Tumor autocrine motility factor is an angiogenic factor hat stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment.     

Autocrine motility factor stimulates a three-fold increase in inositol trisphosphate in human melanoma cells

The biochemical pathways through which tumor cell locomotion is mediated are poorly understood. Autocrine motility factor (AMF), which is produced by and stimulates motility in A2058 human melanoma cells, was used to characterize phosphoinositide (PtdIns) metabolism activated in association with tumor cell motility. AMF stimulated up to a 400% increase in de novo incorporation of 3H-myo-inositol into cellular lipids beginning 40 minutes after exposure. In cells prelabeled with 3H-myo-inositol, AMF stimulated a 200% increase in total inositol phosphates (inositol monophosphate, InsP1; inositol bisphosphate, InsP2; inositol trisphosphate, InsP3) after 90 minutes of exposure, with a 300% maximal increase in InsP3 at 120 minutes. InsP1 and InsP2 were maximally increased 130% of control values. Treatment with AMF stimulated a parallel dose-dependent increase in both motility and PtdIns levels. We have shown previously that the A2058 motile response to AMF is inhibited markedly by cell pretreatment with pertussis toxin (PT). Inositol phosphate production was inhibited by a 2-hour pretreatment of cells with PT (0.5 microgram/ml). PT treatment of A2058 membranes was associated with ADP-ribosylation of a 40-kDa protein consistent with the presence of an alpha subunit of a guanine nucleotide-binding protein (G protein). These data indicate that AMF elicits increases in cell motility and phosphoinositide metabolism via a PT-sensitive G protein signal transduction pathway.     

Tumor autocrine motility factor induces hyperpermeability of endothelial and mesothelial cells leading to accumulation of ascites fluid

Accumulation of ascites fluid often observed in some solid tumors is one of the most devastating conditions of a patient's difficulty in responding to treatment, and to a decrease in the quality of life. Various factors are thought to be associated with the formation of cancer-induced fluid accumulation and hyperpermeability of a blood vessel is thought to go with this process. Here, we report a new factor that is involved in this process, e.g., autocrine motility factor (AMF). AMF is a tumor-related cytokine which stimulates the tumor cell locomotion and migration and promotes tumor cell invasion during metastasis. AMF secretion and its receptor (AMFR) expression in tumor cells are closely correlated with disease aggravation of convalescence. The response of endothelial or mesothelial cellular morphological alternation to AMF leads to motile enhancement and vascular permeability. Tumor AMF induces gaps in an endothelial or mesothelial monolayer by stimulating a cellular movement, and accelerates the ascites accumulation. And treatment experiment with anti-AMF antibody succeeded in the reduction of the ascites accumulation, which renders AMF to the target molecule. It is suggested that AMF is one of the significant factors which relates to various pathological malignancies induced by tumor mass, and understanding of its function could benefit prognosis and treatment.     

Phosphohexose isomerase/autocrine motility factor/neuroleukin/maturation factor is a multifunctional phosphoprotein

Phosphohexose isomerase (PHI) is a member of the ectoenzyme/exoenzyme family and plays a key role in both glycolysis and gluconeogenesis pathways. Upon secretion PHI acts as a cytokine with tumor autocrine motility factor (AMF), neuroleukin (NLK) and maturation factor (MF) functions. Signaling is initiated by its binding to a cell surface 78 kDa glycoprotein (gp78). However, since PHI protein is a 'leaderless' secretory protein, released from cells via a non-classical route(s), we questioned whether the molecule undergoes post-translation modification while retaining proper folding and maintaining intact enzymatic and motogenic activities. To address this, we have generated, expressed and isolated a recombinant human AMF (rhAMF). The rhAMF retained the biological activities of the native AMF, i.e., catalyzes phosphohexose isomerization and stimulated cell motility. Additionally, we show here that human PHI is phosphorylated at serine 185 by casein kinase II (CK II) and we provide experimental evidence suggesting that this phosphorylation is associated with secretion, thus providing insights for elucidating the intracellular signal transmission of cell response to stimulation by AMF/NLK/MF.     

Raft-dependent endocytosis of autocrine motility factor is phosphatidylinositol 3-kinase-dependent in breast carcinoma cells

Autocrine motility factor (AMF) is internalized via a receptor-mediated, dynamin-dependent, cholesterol-sensitive raft pathway to the smooth endoplasmic reticulum that is negatively regulated by caveolin-1. Expression of AMF and its receptor (AMFR) is associated with tumor progression and malignancy; however, the extent to which the raft-dependent uptake of AMF is tumor cell-specific has yet to be addressed. By Western blot and cell surface fluorescence-activated cell sorter (FACS) analysis, AMFR expression is increased in tumorigenic MCF7 and metastatic MDA-231 and MDA-435 breast cancer cell lines relative to dysplastic MCF10A mammary epithelial cells. AMF uptake, determined by FACS measurement of protease-insensitive internalized fluorescein-conjugated AMF, was increased in MCF7 and MDA-435 cells relative to MCF-10A and caveolin-1-expressing MDA-231 cells. Uptake of fluorescein-conjugated AMF was dynamin-dependent, methyl-beta-cyclodextrin- and genistein-sensitive, reduced upon overexpression of caveolin-1 in MDA-435 cells, and increased upon short hairpin RNA reduction of caveolin-1 in MDA-231 cells. Tissue microarray analysis of invasive primary human breast carcinomas showed that AMFR expression had no impact on survival but did correlate significantly with expression of phospho-Akt. Phospho-Akt expression was increased in AMF-internalizing MCF7 and MDA-435 breast carcinoma cells. AMF uptake in these cells was reduced by phosphatidylinositol 3-kinase inhibition but not by regulators of macropinocytosis such as amiloride, phorbol ester, or actin cytoskeleton disruption by cytochalasin D. The raft-dependent endocytosis of AMF therefore follows a distinct phosphatidylinositol 3-kinase-dependent pathway that is up-regulated in more aggressive tumor cells.     

Down-regulation of phosphoglucose isomerase/autocrine motility factor expression sensitizes human fibrosarcoma cells to oxidative stress leading to cellular senescence

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.     

Protein factors which regulate cell motility

Summary Cell motility (i.e., movement) is an essential component of normal development, inflammation, tissue repair, angiogenesis, and tumor invasion. Various molecules can affect the motility and positioning of mammalian cells, including peptide growth factors, (e.g., EGF, PDGF, TGF-beta), substrate-adhesion molecules (e.g., fibronectin, laminin), cell adhesion molecules (CAMs), and metalloproteinases. Recent studies have demonstrated a group of motility-stimulating proteins which do not appear to fit into any of the above categories. Examples include: 1)scatter factor (SF), a mesenchymal cell-derived protein which causes contiguous sheets of epithelium to separate into individual cells and stimulates the migration of epithelial as well as vascular endothelial cells; 2)autocrine motility factor (AMF), a tumor cell-derived protein which stimulates migration of the producer cells; and 3)migration-stimulating factor (MSF), a protein produced by fetal and cancer patient fibroblasts which stimulates penetration of three-dimensional collagen gels by non-producing adult fibroblasts. SF, AMF, and MSF are soluble and heat labile proteins with Mr of 77, 55, and 70 kd by SDS-PAGE, respectively, and may be members of a new class of cell-specific regulators of motility. Their physiologic functions have not been established, but available data suggest that they may be involved in fetal development and/or tissue repair.     

Raft-dependent endocytosis of autocrine motility factor/phosphoglucose isomerase: a potential drug delivery route for tumor cells

Background

Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) is the extracellular ligand for the gp78/AMFR receptor overexpressed in a variety of human cancers. We showed previously that raft-dependent internalization of AMF/PGI is elevated in metastatic MDA-435 cells, but not metastatic, caveolin-1-expressing MDA-231 cells, relative to non-metastatic MCF7 and dysplastic MCF10A cells suggesting that it might represent a tumor cell-specific endocytic pathway.

Methodology/Principal Findings

Similarly, using flow cytometry, we demonstrate that raft-dependent endocytosis of AMF/PGI is increased in metastatic HT29 cancer cells expressing low levels of caveolin-1 relative to metastatic, caveolin-1-expressing, HCT116 colon cells and non-metastatic Caco-2 cells. Therefore, we exploited the raft-dependent internalization of AMF/PGI as a potential tumor-cell specific targeting mechanism. We synthesized an AMF/PGI-paclitaxel conjugate and found it to be as efficient as free paclitaxel in inducing cytotoxicity and apoptosis in tumor cells that readily internalize AMF/PGI compared to tumor cells that poorly internalize AMF/PGI. Murine K1735-M1 and B16-F1 melanoma cells internalize FITC-conjugated AMF/PGI and are acutely sensitive to AMF/PGI-paclitaxel mediated cytotoxicity in vitro. Moreover, following in vivo intratumoral injection, FITC-conjugated AMF/PGI is internalized in K1735-M1 tumors. Intratumoral injection of AMF/PGI-paclitaxel induced significantly higher tumor regression compared to free paclitaxel, even in B16-F1 cells, known to be resistant to taxol treatment. Treatment with AMF/PGI-paclitaxel significantly prolonged the median survival time of tumor bearing mice. Free AMF/PGI exhibited a pro-survival role, reducing the cytotoxic effect of both AMF/PGI-paclitaxel and free paclitaxel suggesting that AMF/PGI-paclitaxel targets a pathway associated with resistance to chemotherapeutic agents. AMF/PGI-FITC uptake by normal murine spleen and thymus cells was negligible both in vitro and following intravenous injection in vivo where AMF/PGI-FITC was selectively internalized by subcutaneous B16-F1 tumor cells.

Conclusions/Significance

The raft-dependent endocytosis of AMF/PGI may therefore represent a tumor cell specific endocytic pathway of potential value for drug delivery to tumor cells.     

Autocrine motility factor stimulates the invasiveness of malignant cells as well as up-regulation of matrix metalloproteinase-3 expression via a MAPK pathway

The autocrine motility factor (AMF) is a multifunctional protein that is involved in tumor progression including enhanced invasiveness via induction of matrix metalloproteinase-3 (MMP3). The increase in MMP3 was found in an AMF-high production tumor cell line, and c-Jun, c-Fos and mitogen-activated protein kinases (MAPKs) were also highly phosphorylated compared with the parent line. AMF stimulation induced the rapid phosphorylation of the cellular MAPK cascade and MMP3 secretion, which was blocked using a specific MAPK inhibitor. Results of this study suggest that AMF stimulation stimulates MMP3 expression via a MAPK signaling pathway.     

Regulation of cell proliferation by autocrine motility factor/phosphoglucose isomerase signaling

Autocrine motility factor (AMF)/phosphoglucose isomerase (PGI; EC 5.3.1.9) is a housekeeping cytosolic enzyme that plays a key role in both glycolysis and gluconeogenesis pathways. AMF/PGI is also a multifunctional protein that displays cytokine properties, eliciting mitogenic, motogenic, and differentiation activities, and has been implicated in tumor progression and metastasis. Because little is known about AMF/PGI-dependent signaling in general and during tumorigenesis in particular, we sought to study its effect on the cell cycle. To elucidate the functional role of PGI, we stably transfected its cDNA into NIH/3T3 and BALB/c 3T3-A31 fibroblasts. Ectopic overexpression of PGI results in the acquisition of a transformed phenotype associated with an acceleration of G1 to S cell cycle transition. These were manifested by up-regulation of cyclin D1 expression and cyclin-dependent kinase activity and down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. The reduced p27Kip1 protein expression level in PGI-overexpressing cells could be restored to control levels by treatment with proteasome inhibitor. PGI-overexpressing cells also exhibited elevated expression of Skp2 involved in p27Kip1 ubiquitination and elevation in the levels of retinoblastoma protein hyperphosphorylation. Thus, we may conclude that the overexpression of AMF/PGI enhances cell proliferation together with up-regulation of cyclin/cyclin-dependent kinase activities and down-regulation of p27Kip1, whereas the induction of 3T3 fibroblast transformation by PGI is regulated by the retinoblastoma protein pathway.     

Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor.

Tumor autocrine motility factor (AMF) has been detected in and purified from serum-free conditioned medium of human HT-1080 fibrosarcoma cells. Under nonreducing conditions, AMF migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of 55 kDa but under reducing conditions as a band of 64 kDa. Two-dimensional polyacrylamide gel electrophoresis of the purified AMF resolved two groups of polypeptides with isoelectric points of 6.1 and 6.2 (majors), 6.35 and 6.4 (minors). Purified AMF stimulated HT-1080 cell migration in a dose-dependent fashion. The motility stimulation of the fibrosarcoma cells with AMF is associated with the phosphorylation of the AMF receptor, a 78-kDa cell surface glycoprotein (gp78), suggesting protein kinase participation in migratory signal transduction. The gene encoding gp78 was cloned from an HT-1080 fibrosarcoma complementary DNA library. The deduced sequence encodes a polypeptide of 323 amino acids. The nucleotide and predicted amino acid sequence of the gp78 reveals significant homology with the human suppressor/oncogene p53 protein.     

Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor

Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin receptor points to a role for the integrins in the interaction of endothelial cells with TSP. We also found that TSP modulates endothelial cell growth. When added to quiescent LE-II cells, it inhibited the mitogenic effects of serum and the angiogenic factor bFGF, in a dose-dependent manner. The inhibition of DNA synthesis detected in the mitogenic assay resulted in a true inhibition of BAEC and LE-II cell growth, as assessed by proliferation assay. This work indicates that TSP affects endothelial cell adhesion, spreading, motility and growth. TSP, therefore, has the potential to modulate the angiogenic process.     

Autocrine motility factor/phosphoglucose isomerase regulates ER stress and cell death through control of ER calcium release

Autocrine motility factor/ phosphoglucose isomerase (AMF/PGI) promotes cell survival by the pAkt survival pathway. Its receptor, gp78/AMFR, is an E3 ubiquitin ligase implicated in endoplasmic reticulum (ER)-associated protein degradation. We demonstrate here that AMF/PGI also protects against thapsigargin (TG)- and tunicamycin (TUN)-induced ER stress and apoptosis. AMF/PGI protection against the ER stress response is receptor mediated as it is not observed in gp78/AMFR-knockdown HEK293 cells. However, AMF/PGI protection against the ER stress response by TG and TUN was mediated only partially through PI3K/Akt activation. AMF/PGI reduction of the elevation of cytosolic calcium in response to either TG or inositol 1,4,5-trisphosphate receptor activation with ATP was gp78/AMFR-dependent, independent of mitochondrial depolarization and not associated with changes in ER calcium content. These results implicate regulation of ER calcium release in AMF/PGI protection against ER stress and apoptosis. Indeed, sequestration of cytosolic calcium with BAPTA-AM limited the ER stress response. Importantly, elevation of cytosolic calcium upon treatment with the calcium ionophore ionomycin, while not inducing an ER stress response, did prevent AMF/PGI protection against ER stress. By regulating ER calcium release, AMF/PGI interaction with gp78/AMFR therefore protects against ER stress identifying novel roles for these cancer-associated proteins in promoting tumor cell survival.     

SNX9 determines the surface levels of integrin β1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9

Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin β1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin β1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.     

The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF

The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.     

Insulin-like growth factor II acts as an autocrine growth and motility factor in human rhabdomyosarcoma tumors

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and appears to arise from developing striated muscle-forming cells. Since insulin-like growth factor II (IGF-II) is involved in normal muscle growth and maturation and elevated IGF-II mRNA levels have previously been reported in rhabdomyosarcomas, we have been studying the possible role of IGF-II in the unregulated growth and invasive potential of these embryonal tumors. In this study, we demonstrate that 13 of 14 rhabdomyosarcoma tumors express high levels of IGF-II mRNA relative to normal adult muscle and also express mRNA for the type I IGF receptors on their cell surface, the receptor thought to mediate the effects of IGF-II on muscle cells. We have established several rhabdomyosarcoma cell lines in mitogen-free media and demonstrate that these cells express type I IGF receptors on their cell surface and secrete IGF-II into the media. Exogenous IGF-II is able to stimulate cellular motility in these cell lines as assayed in a modified Boyden chamber. Finally, alpha IR-3, a type I receptor antagonist, inhibits the growth of these cell lines in serum-free media but does not inhibit IGF-II-induced motility of these cells. These data suggest that endogenously produced IGF-II functions as an autocrine growth and motility factor in many rhabdomyosarcoma tumors. The mitogenic actions of IGF-II are mediated through a domain of the type I IGF receptor that is blocked by alpha IR-3. IGF-II-induced motility may be mediated through an alternative signaling pathway.     

Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule- associated tubular organelles.     

Membrane microviscosity regulates endothelial cell motility

Endothelial cell (EC) movement is an initiating and rate-limiting event in the neogenesis and repair of blood vessels. Here, we explore the hypothesis that microviscosity of the plasma membrane (PM) is a key physiological regulator of cell movement. Aortic ECs treated with membrane-active agents, such as alpha-tocopherol, cholesterol and lysophospholipids, exhibited a biphasic dependency on membrane microviscosity, in which moderate increases enhanced EC migration, but increases beyond a threshold markedly inhibited migration. Surprisingly, angiogenic growth factors, that is, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), also increased membrane microviscosity, as measured in live cells by fluorescence recovery after photobleaching (FRAP). The localization of Rac to the PM was modified in cells treated with membrane-active agents or growth factors, suggesting a molecular mechanism for how membrane microviscosity influences cell movement. Our data show that angiogenic growth factors, as well as certain lipophilic molecules, regulate cell motility through alterations in membrane properties and the consequent relocalization of critical signalling molecules to membranes.