Cell Surface Actin Research Articles

An interplay between cellular growth and atypical fusion defines morphogenesis of a modular glial niche in Drosophila

Neural stem cells (NSCs) live in an intricate cellular microenvironment supporting their activity, the niche. Whilst shape and function are inseparable, the morphogenetic aspects of niche development are poorly understood. Here, we use the formation of a glial niche to investigate acquisition of architectural complexity. Cortex glia (CG) in Drosophila regulate neurogenesis and build a reticular structure around NSCs. We first show that individual CG cells grow tremendously to ensheath several NSC lineages, employing elaborate proliferative mechanisms which convert these cells into syncytia rich in cytoplasmic bridges. CG syncytia further undergo homotypic cell–cell fusion, using defined cell surface receptors and actin regulators. Cellular exchange is however dynamic in space and time. This atypical cell fusion remodels cellular borders, restructuring the CG syncytia. Ultimately, combined growth and fusion builds the multi-level architecture of the niche, and creates a modular, spatial partition of the NSC population. Our findings provide insights into how a niche forms and organises while developing intimate contacts with a stem cell population.

Multiplexed two-color imaging and analysis reveals the influence of actin cortex dynamics on CD36 receptor organization

CD36 is a cell surface receptor whose organization and signaling in multiple cell types have been shown to depend on the actin cortex, although largely through actin perturbation experiments. Perturbation experiments raise concerns of nonspecific effects, and preclude any quantification of actin dynamics. Thus, to derive explicit relationships between cell surface CD36 organization and actin cortex dynamics simultaneously in live unperturbed cells, we developed a novel imaging and computational analysis framework capable of integrating single-molecule imaging of receptor behavior and fluorescent speckle microscopy of actin cortex dynamics (SMI-FSM).

Restraint of Human Skin Fibroblast Motility, Migration, and Cell Surface Actin Dynamics, by Pannexin 1 and P2X7 Receptor Signaling.

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.

Quantification of Surface Receptor - Actin Cortex Interplay via Multiplexed Two-Color Imaging

The dynamic organization of receptors in the plasma membrane is crucial for ligand binding and downstream signaling. The actin cortex plays an important role in regulating cell surface receptor organization. However its role has been almost exclusively deduced via cell-wide actin perturbation experiments, which suffer from various caveats, such as lack of information about cortex dynamics and potential nonspecific effects. We have developed a quantitative imaging and computational analysis framework to derive perturbation free, explicit relationships between cell surface receptor organization and actin cortex dynamics. We use the receptor CD36 in endothelial cells as a model system, because its organization and signaling on the endothelial cell surface are thought to depend on actin. Briefly, to monitor both CD36 and actin cortex behavior at the same time in the same cell, we perform TIRF-based simultaneous single-molecule imaging of CD36 and fluorescent speckle microscopy (FSM) of actin, in live endothelial cells. Because of the disparate timescales of their dynamics, we image each at its relevant frame rate. Next, CD36 molecules are tracked to characterize their movement and clustering state to yield information about their dynamic organization, while neighboring actin behavior is characterized in the form of speckle density, movement and lifetime (reflecting actin turnover kinetics). Machine learning approaches are then applied to this multidimensional dataset to derive relationships between CD36 and actin cortex properties. Using this framework, we indeed find interdependence between CD36 and actin cortex properties. Interestingly, these inter-relationships vary based on subcellular location (cell side vs tip) and cell edge activity (actively ruffling vs quiescent). These observations highlight further the need for direct observation of these inter-relationships, instead of indirect deduction based on cell-wide perturbations that alter cell morphology and activity, leading to potentially difficult-to-interpret results.

Alpha-mangostin inhibits the migration and invasion of A549 lung cancer cells.

Several studies have indicated that α-mangostin exerts anti-metastasis and anti-subsistence effects on several types of cancer cells. Especially, the anti-metastatic effect of α-mangostin on cancer cells is a prospective function in cancer treatment. However, the metastasis process is complicated, and includes migration, invasion, intravasation, and extravasation; thus, the main target of anti-metastatic effect of α-mangostin is not known. In this study, we investigated the effects of α-mangostin on the invasion, subsistence, and migration of lung cancer cells under co-culture conditions with normal cells and regular mono-culture conditions. We found that α-mangostin killed the lung cancer and normal cells in a dose-dependent manner. Furthermore, the alteration in the surface mechanical properties of cells was examined by using atomic force microscopy. Although the α-mangostin concentrations of 5 and 10 µM did not affect the short-term cell viability, they considerably decreased the Young’s modulus of lung cancer cells implying a decline in cell surface actin cytoskeletal properties. Additionally, these concentrations of α-mangostin inhibited the migration of lung cancer cells. In co-culture conditions (cancer cells with normal cells), the invasive activities of cancer cells on normal cells were discernibly observed, and was inhibited after treatment with 5 and 10 µM of α-mangostin. Taken together, α-mangostin suppressed the subsistence of lung cancer cells and displayed anti-metastatic activities by inhibiting the migration and invasion, and reducing the actin cytoskeleton of cancer cells. Our findings suggest that α-mangostin could be a potential therapeutic agent for cancer treatment.

Abstract B094: The dual Rac/Cdc42 inhibitor MBQ-167 and derivatives as anticancer compounds

Abstract Metastatic disease lacks effective treatments, and remains the primary cause of mortality from epithelial cancers. The Rho family GTPases Rac and Cdc42 and their downstream effector p21-activated kinase (PAK) are pivotal regulators of metastatic cancer cell migration and invasion; thus, overexpression of Rac/Cdc42/PAK has been correlated with reduced patient survival in breast, gastric, and lung cancer. Our studies on the efficacy and pharmacokinetics of the Rac inhibitor EHop-016 (US patents # 8,884,006 B2; 9,278,956B1) validated the development of Rac and Cdc42 inhibitors as anti-metastatic cancer therapeutics. To improve the pharmacologic utility of EHop-016, structural derivatives were screened for enhanced efficacy and bioavailability. We recently identified a dual Rac and Cdc42 inhibitor, MetaBloq(MBQ)-167, which inhibits Rac and Cdc42 activation with IC50s of 103 nM for Rac inhibition and 78 nM for Cdc42 inhibition. Moreover, MBQ-167 is specific to cancer cells that have undergone epithelial-to-mesenchymal transition (EMT), but not epithelial cancer cells or noncancer cells. In metastatic cancer cells, MBQ-167 induces a unique phenotype of loss in cell polarity, cell surface actin extensions, and cell-substrate attachments to undergo anoikis (apoptosis due to inadequate cell-matrix interactions). In vivo, MBQ-167 inhibits HER2 type mammary tumor growth and metastasis in immunocompromised mice by ~90-100% (Humphries-Bickley et al., Mol Cancer Ther 2017; patent applications US 15/499532, PCT/US17/29921). In this study, we have further tested the pharmacokinetics and utility of MBQ-167 in additional models of cancer and screened MBQ-167 derivatives for their utility as Rac/Cdc42 inhibitors. Similar to the results with HER2 type breast cancer, in severe combined immunodeficiency (SCID) mice with mammary fat pad tumors from the triple-negative MDA-MB-231 human breast cancer cell line, the effect of MBQ-167 saturated at 1 mg/kg BW with a 80% reduction in tumor growth and a 100% reduction in metastasis. In gastric cancer, MBQ-167 reduced the viability of metastatic NCI-N87 gastric cancer cells, induced anoikis without affecting the AGS nonmetastatic gastric cancer cells, and inhibited tumor growth in SCID mice. We have screened a range of MBQ-167 derivatives for cell viability and the “anoikis” phenotype and isolated 4 compounds that reduced breast cancer cell viability with GI50s in the nM range. From this screen, MBQ-14 acts similar to MBQ-167, and thus is considered to be a viable inhibitor for further investigation. We have developed a quantitative method for identifying MBQ-167 and MBQ-14 from mouse plasma using ultra-performance convergence chromatography (UPC2)/MS/MS, and find that MBQ-167 is bioavailable in mouse plasma with a half-life of ~4h following oral or intraperitoneal administration. Overall our data demonstrate the utility of further developing MBQ-167 and derivatives as anti-metastatic cancer therapeutics. Citation Format: Linette Castillo-PIchardo, Maria Del Mar Maldonado, Jean Ruiz-Calderon, Jose F. Rodriguez-Orengo, Eliud Hernandez, Cornelis Vlaar, Surangani F. Dharmawardhane Flanagan. The dual Rac/Cdc42 inhibitor MBQ-167 and derivatives as anticancer compounds [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B094.

Transcriptomic analysis of purified human cortical microglia reveals age-associated changes

Microglia are essential for CNS homeostasis and innate neuroimmune function, and play important roles in neurodegeneration and brain aging. Here we present gene expression profiles of purified microglia isolated at autopsy from the parietal cortex of 39 human subjects with intact cognition. Overall, genes expressed by human microglia were similar to those in mouse, including established microglial genes CX3CR1, P2RY12 and ITGAM (CD11B). However, a number of immune genes, not identified as part of the mouse microglial signature, were abundantly expressed in human microglia, including TLR, Fcγ and SIGLEC receptors, as well as TAL1 and IFI16, regulators of proliferation and cell cycle. Age-associated changes in human microglia were enriched for genes involved in cell adhesion, axonal guidance, cell surface receptor expression and actin (dis)assembly. Limited overlap was observed in microglial genes regulated during aging between mice and humans, indicating that human and mouse microglia age differently.

Astrocytes regulate the balance between plasminogen activation and plasmin clearance via cell-surface actin

Plasminogen activation is involved in many processes within the central nervous system, including synaptic plasticity, neuroinflammation and neurodegeneration. However, the mechanisms that regulate plasminogen activation in the brain still remain unknown. Here we demonstrate that astrocytes participate in this regulation by two mechanisms. First, the astrocyte plasma membrane serves as a surface for plasminogen activation by tissue-type plasminogen activator. This activation triggers downstream plasmin-dependent processes with important impacts in brain health and disease, such as fibrinolysis and brain-derived neurotrophic factor conversion. Second, astrocytes take up plasminogen and plasmin in a regulated manner through a novel mechanism involving endocytosis mediated by cell-surface actin and triggered by extracellular plasmin activity at the surface of astrocytes. Following endocytosis, plasminogen and plasmin are targeted to lysosomes for degradation. Thus, cell-surface actin acts as a sensor of plasmin activity to induce a negative feedback through plasmin endocytosis. This study provides evidence that astrocytes control the balance between plasmin formation and plasmin elimination in the brain parenchyma.

Abstract 3076: Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in breast cancer

Abstract The expression and activities of the Rho GTPases Rac and Cdc42, and their downstream effector P21-activated kinase (PAK), have been correlated with metastatic cancer. Our published studies with the Rac/PAK inhibitor EHop-016 demonstrate the validity of using Rac inhibitors as anti metastatic cancer therapeutics. However, the relatively high effective concentrations (Rac activity IC50 1μM, tumor inhibition at 25 mg/kg body weight (BW)), and the moderate bioavailability (∼30%, t1/2 4.5h) of EHop-016 need improvement. Therefore, we developed EHop-016 derivatives and identified EHop-167 as a Rac inhibitor at nM concentrations. Unlike EHop-016, which does not substantially change breast cancer cell shape, but only reduces cell surface actin based invadopodia, EHop-167 induced a marked decrease in breast cancer cell polarity, cell surface extensions, and cell-extracellular matrix (ECM) attachments (focal adhesions). This phenotype of cell rounding and detachment in response to EHop-167 was demonstrated only by breast cancer cell lines that have undergone epithelial to mesenchymal transition, but not by epithelial breast cancer cells, or MCF-10A mammary epithelial cells. As assessed by pulldown assays and western blotting, Rac and PAK activities were reduced by 80-90% in response to 250 nM EHop-167, in the detached cells. As demonstrated by caspase assays, the cell rounding and detachment from the ECM ultimately resulted in anoikis (cell death due to loss of focal adhesions). Accordingly, Transwell assays of mesenchymal breast cancer cells following 250 nM Ehop-167 showed a ∼90% reduction in cell migration in the detached breast cancer cells, and a ∼60% inhibition in the attached cells. EHop-167 also reduced the mammosphere formation efficiency of metastatic cancer cells by 50%, indicating an inhibitory effect on cancer stem cells. To determine the in vivo efficacy of EHop-167, athymic nude mice, bearing mammary fatpad tumors of MDA-MB-435 metastatic cancer cells, were treated 3X a week with 0, 1, or 10 mg/kg BW EHop-167 for 50 days. Treatment with 1.0 mg/kg BW EHop-167 resulted in a 50% reduction in tumor growth, while 10.0 mg/kg BW EHop-167 induced an ∼95% reduction in tumor growth, compared to controls. Additionally, these mice did not show gross signs of toxicity or significant weight loss. Since the parental compound EHop-016 has no anticancer effects at similar concentrations, we conclude that EHop-167 is an improved Rac/PAK inhibitor that holds promise as an anti metastatic breast cancer therapeutic. Citation Format: Linette Castillo-Pichardo, Tessa Humphries-Bickley, Ingrid Forrestier-Roman, Luis Borrero-Garcia, Fabiola Pagan Melendez, Eliud Hernandez, Cornelis Vlaar, Luis A. Cubano, Surangani Dharmawardhane. Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3076.

SAR650984 directly induces multiple myeloma cell death via lysosomal-associated and apoptotic pathways, which is further enhanced by pomalidomide.

The anti-CD38 monoclonal antibody SAR650984 (SAR) is showing promising clinical activity in treatment of relapsed and refractory multiple myeloma (MM). Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define molecular mechanisms of SAR-directed MM cell death and enhanced anti-MM activity triggered by SAR with Pomalidomide (Pom). Without Fc-cross-linking agents or effector cells, SAR specifically induces homotypic aggregation (HA)-associated cell death in MM cells dependent on the level of cell surface CD38 expression, actin cytoskeleton and membrane lipid raft. SAR and its F(ab)'2 fragments trigger caspase 3/7-dependent apoptosis in MM cells highly expressing CD38, even with p53 mutation. Importantly, SAR specifically induces lysosome-dependent cell death (LCD) by enlarging lysosomes and increasing lysosomal membrane permeabilization associated with leakage of cathepsin B and LAMP-1, regardless of the presence of interleukin-6 or bone marrow stromal cells. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR further upregulates reactive oxygen species. Pom enhances SAR-induced direct and indirect killing even in MM cells resistant to Pom/Len. Taken together, SAR is the first therapeutic monoclonal antibody mediating direct cytotoxicity against MM cells via multiple mechanisms of action. Our data show that Pom augments both direct and effector cell-mediated MM cytotoxicity of SAR, providing the framework for combination clinical trials.

Caspase-1-mediated pathway promotes generation of thromboinflammatory microparticles.

Extracellular ATP is a signal of tissue damage and induces macrophage responses that amplify inflammation and coagulation. Here we demonstrate that ATP signaling through macrophage P2X7 receptors uncouples the thioredoxin (TRX)/TRX reductase (TRXR) system and activates the inflammasome through endosome-generated ROS. TRXR and inflammasome activity promoted filopodia formation, cellular release of reduced TRX, and generation of extracellular thiol pathway-dependent, procoagulant microparticles (MPs). Additionally, inflammasome-induced activation of an intracellular caspase-1/calpain cysteine protease cascade degraded filamin, thereby severing bonds between the cytoskeleton and tissue factor (TF), the cell surface receptor responsible for coagulation activation. This cascade enabled TF trafficking from rafts to filopodia and ultimately onto phosphatidylserine-positive, highly procoagulant MPs. Furthermore, caspase-1 specifically facilitated cell surface actin exposure, which was required for the final release of highly procoagulant MPs from filopodia. Together, the results of this study delineate a thromboinflammatory pathway and suggest that components of this pathway have potential as pharmacological targets to simultaneously attenuate inflammation and innate immune cell-induced thrombosis.

A Crucial Role for Caspase 1 in Thrombo-Inflammatory Microparticle Release

Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell injury signals trigger the macrophages P2X7 receptor and thereby induce the release both proinflammatory IL-1β and prothrombotic MP. Prothrombotic MP carry tissue factor (TF) and high content of phosphatidylserine (PS), and can induce thrombosis causing major clinical complications in patients. We previously identified the P2X7 receptor as a crucial component of thrombosis in mice, but the mechanistic details of macrophage MP release in this thrombo-inflammatory pathway remain incompletely understood.The the generation of these MP requires thiol-disulfide exchange-dependent activation of the inflammasome and is accompanied by the release of various soluble proteins into the extracellular space. We hypothesized that the released proteome presents regulators and structural components of the MP generation pathway and employed proteomics to unveil their identity. Amongst the most abundant proteins were γ-actin and actin cytoskeleton associated proteins, including PS-binding proteins annexin 1 and annexin 5. Cytoskeletal remodeling processes leading to formation of filopodia downstream of P2X7 receptor activation were crucial for the biogenesis of thrombo-inflammatory MP, since pharmacological inhibitors of inflammasome activation, cytoskeletal remodeling and the thioredoxin system attenuated both, filopodia formation and the release of highly procoagulant MP. Confocal microscopy demonstrated raft dependent translocation of TF onto filopodia that was prevented by the same inhibitory strategies. Surprisingly, phalloidin-staining of non-permeabilized macrophages revealed that F-actin is exposed to the cell surface decorating the base of filopodia. Positive phalloidin-staining of thrombo-inflammatory MP further demonstrated that F-actin remained associated with the MP surface. Strikingly, blocking surface actin by incubation with high concentration of phalloidin prevented the release of PS-rich MP, demonstrating that exposure of F-actin during filopodia formation is functionally linked to the biogenesis of thrombo-inflammatory MP.As the underlying common mechanism for thiol-disulfide exchange-dependent cell surface actin exposure and MP release, we showed that blockade of the cysteine protease caspase 1, which mediates processing and release of IL-1β downstream of inflammasome activation is also required for the release of thrombo-inflammatory MP. Although caspase 1-mediated activation of caplain was required for the release of filamin A localized TF to the cell cortex, calpain was not involved in the release of thrombo-inflammatory MP release. The N-terminus of γ-actin harbors a recognition and cleavage motif for caspase 1. Residual γ-actin released from caspase 1-blocked macrophages showed decreased electrophoretic mobility, indicating prior cleavage of actin that becomes exposed on the cell surface.We show here that the proteome released during thrombo-inflammatory activation of macrophages includes critical players in the biogenesis of MP and may provide diagnostic fingerprints in complex biological samples. Our data demonstrate an entirely unexpected role for caspase 1 and surface exposure of polymerized actin in the severing of prothrombotic MP from filopodia and thus position this protease upstream of both IL-1β processing and thrombo-inflammatory MP in cardiovascular diseases. DisclosuresNo relevant conflicts of interest to declare.

Human recombinant truncated RNASET2, devoid of RNase activity; A potential cancer therapeutic agent

Human RNASET2 has been implicated in antitumorigenic and antiangiogenic activities, independent of its ribonuclease capacities. We constructed a truncated version of human RNASET2, starting at E50 (trT2-50) and devoid of ribonuclease activity. trT2-50 maintained its ability to bind actin and to inhibit angiogenesis and tumorigenesis. trT2-50 binds to cell surface actin and formed a complex with actin in vitro. The antiangiogenic effect of this protein was demonstrated in human umbilical vein endothelial cells (HUVECs) by its ability to arrest tube formation on Matrigel, induced by angiogenic factors. Immunofluorescence staining of HUVECs showed nuclear and cytosolic RNASET2 protein that was no longer detectable inside the cell following trT2-50 treatment. This effect was associated with disruption of the intracellular actin network. trT2-50 co-localized with angiogenin, suggesting that both molecules bind (or compete) for similar cellular epitopes. Moreover, trT2-50 led to a significant inhibition of tumor development. Histological analysis demonstrated abundant necrotic tissue and a substantial loss of endothelial structure in trT2-50-treated tumors. Collectively, the present results indicate that trT2-50, a molecule engineered to be deficient of its catalytic activity, still maintained its actin binding and anticancer-related biological activities. We therefore suggest that trT2-50 may serve as a potential cancer therapeutic agent.

Dynamics of thrombin-induced exposition of actin on the platelet surface.

Platelets play the key role in thrombosis and are also involved in angiogenesis as well as immune and reparative responses. In the function cascade, platelets undergo a complex cell processing, and subcellular fragments, not detectable in the resting state, are exposed on platelet surface after stimulation with agonists. This study has been performed to evaluate dynamic characteristics of actin exposition on the surface of plasma membrane of thrombin-activated platelets. Using flow-cytometric assay, it has been observed that the level of actin presented on activated platelets directly depends on agonist concentration. In the case of platelet stimulation with thrombin in the highest concentration (1.0 U/ml) taken for this study, the level of actin exposed on activated platelets was up to 4.4 times higher as compared with resting cells. Confirmation of the flow cytometry data for cell-surface actin on thrombin-activated platelets was achieved by direct visualization using a confocal laser scanning microscopy. Period of actin exposition appeared to be longer than the time phase corresponding to platelet secretion stage. Functional role of platelet surface actin has required further detailed studying, however, it is thought that superficial actin could interact with various blood plasma proteins, including plasminogen and its activators, serving as a binding site and/or center for their pericellular processing.

Progress of dendritic spines in the nervous system diseases

Dendritic spines are functional protrusions on neuron dendrites and formation of excitatory synaptic sites.Dendritic spines contain a variety of cell surface receptors,actin cytoskeleton,scaffolding proteins and other components,which regulate the growth of dendritic spines maturation and structure.Multiple components within dendritic spines not only have effects on expression of its shape and structure,but also on the function.Morphology of dendritic spines is closely related with its function.Its shape is affected by many factors in the changing process.So dendritic spine plasticity is an important aspect of synaptic function in the central nervous system,and is closely related with learning and memory function and central nervous system diseases. Key words: Dendritic spines; Plasticity; Learning and memory; Aging

Role of ROS in Aβ42 mediated cell surface P-selectin expression and actin polymerization

P-selectin and actin cytoskeleton reorganization play an important role in vascular inflammation. In turn, there is increasing evidence that cerebrovascular factors contribute significantly to the development and progression of Alzhemer’s disease. In this study we have evaluated the effects of Aβ42 oligomers on P-selectin expression and actin polymerization in mouse endothelial cells (bEnd3). Our results indicated that Aβ42 induced plasma membrane accumulation of P-selectin and promoted actin polymerization, and these events were correlated with increased reactive oxygen species (ROS) generation. The rapid, posttranslational cell signaling response mediated by ROS may well represent an important physiological trigger of the microvascular inflammation in Alzheimer disease.

Abstract 497: Loss of Notch-1 expression in human prostate epithelial cells results in reduced PTEN expression and altered differentiative capacity

Abstract Notch-1 is part of an evolutionarily ancient cell surface receptor signaling pathway that functions in the determination of stem/progenitor cell fate. The human genome encodes four Notch-receptors (Notch-1, -2, -3, and -4) and at least five Notch-ligands (Delta-1, -3, -4, Jagged-1, and-2). Signaling is initiated upon ligand binding to the Notch receptor, which results in a series of proteolytic cleavages that release constitutively active intracytoplamsic Notch (ICN). Nuclear translocation of ICN results in activation of the CBF-1 transcription factor which in turn increases expression of developmentally regulated transcription factors such as Hes-1 and Hey-1. The PTEN tumor suppressor gene is a recently identified Notch-1 target. We have reported that Notch-1 signaling increases expression of the PTEN tumor suppresser gene in prostate tumor cell lines and that Notch-1 signaling is lost in the tumor foci of prostate adenocarcinoma patients. To test the hypothesis that loss of Notch-1 will result in deregulation of normal prostate epithelial cell differentiation and promote tumorigenesis, a lentiviral vector bearing a Notch-1 shRNA (shN1) was used to stably knockdown Notch-1 expression in the benign human prostate epithelial cell line, RWPE-1. Western blotting revealed that RWPE-1/shN1 cells lacked Notch-1 expression, and exhibited reduced expression of Hes-1, confirming that loss of Notch-1 expression resulted in loss of Notch signaling. PTEN protein expression was also reduced, consistent with our previous studies demonstrating that Notch-1 signaling regulated PTEN expression. RT-PCR experiments verified that loss of PTEN expression was mediated at the level of transcription. RWPE-1/shN1 cells lacking Notch-1 expression exhibited a more rounded morphology, as compared with the parental cell line. Rhodamine-phalloidin staining revealed that RWPE-1/shN1 cells had less numerous and shorter cell surface actin projections. RWPE-1/shN1 cells lacking Notch-1 expression exhibited a 2-fold increase in acinar differentiation upon 3-D culture in matrigel as compared with the parental cell line. Flow cytometric analysis revealed that RWPE-1 cells without Notch-1 expression had reduced surface expression of TROP2 and CD149f. Preliminary RT-PCR results indicate that expression of the basal cell marker keratin-5 is reduced in RWPE-1/shN1 cells as compared with parental RWPE-1 cells. Our data suggest that loss Notch-1 expression, and hence loss of signaling, may promote developmental plasticity in prostate epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 497. doi:10.1158/1538-7445.AM2011-497

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenin’s propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.

Cell-Surface Actin Binds Plasminogen and Modulates Neurotransmitter Release from Catecholaminergic Cells

An emerging area of research has documented a novel role for the plasminogen activation system in the regulation of neurotransmitter release. Prohormones, secreted by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that feed back to inhibit secretagogue-stimulated release. Catecholaminergic cells of the sympathoadrenal system are prototypic prohormone-secreting cells. Processing of prohormones by plasmin is enhanced in the presence of catecholaminergic cells, and the enhancement requires binding of plasmin(ogen) to cellular receptors. Consequently, modulation of the local cellular fibrinolytic system of catecholaminergic cells results in substantial changes in catecholamine release. However, mechanisms for enhancing prohormone processing and cell-surface molecules mediating the enhancement on catecholaminergic cells have not been investigated. Here we show that plasminogen activation was enhanced >6.5-fold on catecholaminergic cells. Carboxypeptidase B treatment decreased cell-dependent plasminogen activation by approximately 90%, suggesting that the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote plasminogen activation. We identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation, using a novel strategy combining targeted specific proteolysis using carboxypeptidase B with a proteomics approach using two-dimensional gel electrophoresis, radioligand blotting, and tandem mass spectrometry. Two major plasminogen-binding proteins that exposed C-terminal lysines on the cell surface contained amino acid sequences corresponding to beta/gamma-actin. An anti-actin monoclonal antibody inhibited cell-dependent plasminogen activation and also enhanced nicotine-dependent catecholamine release. Our results suggest that cell-surface-expressed forms of actin bind plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to inhibition of neurotransmitter release.

Cell surface actin remodeling

Actin filament remodeling at cell surfaces is a fundamental aspect of cellular life. Except for minor sequence variations, actin proteins are structurally identical. They have similar self-association properties and ATPase activities that accommodate their assembly into polarized semi-flexible