Formation Of Cell Protrusions Research Articles

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial–mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-β1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-β1 and may contribute to EMT.

Phosphorylation of E3 Ligase Smurf1 Switches Its Substrate Preference in Support of Axon Development

Ubiquitin E3 ligases serve for ubiquitination of specific substrates, and its ligase efficacy is regulated by interacting proteins or substrate modifications. Whether and how the ligases themselves are modified by cellular signaling is unclear. Here we report that protein kinase A (PKA)-dependent phosphorylation of Smad Ubiquitin Regulatory Factor 1 (Smurf1) can switch its substrate preference between two proteins of opposing actions on axon development. Extracellular factors that promote axon formation elevated Smurf1 phosphorylation at a PKA site Thr³⁰⁶, and preventing this phosphorylation reduced axon formation in cultured hippocampal neurons and impaired polarization of cortical neurons in vivo. Thr³⁰⁶-phosphorylation changed the relative affinities of Smurf1 for its substrates, leading to reduced degradation of polarity protein Par6 and increased degradation of growth-inhibiting RhoA. Thus, PKA-dependent phosphorylation of the E3 ligase could switch its substrate preference, contributing to selective protein degradation required for localized cellular function.

Control of Directed Cell Migration In Vivo by Membrane-to-Cortex Attachment

Author SummaryCell migration, like any event involving shape changes, is a mechanical process controlled by complex biochemical pathways. Here, we examine cell migration in developing embryos with a combination of cell biological tools and atomic force microscopy, so as to investigate how cellular mechanical properties control migration. A fundamental step during migration is the formation of a protrusion at the leading edge of the cell. In three-dimensional environments, and particularly in vivo, cells use different protrusion types: spike-like filopodia and flattened lamellipodia, whose growth is driven by actin polymerization, and spherical blebs, which grow because of intracellular pressure pushing on the membrane. It is important to understand how the formation of different protrusion types is mechanically and molecularly controlled, and how the different protrusions specifically contribute to migration. We have addressed this using the zebrafish embryo as a model system. We show that reducing the strength of the attachment between the plasma membrane and the underlying cortical network of actin filaments, or increasing intracellular pressure, increases the proportion of cellular blebs and reduces the directionality of cell migration. Our work reveals that blebs, lamellipodia, and filopodia are not interchangeable and that the relative proportion of each type of protrusion, under the control of mechanical parameters, determines migration directionality during zebrafish gastrulation.

MicroRNA-200 Family Members Differentially Regulate Morphological Plasticity and Mode of Melanoma Cell Invasion

BackgroundA functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR-200 family has received much attention for potentially regulating tumor progression. The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis. Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others.Principal FindingsWe show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration. Individual tumor cells can invade in either elongated, “mesenchymal-type” or rounded, “amoeboid-like” modes and these two modes of invasion are inter-convertible [1]. In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion. MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion. Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions. In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology.SignificanceOverall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members.

Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases

Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology-pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of approximately 0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of betaPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.

Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. We identified myotubularin (mtm), a Drosophila melanogaster MTM1/MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. We propose that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease.

Control of cell shape and plasticity during development and disease by the actin-binding protein Drebrin.

Drebrin is an actin-binding protein, originally identified in neuronal cells, involved in the regulation of actin filament organisation, especially during the formation of neurites and cell protrusions of motile cells. Drebrin is found in diverse non-neuronal cells, primarily in association with cell processes and intercellular junctions where it again plays a key role in actin remodelling. The downregulation of Drebrin in Alzheimer's Disease and Down Syndrome and conversely its upregulation in various carcinomas indicate that Drebrin is an important component of the pathogenesis of multiple diseases.

Gas7 Functions with N-WASP to Regulate the Neurite Outgrowth of Hippocampal Neurons

Neuritogenesis, or neurite outgrowth, is a critical process for neuronal differentiation and maturation in which growth cones are formed from highly dynamic actin structures. Gas7 (growth arrest-specific gene 7), a new member of the PCH (Pombe Cdc15 homology) protein family, is predominantly expressed in neurons and is required for the maturation of primary cultured Purkinje neurons as well as the neuron-like differentiation of PC12 cells upon nerve growth factor stimulation. We report that Gas7 co-localizes and physically interacts with N-WASP, a key regulator of Arp2/3 complex-mediated actin polymerization, in the cortical region of Gas7-transfected Neuro-2a cells and growth cones of hippocampal neurons. The interaction between Gas7 and N-WASP is mediated by WW-Pro domains, which is unique in the PCH protein family, where most interactions are of the SH3-Pro kind. The interaction contributes to the formation of membrane protrusions and processes by recruiting the Arp2/3 complex in a Cdc42-independent manner. Importantly, specific interaction between Gas7 and N-WASP is required for regular neurite outgrowth of hippocampal neurons. The data demonstrate an essential role of Gas7 through its interaction with N-WASP during neuronal maturation/differentiation.

Mammalian arylsulfatase A functions as a novel component of the extracellular matrix

Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2β1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes.

The PAR-6 Polarity Protein Regulates Dendritic Spine Morphogenesis through p190 RhoGAP and the Rho GTPase

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. Here we show that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, we further show that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity.

EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Foreign Body Giant Cell Formation Is Preceded by Lamellipodia Formation and Can Be Attenuated by Inhibition of Rac1 Activation

Macrophages that are recruited to the site of implanted biomaterials undergo fusion to form surface-damaging foreign body giant cells. Exposure of peripheral blood monocytes to interleukin-4 can recapitulate the fusion process in vitro. In this study, we used interleukin-4 to induce multinucleation of murine bone marrow-derived macrophages and observed changes in cell shape, including elongation and lamellipodia formation, before fusion. Because cytoskeletal rearrangements are regulated by small GTPases, we examined the effects of inhibitors of Rho kinase (Y-32885) and Rac activation (NSC23766) on fusion. Y-32885 did not prevent cytoskeletal changes or fusion but limited the extent of multinucleation. NSC23766, on the other hand, inhibited lamellipodia formation and fusion in a dose-dependent manner. In addition, we found that in control cells, these changes were preceded by Rac1 activation. However, NSC23766 did not block the uptake of polystyrene microspheres. Likewise, short interfering RNA knockdown of Rac1 limited fusion without limiting phagocytosis. Thus, phagocytosis and fusion can be partially decoupled based on their susceptibility to NSC23766. Furthermore, poly(ethylene-co-vinyl acetate) scaffolds containing NSC23766 attenuated foreign body giant cell formation in vivo. These observations suggest that targeting Rac1 activation could protect biomaterials without compromising the ability of macrophages to perform beneficial phagocytic functions at implantation sites.

Effects of temperature and light on the formation of chloroplast protrusions in leaf mesophyll cells of high alpine plants

Chloroplasts of many alpine plants have the ability to form marked, stroma-filled protrusions that do not contain thylakoids. Effects of temperature and light intensity on the frequency of chloroplasts with such protrusions in leaf mesophyll cells of nine different alpine plant species (Carex curvula All., Leontodon helveticus Merat., Oxyria digyna (L.) Hill., Poa alpina L. ssp. vivipara, Polygonum viviparum L., Ranunculus glacialis L., Ranunculus alpestris L., Silene acaulis L. and Soldanella pusilla Baumg.) covering seven different families were studied. Leaves were exposed to either darkness and a stepwise increase in temperature (10-38 degrees C) or to different light intensities (500 and 2000 micromol photons m(-2) s(-1)) and a constant temperature of 10 or 30 degrees C in a special temperature-regulated chamber. A chloroplast protrusions index characterising the relative proportion of chloroplasts with protrusions was defined. Seven of the nine species showed a significant increase in chloroplast protrusions when temperature was elevated to over 20 degrees C. In contrast, the light level did not generally affect the abundance of chloroplasts with protrusions. Chloroplast protrusions lead to a dynamic enlargement of the chloroplast surface area. They do not appear to be directly connected to a distinct photosystem II (PSII) (F(v)/F(m)) status and thus seem to be involved in secondary, not primary, photosynthetic processes.

ANR5, an FGF Target Gene Product, Regulates Gastrulation in Xenopus

Gastrulation is a morphogenetic process in which tightly coordinated cell and tissue movements establish the three germ layers (ectoderm, mesoderm, and endoderm) to define the anterior-to-posterior embryonic organization [1]. To elicit this movement, cells modulate membrane protrusions and undergo dynamic cell interactions. Here we report that ankyrin repeats domain protein 5 (xANR5), a novel FGF target gene product, regulates cell-protrusion formation and tissue separation, a process that develops the boundary between the ectoderm and mesoderm [2, 3], during Xenopus gastrulation. Loss of xANR5 function by antisense morpholino oligonucleotide (MO) caused a short trunk and spina bifida without affecting mesodermal gene expressions. xANR5-MO also blocked elongation of activin-treated animal caps (ACs) and tissue separation. The dorsal cells of xANR5-MO-injected embryos exhibited markedly reduced membrane protrusions, which could be restored by coinjecting active Rho. Active Rho also rescued the xANR5-MO-inhibited tissue separation. We further demonstrated that xANR5 interacted physically and functionally with paraxial protocadherin (PAPC), which has known functions in cell-sorting behavior, tissue separation, and gastrulation cell movements [4-6], to regulate early morphogenesis. Our findings reveal for the first time that xANR5 acts through Rho to regulate gastrulation and is an important cytoplasmic partner of PAPC, whose cytoplasmic partner was previously unknown.

Cellular Cholesterol Modulates VEGFR-1 Function on Acute Leukemia Cells.

Vascular endothelial growth factor (VEGF) and its receptors play a crucial role in malignancy and in disease, regulating the survival, proliferation, and migration of several cell types, such as endothelium and also leukemia cells. Following our recent report on the role of VEGFR-1 (FLT-1) in ALL (Fragoso R et al, 2006), in the present study we analyzed the molecular mechanisms whereby it modulates acute leukemia cell migration in response to VEGF/Placental Growth Factor (PLGF). First, we observed the formation of cell protrusions on ALL cells after VEGF/PLGF stimulation, with evidence for polymerized actin and FLT-1 co-localization (as determined by phalloidin, immunofluorescence staining, and confocal microscopy). Western blot analysis revealed that PLGF/VEGF stimulation resulted in increased RhoA and Rac1 GTPases expression. Co-treatment with LY200942 significantly decreased RhoA and Rac1 induction and cell migration by PLGF/VEGF, demonstrating this effect is modulated via Pi3 kinase. Next, we investigated the mechanisms whereby FLT-1 and actin co-localize at the cell “leading edge” (protrusions), after VEGF/PLGF stimulation, and the relevance of such co-localization for cell migration. We addressed this question by impairing the formation of lipid rafts/caveolae using drugs that either sequester (nystatin) or deplete (methyl-β-ciclodextrin) total cholesterol. Accordingly, co-treatment of leukemia cells with nystatin or MβCD and PLGF/VEGF blocked cell migration, an effect that was associated with a decrease in FLT-1 polarization and co-localization with actin filaments. Instead, FLT-1 was now found mostly in the cell cytosol. Given that leukemia cells have an increased rate of cholesterol up-take we sought to understand if increased cholesterol levels affected FLT-1 function in leukemia cells. Cholesterol repletion in leukemia cells enhanced leukemia cells migration in response to VEGF/PlGF (about 3 folds). This significant increase was associated with an increase in FLT-1 protein expression that, very interestingly, was particularly concentrated intracellulary in the cytoplasm. At this time we are trying to understand if this increase in FLT-1 expression after cholesterol repletion is associated with increase protein translation or impairment in proteasome activity. Finally, our preliminary in vivo experiments using Nod-Scid mice subjected (n=3) or not (n=3) to high fat diet (that results in increased cholesterol levels in the BM and in the spleen), showed this metabolic condition worsens disease symptoms and significantly decreases mouse survival. These results reveal for the first time some of the molecular mechanisms involved in FLT-1-mediated leukemia migration, namely the involvement of cholesterol metabolism, which may be crucial for new therapeutics delineation.

E-Cadherin Regulates Human Nanos1, which Interacts with p120ctn and Induces Tumor Cell Migration and Invasion

Down-regulation of the epithelial cell-cell adhesion molecule E-cadherin is frequently associated with tumor formation and progression. Besides its role in physical cell-cell adhesion, E-cadherin is also thought to be involved in intracellular signaling in normal epithelial cells. In these cells, the Armadillo catenin p120ctn binds to the cytoplasmic domain of E-cadherin and stabilizes the adhesion complexes. On loss of E-cadherin, cytoplasmic p120ctn might accumulate and contribute to tumor malignancy. We used suppression subtractive hybridization to search for genes regulated by E-cadherin expression. We isolated human Nanos1 as a transcript of which levels decrease on E-cadherin reexpression in a human breast cancer cell line. The hNanos1 protein bears a COOH-terminal (CCHC)(2) zinc finger domain and belongs to an evolutionarily conserved protein family sharing functions in germ cell development in both vertebrates and invertebrates. We found an inverse correlation between E-cadherin and hNanos1 expression in various cell lines and under diverse conditions. Conditional expression of hNanos1 in human colorectal DLD1 cancer cells functionally abolished cell-cell adhesion. It induced cytoplasmic translocation of p120ctn, as well as strong migratory and invasive properties. We also found that the NH(2)-terminal domain of hNanos1, which is conserved only among mammals, interacts with p120ctn. hNanos1 counteracted the stimulatory effect of p120ctn on cell protrusion formation. Together, these findings describe a new function for hNanos1 as a downstream effector of E-cadherin loss contributing to tumor progression. Targeting hNanos1 might be a promising strategy in the treatment of E-cadherin-negative tumors in particular.

Prognostic significance of fascin expression in advanced colorectal cancer: an immunohistochemical study of colorectal adenomas and adenocarcinomas

BackgroundFascin is an actin bundling protein with roles in the formation of cell protrusions and motility of mesenchymal and neuronal cells. Fascin is normally low or absent from epithelia, but is upregulated in several epithelial neoplasms where it may contribute to an invasive phenotype. Here, we report on the prevalence and potential clinical significance of fascin expression in relation to the progression of colorectal adenocarcinoma and to tumor cell proliferation as measured by Ki67 index.MethodsConventional tissue sections of 107 colorectal adenomas and 35 adenocarcinomas were analyzed by immunohistochemistry for fascin and Ki67 expression.Fascin expression and Ki67 proliferation index were also investigated by use of a tissue microarray containing cores from a further 158 colorectal adenocarcinomas and 15 adenomas linked to a CCF, IRB-approved database with a mean of 38 months of clinical follow-up. Survival analysis was carried out by the Kaplan-Meier and Cox regression methods.ResultsFascin was not expressed by the normal colonic epithelium. In conventional sections, 16% of adenomas and 26% of adenocarcinomas showed fascin expression in greater than 10% of the tumor cells. In the clinically-annotated tumors, fascin immunoreactivity was more common in tumors located in the proximal colon (p = 0.009), but was not associated with age, gender, or TNM stage. Patients with stage III/IV adenocarcinomas (n = 62) with strong fascin immunoreactivity had a worse prognosis than patients with low or absent fascin, (3-year overall survival of 11% versus 43% for fascin-negative patients; p = 0.023). In adenomas, fascin and Ki67 tended to be inversely correlated at the cellular level; this trend was less apparent in adenocarcinomas.ConclusionFascin is upregulated in a proportion of adenomas, where its expression is often focal. Strong and diffuse expression was seen in a subset of advanced colorectal adenocarcinomas that correlated with shorter survival in stage III and IV patients. Fascin may have prognostic value as an early biomarker for more aggressive colorectal adenocarcinomas.

Ermin, A Myelinating Oligodendrocyte-Specific Protein That Regulates Cell Morphology

Oligodendrocytes form an insulating multilamellar structure of compact myelin around axons, thereby allowing rapid propagation of action potentials. Despite the considerable clinical importance of myelination, little is known about the molecular mechanisms that enable oligodendrocytes to generate their specialized membrane wrapping. Here, we used microarray expression profiling of oligodendrocyte-ablated mutant mice to identify new glial molecules that are involved in CNS myelination. This effort resulted in the identification of Ermin, a novel cytoskeletal molecule that is exclusively expressed by oligodendrocytes. Ermin appears at a late stage during myelination, and in the mature nerves, it is localized to the outer cytoplasmic lip of the myelin sheath and the paranodal loops. In cultured oligodendrocytes, Ermin becomes visible in well differentiated MBP-positive cells, where it is concentrated at the tip of F-actin-rich processes (termed "Ermin spikes"). Ectopic expression of Ermin, but not of a mutant protein lacking its actin-binding domain, induced the formation of numerous cell protrusions and a pronounced change in cell morphology. Our results demonstrate that Ermin is a novel marker of myelinating oligodendroglia and suggest that it plays a role in cytoskeletal rearrangements during the late wrapping and/or compaction phases of myelinogenesis.

VEGF Regulates Leukemia Migration Via FLT-1, Involving Pi3 Kinase, RhoA and Rac1 Activation and Lipid Rafts Formation.

Vascular endothelial growth factor (VEGF) and its receptors play a crucial role in malignancy and in disease, regulating the survival, proliferation, and migration of several cell types, such as endothelium and also leukemia cells. We previously demonstrated crucial roles for VEGFR-2 in acute myeloid leukemia, where its blockade showed clinical potential in murine models, by affecting leukemia survival and proliferation. In the present study we focused on a different VEGF receptor, FLT-1, and studied the molecular mechanisms whereby it modulates acute leukemia cell migration in response to VEGF/Placental Growth Factor (PLGF). First, we observed the formation of cell protrusions on ALL cells after VEGF/PLGF stimulation, with evidence for polimerized actin and FLT-1 co-localization (as determined by phalloidin, immunofluorescence staining, and confocal microscopy). Western blot analysis revealed that PLGF/VEGF stimulation resulted in increased RhoA and Rac1 GTPases expression. Co-treatment with LY200942 significantly decreased RhoA and Rac1 induction and cell migration by PLGF/VEGF, demonstrating this effect is modulated via Pi3 kinase. Next, we investigated the mechanisms whereby FLT-1 and actin co-localize at the cell “leading edge” (protrusions), after VEGF/PLGF stimulation, and the relevance of such co-localization for cell migration. We addressed this question using a drug (nystantin) that impairs the formation of lipid rafts, cholesterol and sphingolipid rich membrane domains that modulate the interaction of several proteins (Rho GTPases) with downstream effectors, an event essential to cell migration. Accordingly, co-treatment of leukemia cells with nystantin and PLGF/VEGF blocked cell migration, an effect that was associated with a decrease in FLT-1 polarization and co-localization with actin filaments. Instead, FLT-1 was now found mostly in the cell cytosol (possibly undergoing lysosomal degradation). Taken together, we hypothesize that FLT-1 localization in lipid raft-rich membrane domains allows interaction with the actin cytoskeleton and downstream effectors, resulting in cell migration. Our data reveal for the first time some of the molecular mechanisms involved in VEGF-mediated leukemia migration, which may be crucial for determining the onset of extramedullary disease (the exit of leukemia cells from the bone marrow).

JAM4 enhances hepatocyte growth factor-mediated branching and scattering of Madin-Darby canine kidney cells.

Junctional adhesion molecule (JAM) 4 is a member of immunoglobulin superfamily that interacts with MAGI-1, a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. We prepared Madin-Darby canine kidney II (MDCK) cells expressing JAM4 (MDCK-JAM4) and compared them with wild MDCK cells. The treatment of hepatocyte growth factor (HGF) induced more prominent branching and scattering in MDCK-JAM4 cells. Subsequently we attempted to identify signalling pathways modified by JAM4. The over-expression of JAM4 induced the formation of protrusions in COS-7 cells. Although those protrusions were different from typical lamellipodia, the dominant negative mutant of Rac suppressed them. The pull-down assay using CDC42 and Rac interactive binding domain of PAK also supports that Rac is activated in COS-7 cells expressing JAM4. Taken together, JAM4 itself activates Rac and may augment Rac activation by HGF, resulting in the enhancement of branching and scattering.