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At least 1,477 records · Page 82Linked to original sources
PubMed2004

Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro.

The Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood-derived HSCs cultured on Delta-like 1-expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 alpha beta-T-cell receptor (TCR)-bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro-differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.

Animals↗
PubMed2005

SAP controls the cytolytic activity of CD8+ T cells against EBV-infected cells.

The adaptor protein SAP regulates signaling through signaling lymphocytic activation molecule (SLAM)-family receptors expressed on T and natural killer (NK) cells. In patients affected by X-linked lymphoproliferative (XLP) disease, mutations in the SH2D1A gene result in defective lytic activity. However, the mechanism by which SAP controls cytotoxic activity remains unclear. T-cell-receptor (TCR) activation of CD8(+) cytotoxic T cells (CTLs) results in down-regulation of SAP, suggesting that this protein is involved in early activation events. Here, we show that SAP-deficient CTLs from patients with XLP and hemophagocytic lymphohistiocytosis (HLH) display a specific lytic defect against autologous and allogeneic Epstein-Barr virus (EBV)-positive B cells. This defect is associated with the defective polarization of 2B4, perforin, and lipid rafts at the contact area of CTLs with EBV-positive targets. Blockade of 2B4 in normal CTLs reproduces the defects in lysis and polarization observed in SAP-deficient CTLs. Expression and regulation of the SLAM-family receptors SLAM, CD84, and 2B4, as well as the lytic effectors perforin and granzyme-B are normal in SAP-deficient CTLs. In addition, TCR stimulation leads to normal proliferation and production of interleukin 2 (IL-2), IL-4, and interferon-gamma (IFN-gamma). These results demonstrate that the SAP/2B4 pathway plays a key role in CTL lytic activity against EBV-positive targets by promoting the polarization of the lytic machinery.

Antigens, CD↗
PubMed2006

Oxidative stress and redox regulation of lung inflammation in COPD.

Reactive oxygen species, either directly or via the formation of lipid peroxidation products, may play a role in enhancing inflammation through the activation of stress kinases (c-Jun activated kinase, extracellular signal-regulated kinase, p38) and redox-sensitive transcription factors, such as nuclear factor (NF)-kappaB and activator protein-1. This results in increased expression of a battery of distinct pro-inflammatory mediators. Oxidative stress activates NF-kappaB-mediated transcription of pro-inflammatory mediators either through activation of its activating inhibitor of kappaB-alpha kinase or the enhanced recruitment and activation of transcriptional co-activators. Enhanced NF-kappaB-co-activator complex formation results in targeted increases in histone modifications, such as acetylation leading to inflammatory gene expression. Emerging evidence suggests the glutathione redox couple may entail dynamic regulation of protein function by reversible disulphide bond formation on kinases, phosphatases and transcription factors. Oxidative stress also inhibits histone deacetylase activity and in doing so further enhances inflammatory gene expression and may attenuate glucocorticoid sensitivity. The antioxidant/anti-inflammatory effects of thiol molecules (glutathione, N-acetyl-L-cysteine and N-acystelyn, erdosteine), dietary polyphenols (curcumin-diferuloylmethane, cathechins/quercetin and reserveratol), specific spin traps, such as alpha-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (extracellular superoxide dismutase (SOD) mimetic, SOD mimetic M40419 and SOD, and catalase manganic salen compound, eukarion-8), porphyrins (AEOL 10150 and AEOL 10113) and theophylline have all been shown to play a role in either controlling NF-kappaB activation or affecting histone modifications with subsequent effects on inflammatory gene expression in lung epithelial cells. Thus, oxidative stress regulates both key signal transduction pathways and histone modifications involved in lung inflammation. Various approaches to enhance lung antioxidant capacity and clinical trials of antioxidant compounds in chronic obstructive pulmonary disease are also discussed.

Antioxidants↗
PubMed2006

Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2.

BACKGROUND: Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined. METHODS: We constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system. Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line. RESULTS: Eighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice. CONCLUSION: This is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines.

Animals↗
PubMed2024

Nanopore-based full-length transcriptome sequencing for understanding the underlying molecular mechanisms of rapid and slow progression of diabetes nephropathy.

BACKGROUND: Diabetic nephropathy (DN) has been a major factor in the outbreak of end-stage renal disease for decades. As the underlying mechanisms of DN development remains unclear, there is no ideal methods for the diagnosis and therapy. OBJECTIVE: We aimed to explore the key genes and pathways that affect the rate progression of DN. METHODS: Nanopore-based full-length transcriptome sequencing was performed with serum samples from DN patients with slow progression (DNSP, n = 5) and rapid progression (DNRP, n = 6). RESULTS: Here, transcriptome proclaimed 22,682 novel transcripts and obtained 45,808 simple sequence repeats, 1,815 transcription factors, 5,993 complete open reading frames, and 1,050 novel lncRNA from the novel transcripts. Moreover, a total of 341 differentially expressed transcripts (DETs) and 456 differentially expressed genes (DEGs) between the DNSP and DNRP groups were identified. Functional analyses showed that DETs mainly involved in ferroptosis-related pathways such as oxidative phosphorylation, iron ion binding, and mitophagy. Moreover, Functional analyses revealed that DEGs mainly involved in oxidative phosphorylation, lipid metabolism, ferroptosis, autophagy/mitophagy, apoptosis/necroptosis pathway. CONCLUSION: Collectively, our study provided a full-length transcriptome data source for the future DN research, and facilitate a deeper understanding of the molecular mechanisms underlying the differences in fast and slow progression of DN.

Humans↗
PubMed2000

Lipoprotein(a) oxidation and autoantibodies: a new path in atherothrombosis.

Lipoprotein(a) (Lp(a)) is considered a vascular pathogen of outstanding importance. High plasma levels of this lipoprotein are associated with premature arterial disease; however, the mechanisms involved have not been clarified. The atherosclerotic process is increasingly regarded as a chronic inflammatory reaction in the arterial wall where oxidation-mediated endothelial injury involving modified forms of low-density lipoprotein (LDL) seems to be a key event. Autoimmune pathways are involved in the progression of atherosclerosis and humoral response to oxidatively modified LDL can be considered among these pathways. A number of factors can be encountered in the pathogenesis of the accelerated arterial disease seen in patients with antiphospholipid (Hughes) syndrome (APS) and systemic lupus erythematosus (SLE). Among these, high levels of Lp(a) have been described in both and increasing evidence indicates that patients with antiphospholipid antibodies (aPL) are under oxidative stress. Recent studies suggest that the so-called 'oxidation theory of atherosclerosis' may also be applied to Lp(a). This fact makes this lipoprotein potentially suitable as a target of the immune system and antibodies reacting against oxidatively-modified Lp(a) by malondialdehyde have been recently described in APS and SLE. It is therefore likely that an immune response to the oxidized moiety of Lp(a) might be influential in the pathogenicity of this lipoprotein and, subsequently, of atherosclerosis.

Antiphospholipid Syndrome↗
PubMed2006

Reduced expression of focal adhesion kinase disrupts insulin action in skeletal muscle cells.

Integrins mediate interactions between cells and extracellular matrix proteins that modulate growth factor signaling. Focal adhesion kinase (FAK) is a key multifunctional integrin pathway protein. We recently reported that disruption of FAK impairs insulin-mediated glycogen synthesis in hepatocytes. To test the hypothesis that FAK regulates skeletal muscle insulin action, we reduced FAK expression in L6 myotubes using FAK antisense. In untransfected myotubes, insulin stimulated both FAK tyrosine phosphorylation and kinase activity. Cells treated with antisense FAK showed 78 and 53% reductions in FAK mRNA and FAK protein, respectively, whereas insulin receptor substrate 1/2 and paxillin abundance were unaffected. Insulin-stimulated U-(14)C-glucose incorporation into glycogen was abolished by FAK antisense, and 2-deoxy-glucose uptake and glucose transporter 4 (GLUT4) translocation were both markedly attenuated. Antisense FAK did not alter GLUT1 or GLUT3 protein abundance. Immunofluorescence staining showed decreased FAK Tyr(397) phosphorylation and reduced actin stress fibers. Thus, in skeletal myotubes, FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. Integrin signaling may play an important regulatory role in muscle insulin action.

Actins↗
PubMed2006

Activin inhibits the human Pit-1 gene promoter through the p38 kinase pathway in a Smad-independent manner.

The pituitary transcription factor Pit-1 regulates hormonal production from the anterior pituitary gland. However, the mechanisms by which Pit-1 gene expression is regulated in humans are poorly understood. Activin, a member of the TGFbeta superfamily, acts as a negative regulator of cell growth and prolactin gene expression in lactotrope cells. In this study, we show that activin negatively regulates the human Pit-1 gene promoter. We defined a 117-bp element within the Pit-1 promoter that is sufficient to relay these inhibitory effects. We further investigated the signaling pathways that mediate activin-induced inhibition of Pit-1 gene promoter in pituitary lactotrope cells. We found that the activin effects on Pit-1 gene regulation are Smad independent and require the p38 MAPK pathway. Specifically, blocking p38 kinase activity reverses activin-mediated inhibition of the Pit-1 gene promoter. Together, our results highlight the p38 MAPK pathway as a key regulator of activin function in pituitary lactotrope cells and further emphasizes the critical role played by activin in regulating hormonal production in the pituitary gland.

Activins↗
PubMed2002

Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes.

In both type 1 and type 2 diabetes, the late diabetic complications in nerve, vascular endothelium, and kidney arise from chronic elevations of glucose and possibly other metabolites including free fatty acids (FFA). Recent evidence suggests that common stress-activated signaling pathways such as nuclear factor-kappaB, p38 MAPK, and NH2-terminal Jun kinases/stress-activated protein kinases underlie the development of these late diabetic complications. In addition, in type 2 diabetes, there is evidence that the activation of these same stress pathways by glucose and possibly FFA leads to both insulin resistance and impaired insulin secretion. Thus, we propose a unifying hypothesis whereby hyperglycemia and FFA-induced activation of the nuclear factor-kappaB, p38 MAPK, and NH2-terminal Jun kinases/stress-activated protein kinases stress pathways, along with the activation of the advanced glycosylation end-products/receptor for advanced glycosylation end-products, protein kinase C, and sorbitol stress pathways, plays a key role in causing late complications in type 1 and type 2 diabetes, along with insulin resistance and impaired insulin secretion in type 2 diabetes. Studies with antioxidants such as vitamin E, alpha-lipoic acid, and N-acetylcysteine suggest that new strategies may become available to treat these conditions.

Animals↗
PubMed2000

Loss of expression of the ubiquitous transcription factor cAMP response element-binding protein (CREB) and compensatory overexpression of the activator CREMtau in the human adrenocortical cancer cell line H295R.

The pituitary hormone ACTH, acting through the cAMP pathway, plays a key role in proliferation and differentiation of the adrenal cortex. CAMP response element (CRE)-binding protein (CREB) is an ubiquitous transcription factor that binds to the CRE present in the promoter of numerous genes and mediates transcription stimulation by cAMP. Characterization of CRE-binding proteins was performed in the H295R cell line, which is considered a model for human adrenocortical tumor studies. Western blot and RT-PCR studies demonstrated that CREB is not expressed in the human adrenocortical cancer cell line H295R, whereas it is expressed in normal adrenal. During transient transfection experiments, cAMP stimulation of two reporter genes containing canonical CRE was maintained. Cotransfection of the dominant negative inhibitor A-CREB, which prevents transcription factors containing a CREB-like leucine zipper domain to bind DNA, completely inhibited cAMP-induced stimulation of CRE activity. Western blot and RT-PCR studies showed that activating transcription factor-1 (ATF-1), CRE modulator-alpha/gamma (CREMalpha/gamma), and CREMtau2alpha are expressed in H295R cells. High amounts of CREM proteins were present in H295R, demonstrating an overexpression of this transcription factor in the absence of CREB. Furthermore, expression of the activator isoform CREMtau was very high in H295R compared to normal adrenal cortex. Transfection assays demonstrated that CREMtau2alpha is a potent stimulator of CRE activity in H295R. Finally, gel retardation assays showed that CREM and ATF-1 are the nuclear proteins that specifically bind the CRE in H295R cells, whereas CREM binding to CRE is not observed in a CREB-expressing cell line. H295R cells are the first established nontransgenic cell line that does not express the ubiquitous transcription factor CREB. H295R demonstrates that CREMtau up-regulation can compensate for CREB deficiency to maintain CRE regulation by cAMP and is a model of compensation mechanisms between the members of the CREB/ CREM/ATF-1 family of transcription factors. This loss of CREB expression and the overexpression of CREM could be linked to cellular transformation, as the normal adrenal cortex express high levels of CREB and no or low levels of CREMtau.

Activating Transcription Factor 1↗
PubMed1993

Staurosporine, a protein kinase C inhibitor, decreases the general anesthetic requirement in Rana pipiens tadpoles.

Protein kinase C, the intracellular effector for the inositol phosphate-mediated signal transduction pathway, plays a key role in neurotransmission in the central nervous system. Although the in vitro activity of protein kinase C is inhibited by therapeutic concentrations of volatile anesthetics, the relation of this effect to in vivo obtundation has not been established. If obtundation by volatile anesthetics involves protein kinase C inhibition, then an inhibitor of this enzyme should decrease the anesthetic requirement. To test this hypothesis, we compared the EC50S of halothane and diethylether for loss of the righting reflex in Rana pipiens tadpoles pretreated with staurosporine and in untreated controls. Anesthetic concentrations were confirmed by gas chromatography and staurosporine concentrations by ultraviolet absorbance spectrophotometry. Results obtained in more than 1000 animals indicated that pretreatment with staurosporine concentrations in the nanomolar range significantly decreased the EC50 for both halothane (68% of control; P < 0.035) and diethylether (41% of control; P < 0.001). This finding implies that protein kinase C inhibition may play a role in general anesthetic-induced obtundation.

Alkaloids↗
PubMed2004

Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein.

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.

Animals↗
PubMed2004

Functions of heparan sulfate proteoglycans in cell signaling during development.

Heparan sulfate proteoglycans (HSPGs) are cell-surface and extracellular matrix macromolecules that are composed of a core protein decorated with covalently linked glycosaminoglycan (GAG) chains. In vitro studies have demonstrated the roles of these molecules in many cellular functions, and recent in vivo studies have begun to clarify their essential functions in development. In particular, HSPGs play crucial roles in regulating key developmental signaling pathways, such as the Wnt, Hedgehog, transforming growth factor-beta, and fibroblast growth factor pathways. This review highlights recent findings regarding the functions of HSPGs in these signaling pathways during development.

Animals↗
PubMed1997

Disruption of gastrulation and oral-aboral ectoderm differentiation in the Lytechinus pictus embryo by a dominant/negative PDGF receptor.

Little is known about the cell signaling involved in forming the body plan of the sea urchin embryo. Previous work suggested that PDGF-like and EGF-like receptor-mediated signaling pathways are involved in gastrulation and spiculogenesis in the Lytechinus pictus embryo. Here we show that expression of the human PDGF receptor-beta lacking the cytoplasmic domain disrupted development in a manner consistent with a dominant/negative mechanism. The truncated PDGF receptor-beta inhibited gut and spicule formation and differentiation along the oral-aboral axis. The most severely affected embryos arrested at a developmental stage resembling mesenchyme blastula. Coinjection into eggs of RNA encoding the entire human PDGF receptor-beta rescued development. The truncated PDGF receptor-beta caused the aboral ectoderm-specific genes LpS1 and LpC2 to be repressed while an oral ectoderm-specific gene, Ecto-V, was expressed in all ectoderm cells. The results support the hypothesis that a PDGF-like signaling pathway plays a key role in the intercellular communication required for gastrulation and spiculogenesis, and in cell commitment and differentiation along the oral-aboral axis.

Animals↗
PubMed1999

Signaling through the stromal epidermal growth factor receptor is necessary for mammary ductal development.

Stromal-epithelial interactions are critical in determining patterns of growth, development and ductal morphogenesis in the mammary gland, and their perturbations are significant components of tumorigenesis. Growth factors such as epidermal growth factor (EGF) contribute to these reciprocal stromal-epithelial interactions. To determine the role of signaling through the EGF receptor (EGFR) in mammary ductal growth and branching, we used mice with a targeted null mutation in the Egfr. Because Egfr-/- mice die perinatally, transplantation methods were used to study these processes. When we transplanted neonatal mammary glands under the renal capsule of immuno-compromised female mice, we found that EGFR is essential for mammary ductal growth and branching morphogenesis, but not for mammary lobulo-alveolar development. Ductal growth and development was normal in transplants of mammary epithelium from Egfr-/- mice into wild-type (WT) gland-free fat pads and in tissue recombinants prepared with WT stroma, irrespective of the source of epithelium (StromaWT/Epi-/-, StromaWT/EpiWT). However, ductal growth and branching was impaired in tissue recombinants prepared with Egfr-/- stroma (Stroma-/-/EpiWT, Stroma-/-/Epi-/-). Thus, for ductal morphogenesis, signaling through the EGFR is required only in the stromal component, the mammary fat pad. These data indicate that the EGFR pathway plays a key role in the stromal-epithelial interactions required for mammary ductal growth and branching morphogenesis. In contrast, signaling through the EGFR is not essential for lobulo-alveolar development. Stimulation of lobulo-alveolar development in the mammary gland grafts by inclusion of a pituitary isograft under the renal capsule as a source of prolactin resulted in normal alveolar development in both Egfr-/- and wild-type transplants. Through the use of tissue recombinants and transplantation, we have gained new insights into the nature of stromal-epithelial interactions in the mammary gland, and how they regulate ductal growth and branching morphogenesis.

Animals↗
PubMed2026

Conserved innate immunity components limit transgene expression in adult planarians.

The planarian flatworm Schmidtea mediterranea has become a powerful model for studying whole-body regeneration, tissue patterning, and stem cell regulation. Yet the absence of reliable tools for transgene expression still limits the elucidation of molecular mechanisms in in this system. Here, we establish a proof-of-principle system for plasmid-based expression of NanoLuciferase (NanoLuc) in S. mediterranea, employing commercially available transfection reagents and a panel of endogenous promoter sequences. Despite successful delivery, reporter expression remained low and transient. To identify biological barriers to robust transgene expression, we investigated the role of innate immune pathways. Candidate gene searches and biochemical pull-down of cytoplasmic DNA coupled to mass spectrometry identified several planarian homologs of conserved immune regulators and putative DNA sensors. Through RNA interference screening of conserved innate immune components, we uncover roles for S. mediterranea homologs of Tank-binding kinase 1 (TBK1) and macrophage mannose receptor 1 (MRC1) as potent repressors of transgene expression. Transcriptomic and functional analyses further implicate TBK1 in regulating broad innate immune and stress-response programs, akin to its vertebrate function. Together, our findings demonstrate that innate immune signaling limits transgene expression in S. mediterranea and suggest that modulating these pathways may be key to enabling stable and efficient genetic manipulation in planarians.

Animals↗
PubMed2003

The ins and outs of fibronectin matrix assembly.

Cell phenotype is specified by environmental cues embedded in the architecture and composition of the extracellular matrix (ECM). Much has been learned about matrix organization and assembly through analyses of the ECM protein fibronectin (FN). FN matrix assembly is a cell-mediated process in which soluble dimeric FN is converted into a fibrillar network. Binding of cell surface integrin receptors to FN converts it to an active form, which promotes fibril formation through interactions with other cell-associated FN dimers. As FN fibrils form on the outside of the cell, cytoplasmic domains of integrin receptors organize cytoplasmic proteins into functional complexes inside. Intracellular connections to the actin cytoskeletal network and stimulation of certain key intracellular signaling pathways are essential for FN-integrin interactions and propagation of FN fibril formation. Thus, assembly of native functional ECM depends on exquisite coordination between extracellular events and intracellular pathways.

Animals↗
PubMed2000

Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium.

The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling macropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium.

3T3 Cells↗