Role of Src-family kinases in formation of the cortical actin cap at the dorsal cell surface

Protein-tyrosine phosphorylation is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Src-family tyrosine kinases (SFKs) participate in the regulation of the actin cytoskeleton. Actin filaments can be accumulated in a cap at the dorsal cell surface, which is called the cortical actin cap. Here, we show that SFKs play an important role in formation of the cortical actin cap. HeLa cells normally exhibit the cortical actin cap, one of the major sites of tyrosine phosphorylation. The cortical actin cap is disrupted by SFK inhibitors or overexpression of the Lyn SH3 domain. Csk-knockout cells form the cortical actin cap when the level of tyrosine phosphorylation is increased by Na₃VO₄, a PTP inhibitor, and the formation of the cortical actin cap is inhibited by SFK inactivation with re-introduction of Csk. SYF cells lacking SFKs minimally exhibit the cortical actin cap even in the presence of Na₃VO₄, and transfection with Lyn restores the cortical actin cap in the presence of Na₃VO₄. Disruption of the cortical actin cap by dominant-negative Cdc42 causes loss of tyrosine phosphorylation at the cell top. These results suggest that SFK(s) is involved in formation of the cortical actin cap, which may serve as a platform of tyrosine phosphorylation signaling.     

Differences in cortical actin structure and dynamics document that different types of blebs are formed by distinct mechanisms

Bleb formation has been studied by specifically targeting major factors controlling this process, such as microtubule disassembly, local actin depolymerization, and increased pressure. At least two different types of blebs (types 1 and 2) formed by different mechanisms and possibly a third type (type 3) can be documented at the front of living polarized cells expressing green fluorescent protein-actin and/or in fixed and stained cells. Type 1 blebs (membrane/cortex dissociation blebs) formed by dissociation of the plasma membrane from cortical actin develop cytoplasmic actin layers associated with restriction rings. They can be induced by the microtubule-disassembling agent colchicine. Type 2 blebs (cortical actin disassembly blebs) form after disassembly of the cortical actin layer in the presence of latrunculin A. Restriction rings without a cytoplasmic actin layer occur in a transition zone between the intact cortical actin layer of the cell body and the compromised actin layer of the bleb. Evidence for a third type of bleb (type 3), showing an intact cortical actin layer but no cytoplasmic actin layer and no recognizable relationship between the actin cytoskeleton and the restriction ring, has been obtained by passive cell deformation in micropipettes, which increases pressure. Repolymerization of the cortical actin layer does not necessarily result in bleb retraction. Once formed, restriction rings do not narrow, suggesting that they result from isometric contraction. A simplified classification scheme has been developed to relate the type of bleb to specific signals or cell functions. Its application shows that spontaneously blebbing cells form almost exclusively type 1 blebs.     

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

Bleb-driven chemotaxis of Dictyostelium cells

Blebs and F-actin–driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.     

GPCR signaling is required for blood-brain barrier formation in drosophila

The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. Here, we show that the G protein-coupled receptor (GPCR) Moody, the G protein subunits G alpha i and G alpha o, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. Our data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. Our study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl₂, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl₂. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na⁺, Mg²⁺, or Mn²⁺ are present in addition to Ca²⁺, but not if comparable concentrations of K⁺ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K⁺ but not exogenous Ca²⁺. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl₂ has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca²⁺ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K⁺ antagonizes this Ca²⁺-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca²⁺ to intracellular K⁺ is critical in the control of polar lobe formation and cytokinesis.     

Gβ Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration

For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis. Here, we demonstrate that the strength of coupling between individual actin oscillators controls cell polarization and directional movement. We implement an inducible sequestration system to inactivate the heterotrimeric G protein subunit Gβ and find that this acute perturbation triggers persistent, high-amplitude cortical oscillations of F-actin. Actin oscillators that are normally weakly coupled to one another in wild-type cells become strongly synchronized following acute inactivation of Gβ. This global coupling impairs sensing of internal cues during spontaneous polarization and sensing of external cues during directional motility. A simple mathematical model of coupled actin oscillators reveals the importance of appropriate coupling strength for chemotaxis: moderate coupling can increase sensitivity to noisy inputs. Taken together, our data suggest that Gβ regulates the strength of coupling between actin oscillators for efficient polarity and directional migration. As these observations are only possible following acute inhibition of Gβ and are masked by slow compensation in genetic knockouts, our work also shows that acute loss-of-function approaches can complement and extend the reach of classical genetics in Dictyostelium and likely other systems as well.     

A Mec17-Myosin II Effector Axis Coordinates Microtubule Acetylation and Actin Dynamics to Control Primary Cilium Biogenesis

Primary cilia are specialized, acetylated microtubule-based signaling processes. Cilium assembly is activated by cellular quiescence and requires reconfiguration of microtubules, the actin cytoskeleton, and vesicular trafficking machinery. How these components are coordinated to activate ciliogenesis remains unknown. Here we identify the microtubule acetyltransferase Mec-17 and myosin II motors as the key effectors in primary cilium biogenesis. We found that myosin IIB (Myh10) is required for cilium formation; however, myosin IIA (Myh9) suppresses it. Myh10 binds and antagonizes Myh9 to increase actin dynamics, which facilitates the assembly of the pericentrosomal preciliary complex (PPC) that supplies materials for cilium growth. Importantly, Myh10 expression is upregulated by serum-starvation and this induction requires Mec-17, which is itself accumulated upon cellular quiescence. Pharmacological stimulation of microtubule acetylation also induces Myh10 expression and cilium formation. Thus cellular quiescence induces Mec17 to couple the production of acetylated microtubules and Myh10, whose accumulation overcomes the inhibitory role of Myh9 and initiates ciliogenesis.     

Myosin I is required for hypha formation in Candida albicans

The pathogenic yeast Candida albicans can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the CaMYO5 gene encoding the only myosin I in C. albicans. A strain with a deletion of both copies of CaMYO5 is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the “TEDS-rule” site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing Camyo5^S366D were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.     

Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.     

Actin polymerization and intracellular solvent flow in cell surface blebbing

The cortical actin gel of eukaryotic cells is postulated to control cell surface activity. One type of protrusion that may offer clues to this regulation are the spherical aneurysms of the surface membrane known as blebs. Blebs occur normally in cells during spreading and alternate with other protrusions, such as ruffles, suggesting similar protrusive machinery is involved. We recently reported that human melanoma cell lines deficient in the actin filament cross-linking protein, ABP-280, show prolonged blebbing, thus allowing close study of blebs and their dynamics. Blebs expand at different rates of volume increase that directly predict the final size achieved by each bleb. These rates decrease as the F-actin concentration of the cells increase over time after plating on a surface, but do so at lower concentrations in ABP-280 expressing cells. Fluorescently labeled actin and phalloidin injections of blebbing cells indicate that a polymerized actin structure is not present initially, but appears later and is responsible for stopping further bleb expansion. Therefore, it is postulated that blebs occur when the fluid-driven expansion of the cell membrane is sufficiently rapid to initially outpace the local rate of actin polymerization. In this model, the rate of intracellular solvent flow driving this expansion decreases as cortical gelation is achieved, whether by factors such as ABP-280, or by concentrated actin polymers alone, thereby leading to decreased size and occurrence of blebs. Since the forces driving bleb extension would always be present in a cell, this process may influence other cell protrusions as well.     

Smooth Muscle α Actin (Acta2) and Myofibroblast Function during Hepatic Wound Healing

Smooth muscle α actin (Acta2) expression is largely restricted to smooth muscle cells, pericytes and specialized fibroblasts, known as myofibroblasts. Liver injury, associated with cirrhosis, induces transformation of resident hepatic stellate cells into liver specific myofibroblasts, also known as activated cells. Here, we have used in vitro and in vivo wound healing models to explore the functional role of Acta2 in this transformation. Acta2 was abundant in activated cells isolated from injured livers but was undetectable in quiescent cells isolated from normal livers. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. Inhibition of Acta2 using several different techniques had no effect on cytoplasmic actin isoform expression, but led to reduced cellular motility and contraction. Additionally, Acta2 knockdown was associated with a significant reduction in Erk1/2 phosphorylation compared to control cells. The data indicate that Acta2 is important specifically in myofibroblast cell motility and contraction and raise the possibility that the Acta2 cytoskeleton, beyond its structural importance in the cell, could be important in regulating signaling processes during wound healing in vivo.     

Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner

To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion (P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin _v3-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion (P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior. cytoskeletal remodeling; dynamic blebs     

Bipolar segregation of mitochondria,actin network,and surface in the Tubifex egg: Role of cortical polarity

The role of the cortex in ooplasmic segregation of the yolky eggs of Tubifex has been studied by epifluorescence microscopy. Living eggs labeled with rhodamine 123 and fine carbon particles placed on the surface showed that, following the second polar body formation, the egg surface cosegregates with subcortical mitochondria in a bipolar fashion, viz. toward the animal and vegetal poles in the animal and vegetal hemispheres, respectively. The egg surface of each pole moves spirally while the equatorial surface appears to remain stationary during this process. The rhodamine-phalloidin staining of whole eggs reveals that actin networks cosegregate with mitochondria. Isolated cortices which were stained with rhodamine-phalloidin demonstrated that cortical actin is organized bipolarly and that, during ooplasmic segregation, it undergoes reorganization directed toward both poles of the egg. The cortical polarity expressed as actin organization is not disrupted by centrifugal force sufficient to stratify the egg cytoplasm into five layers. The surface of a centrifuged egg moves according to the original cortical polarity. This surface movement is accompanied by the reorganization of cortical actin which appears to be identical to that in intact eggs. Other centrifugation experiments have demonstrated that the connection of the subcortical cytoplasm to the cortex is resistant to a centrifugal force of up to 650g. The nature of cortical polarity and its role in ooplasmic segregation are discussed in the light of the present results.     

Relation between cell activity and the distribution of cytoplasmic actin and myosin

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.     

The Tension at the Top of the Animal Pole Decreases during Meiotic Cell Division

Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish (Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation.     

ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.     

The Actin Targeting Compound Chondramide Inhibits Breast Cancer Metastasis via Reduction of Cellular Contractility

Background

A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent.

Methods

In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis.

Results

In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction.

Conclusions

Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion.     

Evidence for G-quadruplex in the promoter of vegfr-2 and its targeting to inhibit tumor angiogenesis

Tumor angiogenesis is mainly mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced by cancer cells and active on the endothelium through the VEGF receptor 2 (VEGFR-2). Here we identify a G-rich sequence within the proximal promoter region of vegfr-2, able to form an antiparallel G-quadruplex (G4) structure. This G4 structure can be efficiently stabilized by small molecules with the consequent inhibition of vegfr-2 expression. Functionally, the G4-mediated reduction of VEGFR-2 protein causes a switching off of signaling components that, converging on actin cytoskeleton, regulate the cellular events leading to endothelial cell proliferation, migration and differentiation. As a result of endothelial cell function impairment, angiogenic process is strongly inhibited by G4 ligands both in vitro and in vivo. Interestingly, the G4-mediated antiangiogenic effect seems to recapitulate that observed by using a specific interference RNA against vegfr-2, and it is strongly antagonized by overexpressing the vegfr-2 gene. In conclusion, we describe the evidence for the existence of G4 in the promoter of vegfr-2, whose expression and function can be markedly inhibited by G4 ligands, thereby revealing a new, and so far undescribed, way to block VEGFR-2 as target for anticancer therapy.