cells with a larger amount of active receptors; (4) PLC-γ and Akt are not directly sensitive to cell density but simply transduce downstream the upstream difference in VEGFR-2 protein level and activation; and (5) the mathematical model correctly predicts the existence of at least one protein tyrosine phosphatase directly targeting PLC-γ and counteracting the receptor-mediated signal. Our data-based mathematical model quantitatively describes VEGF signaling in quiescent and angiogenic endothelium and is suitable to identify new molecular determinants and therapeutic targets.

The miR-126 regulates angiopoietin-1 signaling and vessel maturation by targeting p85ß.

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85ß). Down-regulation of miR-126 and over-expression of p85ß in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase.

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

Liver X receptor activation reduces angiogenesis by impairing lipid raft localization and signaling of vascular endothelial growth factor receptor-2.

OBJECTIVE: Liver X receptors (LXRα, LXRß) are master regulators of cholesterol homeostasis. In the endothelium, perturbations of cell cholesterol have an impact on fundamental processes. We, therefore, assessed the effects of LXR activation on endothelial functions related to angiogenesis in vitro and in vivo. METHODS AND RESULTS: LXR agonists (T0901317, GW3965) blunted migration, tubulogenesis, and proliferation of human umbilical vein endothelial cells. By affecting endothelial cholesterol homeostasis, LXR activation impaired the compartmentation of vascular endothelial growth factor receptor-2 in lipid rafts/caveolae and led to defective phosphorylation and downstream signaling of vascular endothelial growth factor receptor-2 upon vascular endothelial growth factor-A stimulation. Consistently, the antiangiogenic actions of LXR agonists could be prevented by coadministration of exogenous cholesterol. LXR agonists reduced endothelial sprouting from wild-type but not from LXRα(-/-)/LXRß(-/-) knockout aortas and blunted the vascularization of implanted angioreactors in vivo. Furthermore, T0901317 reduced the growth of Lewis lung carcinoma grafts in mice by impairing angiogenesis. CONCLUSIONS: Pharmacological activation of endothelial LXRs reduces angiogenesis by restraining cholesterol-dependent vascular endothelial growth factor receptor-2 compartmentation and signaling. Thus, administration of LXR agonists could exert therapeutic effects in pathological conditions characterized by uncontrolled angiogenesis.

Collective directional migration drives the formation of heteroclonal cancer cell clusters.

Metastasisation occurs through the acquisition of invasive and survival capabilities that allow tumour cells to colonise distant sites. While the role of multicellular aggregates in cancer dissemination is acknowledged, the mechanisms that drive the formation of multiclonal cell aggregates are not fully elucidated. Here, we show that cancer cells of different tissue of origins can perform collective directional migration and can actively form heteroclonal aggregates in 3D, through a proliferation-independent mechanism. Coalescence of distant cell clusters is mediated by subcellular actin-rich protrusions and multicellular outgrowths that extend towards neighbouring aggregates. Coherently, perturbation of cytoskeletal dynamics impairs collective migration while myosin II activation is necessary for multicellular movements. We put forward the hypothesis that cluster attraction is mediated by secreted soluble factors. Such a hypothesis is consistent with the abrogation of aggregation by inhibition of PI3K/AKT/mTOR and MEK/ERK, the chemoattracting activity of conditioned culture media and with a wide screening of secreted proteins. Our results present a novel collective migration model and shed light on the mechanisms of formation of heteroclonal aggregates in cancer.

Protein kinase D1 regulates VEGF-A-induced alphavbeta3 integrin trafficking and endothelial cell migration.

The bidirectional communication between integrin alphavbeta3 and vascular endothelial growth factor (VEGF) receptors acts to integrate and coordinate endothelial cell (EC) activity during angiogenesis. However, the molecular mechanisms involved in this signaling crosstalk are only partially revealed. We have found that protein kinase D1 (PKD1) was activated by VEGF-A, but not by other angiogenic factors, and associated with alphavbeta3 integrin. Moreover, knockdown of PKD1 increased endocytosis of alphavbeta3 and reduced its return from endosomes to the plasma membrane leading to accumulation of the integrin in Rab5- and Rab4-positive endosomes. Consistent with this, PKD1 knockdown caused defects in focal complex formation and reduced EC migration in response to VEGF-A. Moreover, knockdown of PKD1 reduced EC motility on vitronectin, whereas migration on collagen I was not PKD1 dependent. These results suggest that PKD1-regulated alphavbeta3 trafficking contributes to the angiogenesis process by integrating VEGF-A signaling with extracellular matrix interactions.

IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism.

Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.

Essential role of PDK1 in regulating endothelial cell migration.

The serine/threonine protein kinase phosphoinositide-dependent kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells (ECs) and that it is involved in the regulation of their chemotaxis. ECs differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to vascular endothelial growth factor-A (VEGF-A). In addition, PDK1(-/-) embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the overexpression of PDK1 increased the EC migration induced by VEGF-A. We propose a model of spatial distribution of PDK1 and Akt in which the synthesis of phosphatidylinositol 3,4,5 triphosphate at plasma membrane by activation of phosphoinositide 3-kinase recruits both proteins at the leading edge of the polarized ECs and promotes cell chemotaxis. These findings establish a mechanism for the spatial localization of PDK1 and its substrate Akt to regulate directional migration.

Iron metabolism in the social amoeba Dictyostelium discoideum: A role for ferric chelate reductases.

Iron is the most abundant transition metal in all living organisms and is essential for several cellular activities, including respiration, oxygen transport, energy production and regulation of gene expression. Iron starvation is used by professional phagocytes, from Dictyostelium to macrophages, as a form of defense mechanism against intracellular pathogens. Previously, we showed that Dictyostelium cells express the proton-driven iron transporter Nramp1 (Natural Resistance-Associated Macrophage Protein 1) and the homolog NrampB (Nramp2) in membranes of macropinosomes and phagosomes or of the contractile vacuole network, respectively. The Nramp-driven transport of iron across membranes is selective for ferrous ions. Since iron is mostly present as ferric ions in growth media and in engulfed bacteria, we have looked for proteins with ferric reductase activity. The Dictyostelium genome does not encode for classical STEAP (Six-Transmembrane Epithelial Antigen of Prostate) ferric reductases, but harbors three genes encoding putative ferric chelate reductase belonging to the Cytochrome b561 family containing a N terminus DOMON domain (DOpamine ß-MONooxygenase N-terminal domain). We have cloned the three genes, naming them fr1A, fr1B and fr1C. fr1A and fr1B are mainly expressed in the vegetative stage while fr1C is highly expressed in the post aggregative stage. All three reductases are localized in the endoplasmic reticulum, but Fr1A is also found in endolysosomal vesicles, in the Golgi and, to a much lower degree, in the plasma membrane, whereas Fr1C is homogeneously distributed in the plasma membrane and in macropinosomal and phagosomal membranes. To gain insight in the function of the three genes we generated KO mutants, but gene disruption was successful only for two of them (fr1A and fr1C), being very likely lethal for fr1B. fr1A- shows a slight delay in the aggregation stage of development, while fr1C- gives rise to large multi-tipped streams during aggregation and displays a strong delay in fruiting body formation. The two single mutants display altered cell growth under conditions of ferric ions overloading and, in the ability to reduce Fe3+, confirming a role of these putative ferric reductases in iron reduction and transport from endo-lysosomal vesicles to the cytosol.