ABSTRACT
BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.
Subject(s)
Fibroblasts/ultrastructure , Myocardium/pathology , Polycystic Kidney, Autosomal Dominant/pathology , 3T3 Cells/ultrastructure , Animals , Animals, Newborn , Atrial Remodeling , Cilia , Fetal Heart/cytology , Fibrosis , Heart Injuries/pathology , Humans , Kinesins/deficiency , Kinesins/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/pathology , Polycystic Kidney, Autosomal Dominant/genetics , Rats , Signal Transduction , Smad3 Protein/physiology , TRPP Cation Channels/deficiency , TRPP Cation Channels/physiology , Transforming Growth Factor beta1/physiology , Ventricular RemodelingABSTRACT
Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as α-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5 kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit α5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for ß1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis.
Subject(s)
3T3 Cells/ultrastructure , Extracellular Matrix , Myoblasts/cytology , Myocytes, Cardiac/cytology , Tissue Engineering/methods , Tissue Scaffolds , Animals , Cell Line , Cell Lineage , Cell Shape , Cellular Microenvironment , Cross-Linking Reagents/pharmacology , Culture Media/pharmacology , Cytoskeletal Proteins/biosynthesis , Extracellular Matrix/drug effects , Extracellular Matrix/ultrastructure , Fibronectins , Gene Expression Profiling , Heart Ventricles/cytology , Iridoids/pharmacology , Mice , Muscle Development , Muscle Proteins/biosynthesis , Rats , Rats, Sprague-DawleyABSTRACT
Ca2+ influx, an immediate consequence of plasma membrane disruption, triggers a resealing mechanism involving exocytosis. Although this has been known for about a decade, a better understanding of the organelles involved and of the molecular machinery controlling membrane repair has been slower to emerge. Recent studies have changed this picture, by identifying lysosomes as exocytotic vesicles involved in membrane resealing and the Ca2+-binding protein synaptotagmin VII as a regulator of this process. New evidence reinforces the role of the C2A and C2B domains of synaptotagmin VII in plasma membrane repair, highlighting the importance of this molecule as a powerful tool for future studies.
Subject(s)
Calcium Signaling , Calcium/physiology , Cell Membrane Permeability , Synaptotagmins/physiology , 3T3 Cells/metabolism , 3T3 Cells/ultrastructure , Animals , Calcium/pharmacology , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Membrane Permeability/drug effects , Eukaryotic Cells/metabolism , Eukaryotic Cells/ultrastructure , Exocytosis , Humans , Lysosomes/physiology , Mice , Neurotoxins/pharmacology , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/physiology , Synaptotagmins/chemistry , Synaptotagmins/genetics , Synaptotagmins/immunologyABSTRACT
Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive imaging in Fourier-space corresponds to a reduction of low spatial frequency contributions leading to a relative enhancement of the high frequencies. Along the direction of the optic axis this also results in an improved sectioning. Image processing can achieve a similar effect. However, a 3D volume dataset must be acquired and processed, yielding a result essentially identical to subtractive imaging but superior in signal-to-noise ratio. The latter can be increased further with the technique of weighted averaging in Fourier-space. A comparison of 2D and 3D experimental data analysed with subtractive imaging, the equivalent Fourier-space processing of the confocal data only, and Fourier-space weighted averaging is presented.
Subject(s)
Image Processing, Computer-Assisted/methods , Microscopy, Confocal/methods , Subtraction Technique , 3T3 Cells/ultrastructure , Animals , Bryopsida/ultrastructure , Fourier Analysis , Mice , Microscopy, Fluorescence/methods , Spores/ultrastructureABSTRACT
In a cell-populated collagen gel, intrinsic fiber structure visible in differential interference contrast images can provide markers for an in situ strain gauge to quantify cell-gel mechanics, while optical sections of fluorescent protein distribution capture cytoskeletal kinematics. Mechanics quantification can be derived automatically from timelapse differential interference contrast images using a Deformation Quantification and Analysis software package accessible online at http://dqa.web.cmu.edu. In our studies, fibroblast contractile machinery was observed to function entirely within pseudopods, while GFP-alpha-actinin concentrated in pseudopod tips and cortex. Complex strain patterns around individual cells showed instances of both elastic and inelastic strain transmission, suggesting a role in observed long-range alignment of cells.
Subject(s)
3T3 Cells/physiology , 3T3 Cells/ultrastructure , Collagen/physiology , Collagen/ultrastructure , Extracellular Matrix/physiology , Extracellular Matrix/ultrastructure , Imaging, Three-Dimensional/methods , 3T3 Cells/drug effects , Animals , Biomimetics/methods , Cell Movement/drug effects , Cell Movement/physiology , Culture Techniques/instrumentation , Culture Techniques/methods , Cytochalasin D/pharmacology , Elasticity , Extracellular Matrix/drug effects , Internet , Mechanotransduction, Cellular/drug effects , Mechanotransduction, Cellular/physiology , Mice , Motion , Nocodazole/pharmacology , Software , Stress, MechanicalABSTRACT
A series of poly(L-lactide)-poly(ethylene glycol) multiblock copolymers (Multi-PLE) with high molecular weight were synthesized and successfully used to fabricate three-dimensional scaffolds. Using mouse NIH 3T3 fibroblasts as model cells, the cell affinity of various Multi-PLE copolymers was evaluated and compared with that of poly(L-lactide) (PLLA) by means of cell attachment efficiency measurement, scanning electron microscopy observation and MTT assay. On one hand, the results showed that the cell attachment efficiency on Multi-PLE 4/1(4/1 refers to the molar ratio of lactidyl units to ethylene oxide units) films was close to that on PLLA film, however, the other Multi-PLE films exhibited much lower cell attachment efficiency than PLLA film, such as Multi-PLE 2/1 and Multi-PLE 1/1, which had higher PEG content. On the other hand, it was interesting to find that cell proliferation on Multi-PLE4/1 and Multi-PLE2/1 scaffolds was better than that on PLLA scaffold, which was closely related to the improved hydrophilicity of Multi-PLE copolymers due to the incorporation of PEG in comparison with pure PLLA. The Multi-PLE copolymer scaffolds with appropriate hydrophilicity were in favor of mass transportation, and then of cell proliferation and cell affinity. It meant that the cell proliferation would be much improved by increasing the hydrophilicity of the three-dimensional scaffolds, which even outweighed the disadvantages of the cell attachment efficiency reduction with the incorporation of PEG.
Subject(s)
3T3 Cells/physiology , 3T3 Cells/ultrastructure , Absorbable Implants , Biocompatible Materials/chemistry , Culture Techniques/methods , Lactates/chemical synthesis , Polyethylene Glycols/chemical synthesis , Tissue Engineering/methods , Animals , Biocompatible Materials/chemical synthesis , Cell Adhesion/physiology , Cell Division , Cell Survival/physiology , Culture Techniques/instrumentation , Lactates/chemistry , Materials Testing , Mice , Polyethylene Glycols/chemistry , Tissue Engineering/instrumentationABSTRACT
It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.
Subject(s)
Cell Transformation, Neoplastic/genetics , MAP Kinase Kinase Kinase 1 , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-raf/genetics , 14-3-3 Proteins , 3T3 Cells/metabolism , 3T3 Cells/transplantation , 3T3 Cells/ultrastructure , Active Transport, Cell Nucleus , Alleles , Animals , Binding Sites , COS Cells/metabolism , COS Cells/ultrastructure , Cell Differentiation/genetics , Cell Division/drug effects , Chlorocebus aethiops , Contact Inhibition , Enzyme Activation , Feedback, Physiological , Genes, Reporter , Mice , Mice, SCID , Mitogen-Activated Protein Kinase 3 , Mutagenesis, Site-Directed , PC12 Cells/cytology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-raf/physiology , Rats , Transfection , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Some cells undergo apoptosis in response to DNA damage, whereas others do not. To understand the biochemical pathways controlling this differential response, we have studied the intracellular localization of cyclin B1 in cell types sensitive or resistant to apoptosis induced by DNA damage. We found that cyclin B1 protein accumulates in the nucleus of cells that are sensitive to gamma radiation-induced apoptosis (thymocytes, lymphoid cell lines), but remains cytoplasmic in apoptosis-resistant cells (primary and transformed fibroblasts). Treatment of both cell types with leptomycin B, an inhibitor of CRM1-dependent cyclin B1 nuclear export, induces apoptosis. Furthermore, ectopic expression of cyclin B1-5xE, a protein that preferentially localizes to the nucleus, is sufficient to trigger apoptosis. Conversely, expression of cyclin B1-5xA, a predominantly cytoplasmic protein, fails to induce apoptosis. This suggests that nuclear accumulation is necessary for cyclin B1-dependent apoptosis. Our observations are consistent with the idea that localization of cyclin B1 is among the factors determining the cellular decision to undergo apoptosis in response to DNA damage.
Subject(s)
Apoptosis/physiology , Cell Nucleus/chemistry , Cyclin B/physiology , DNA Damage , 3T3 Cells/cytology , 3T3 Cells/radiation effects , 3T3 Cells/ultrastructure , Active Transport, Cell Nucleus/drug effects , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Burkitt Lymphoma/pathology , COS Cells/cytology , COS Cells/radiation effects , COS Cells/ultrastructure , Chlorocebus aethiops , Cyclin B/analysis , Cyclin B/genetics , Cyclin B1 , Cytoplasm/chemistry , DNA, Neoplasm/radiation effects , Fatty Acids, Unsaturated/pharmacology , Gamma Rays , Humans , Mice , Microscopy, Confocal , Radiation Tolerance , Recombinant Fusion Proteins/physiology , Transfection , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/radiation effects , Tumor Cells, Cultured/ultrastructureSubject(s)
Bone Substitutes/chemistry , Culture Techniques/methods , Durapatite/chemistry , Manufactured Materials , Materials Testing , Organophosphorus Compounds/chemistry , Polymers/chemistry , Tissue Engineering/methods , 3T3 Cells/physiology , 3T3 Cells/ultrastructure , Absorbable Implants , Animals , Biocompatible Materials/adverse effects , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Bone Substitutes/adverse effects , Bone Substitutes/chemical synthesis , Cell Adhesion/physiology , Cell Division/physiology , Cells, Cultured , Durapatite/chemical synthesis , Elasticity , Extracellular Matrix/physiology , Extracellular Matrix/ultrastructure , Mice , Organophosphorus Compounds/chemical synthesis , Polymers/chemical synthesis , Surface PropertiesABSTRACT
NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.
Subject(s)
3T3 Cells/ultrastructure , Cytoskeleton/ultrastructure , Leukemia Virus, Murine/ultrastructure , Microscopy, Atomic Force/methods , Virion/ultrastructure , 3T3 Cells/drug effects , Animals , Cell Membrane/ultrastructure , Fixatives/pharmacology , Glutaral/pharmacology , Leukemia Virus, Murine/pathogenicity , Mice , Mutation , Osmium Tetroxide/pharmacologyABSTRACT
A new instrument was constructed by combining an objective-type total internal reflection fluorescence microscope with an atomic force microscope (AFM). Our purpose of constructing such an instrument is to detect and confirm the result of cellular level manipulations made with the AFM part through the detection system of the highly sensitive fluorescence microscope part. In this combination, manipulations are now possible from the nanometer to the micrometer scales and the fluorescence detection system is sensitive enough even for localizing single molecules. In this paper, we applied the system as a precise intracellular injector (nanoplanter). Fluorescent beads were first chemically immobilized onto a ZnO whisker that was glued to an AFM tip and were injected into a living BALB/3T3 cell together with the whisker. It was demonstrated that the system could clearly show the result of injection, that is, the presence of a small number of fluorescent beads in the cell.
Subject(s)
3T3 Cells/ultrastructure , Microscopy, Atomic Force/methods , Microscopy, Fluorescence/instrumentation , Nanotechnology/methods , Animals , Cell Line , Mice , Mice, Inbred BALB C , Microscopy, Atomic Force/instrumentation , Microscopy, Fluorescence/methodsABSTRACT
Blocks of two porous synthetic hydroxyapatites (HA) with porosity fraction of 30-40 and 50-60 vol%, respectively and a coralline derived porous HA were evaluated in vitro in presence of the osteogenic line MC3T3-E1 and of L929 fibroblasts. The two tested biomaterials did not affect cellular proliferation (MTT test), but the contact inhibited alkaline phosphatase activity. Porous aggregates resulted perfectly biocompatible in the tests performed, since observations performed by light microscopy did not show any cell morphological change, osteoblast presented a stellar shape and typical pseudopodes. SEM observations showed intercellular matrix containing fibers on HA-based porous aggregates.
Subject(s)
Ceramics/classification , Durapatite/classification , Fibroblasts/cytology , Materials Testing , Osteoblasts/cytology , 3T3 Cells/cytology , 3T3 Cells/drug effects , 3T3 Cells/ultrastructure , Animals , Biocompatible Materials/classification , Biocompatible Materials/pharmacology , Cell Line , Fibroblasts/drug effects , Fibroblasts/ultrastructure , Mice , Osteoblasts/drug effects , Osteoblasts/ultrastructure , Porosity , Sensitivity and SpecificityABSTRACT
A stable connection between the biomaterial surface and the surrounding tissue is one of the most important prerequisites for the long-term success of implants. Therefore, a strong adhesion of the cells on the biomaterial surface is required. Beside the surface composition the surface topography influences the properties of the adherent cells. The quality of the connection between the cell and the biomaterial is-among other factors-determined by the dimensions of the surface topography. Osteoblasts and fibroblast-like cells in contact with a ground biomaterial surface spread in the direction of the surface structures. These aligned cells provide a more favourable adhesion behaviour than a spherically shaped cell. To determine the influence of the surface structure on the cell alignment and cytoskeleton organisation or arrangement, substrate discs of cp-titanium were ground, producing different roughness of the substrates. The oriented cells had a higher density of focal contacts when they were in contact with the edges of the grooves and showed a better organisation of the cytoskeleton and stronger actin fibres. These changes of the aligned cells depend on the peak to valley height of the surface structures.
Subject(s)
3T3 Cells/ultrastructure , Cytoskeleton/ultrastructure , Endothelium, Vascular/ultrastructure , Fibroblasts/ultrastructure , Materials Testing/methods , Titanium/chemistry , 3T3 Cells/physiology , Animals , Aorta/ultrastructure , Biocompatible Materials/chemistry , Biocompatible Materials/classification , Cattle , Cell Adhesion , Cell Line , Cell Movement , Cell Polarity , Chlorocebus aethiops , Endothelium, Vascular/physiology , Fibroblasts/physiology , Gingiva/ultrastructure , Mice , Sensitivity and Specificity , Surface Properties , Titanium/classification , Vero CellsABSTRACT
We investigated the nanometer scale height fluctuations of 3T3 fibroblast cells with the atomic force microscope under physiological conditions. A correlation between these fluctuations and lateral cellular motility can be observed. Fluctuations measured on leading edges appear to be predominantly related to actin polymerization-depolymerization processes. We found fast (5 Hz) pulsatory behavior with 1-2 nm amplitude on a cell with low motility showing emphasized structure of stress fibers. Myosin driven contractions of stress fibers are thought to induce this pulsation.
Subject(s)
Fibroblasts/ultrastructure , Microscopy, Atomic Force , 3T3 Cells/physiology , 3T3 Cells/ultrastructure , Animals , Cell Line , Cell Movement , Central Nervous System Neoplasms/metabolism , Central Nervous System Neoplasms/ultrastructure , Fibroblasts/physiology , Glioma/metabolism , Glioma/ultrastructure , Mice , Microscopy, Atomic Force/methods , Rats , Tumor Cells, CulturedABSTRACT
PR-39, which is an endogenous antimicrobial peptide, can bind to Src homology 3 domains of the NADPH complex protein p47(phox) and the signaling adapter protein p130(Cas). Recently, we have reported that PR-39 gene transduction altered invasive activity and actin structure of human hepatocellular carcinoma cells, suggesting that this peptide affects cellular signaling due to its proline-rich motif. In order to clarify the mechanism of the PR-39 functions, we transfected the PR-39 gene into mouse NIH3T3 cells which had already been transformed with human activated k-ras gene. The PR-39 gene transfectant showed a reorganization of actin structure and suppression of cell proliferation both in vitro and in vivo. Decreases of MAP (mitogen-activated protein) kinase activity, cyclin D1 expression and JNK activity were observed in the PR-39 gene transfectant. Co-immunoprecipitation analysis revealed that PR-39 binds to PI3-kinase p85alpha, which is a regulatory subunit of PI3-kinase and one of the effectors by which ras induces cytoskeletal changes and stimulates mitogenesis. The PI3-kinase activity of the PR-39 gene transfectant was decreased compared with that of the ras transformant. These results suggest that PR-39 alters actin structure and cell proliferation rate by binding to PI3-kinase p85alpha and suppressing the PI3-kinase activity.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Cell Transformation, Neoplastic , Genes, ras , Phosphoinositide-3 Kinase Inhibitors , 3T3 Cells/enzymology , 3T3 Cells/ultrastructure , Actin Cytoskeleton/ultrastructure , Amino Acid Motifs , Animals , Cell Division , Cell Line, Transformed , Cell Transformation, Neoplastic/genetics , Cyclin D1/biosynthesis , Cyclin D1/genetics , Cytoskeleton/ultrastructure , Enzyme Induction , Humans , JNK Mitogen-Activated Protein Kinases , MAP Kinase Signaling System , Mice , Mitogen-Activated Protein Kinases/biosynthesis , Mitogen-Activated Protein Kinases/genetics , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Protein Interaction Mapping , Recombinant Fusion Proteins/metabolism , Swine/genetics , Transfection , src Homology DomainsABSTRACT
The insulin-sensitive glucose transporter GLUT4 is translocated to the plasma membrane in response to insulin and recycled back to the intracellular store(s) after removal of the hormone. We have used clonal 3T3-L1 fibroblasts and adipocyte-like cells stably expressing wild-type GLUT4 to characterize (a) the intracellular compartment where the bulk of GLUT4 is intracellularly stored and (b) the mechanisms involved in the recycling of endocytosed GLUT4 to the store compartment. Surface internalized GLUT4 is targeted to a large, flat, fenestrated saccular structure resistant to brefeldin A that localized to the vicinity of the Golgi complex is sealed to endocytosed transferrin (GLUT4 storage compartment). Recycling of endocytosed GLUT4 was studied by comparing the cellular distributions of antibody/biotin tagged GLUT4 and GLUT4(Ser(5)), a mutant with the Phe(5)-Gln(6)-Gln(7)-Ile(8) inactivated by the substitution of Ser for Phe(5). Ablation of the Phe(5)-Gln(6)-Gln(7)-Ile(8) inhibits the recycling of endocytosed GLUT4 to the GLUT4 store compartment and results in its transport to late endosomes/lysosomes where it is rapidly degraded.
Subject(s)
Endocytosis/physiology , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , 3T3 Cells/ultrastructure , Adipocytes/physiology , Amino Acid Motifs/physiology , Animals , Antibodies/immunology , Antigen-Antibody Complex/metabolism , Biological Transport , Brefeldin A/pharmacology , Cell Membrane/metabolism , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Glucose Transporter Type 4 , Hydrogen-Ion Concentration , Mice , Microsomes/physiology , Monosaccharide Transport Proteins/chemistry , Protein Synthesis Inhibitors/pharmacology , Saccule and Utricle/drug effects , Saccule and Utricle/physiology , Transferrin/metabolismABSTRACT
Cdc42 is a member of the Rho family of GTPase. Cdc42 has been implicated to be involved in the movement, multiplication and transformation of mammalian cells by controlling the rearrangement of actin cytoskeleton and gene expression. But the mechanism of Cdc42 function has not yet been discovered. In this report we present data showing a perinuclear accumulation of Cdc42 in response to fetal bovine serum (FBS). There was no change in the amount of Cdc42 in response to FBS in the cell. It was found that protein component(s) of serum plays a major role in the perinuclear accumulation of Cdc42. Epidermal growth factor has also been found to stimulate the perinuclear accumulation of Cdc42 while NGF has no effect. Kinase inhibitors, quercetin and NDGA were found to block signals for the perinuclear accumulation of Cdc42. This suggests that phosphorylation of cellular proteins is essential for transducing signals generated from the serum component(s) to induce the perinuclear accumulation of Cdc42. These results indicate that redistribution of Cdc42 might be an important step in alteration of gene expression for controlling various functions of the cell including cell division.
Subject(s)
3T3 Cells/drug effects , Culture Media/pharmacology , Fetal Blood/physiology , Mammary Neoplasms, Experimental/pathology , Protein Transport/drug effects , cdc42 GTP-Binding Protein/metabolism , 3T3 Cells/ultrastructure , Animals , Cattle , Cells, Cultured/drug effects , Cells, Cultured/ultrastructure , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Genes, jun/drug effects , Masoprocol/pharmacology , Mice , Phosphorylation/drug effects , Protein Denaturation , Protein Kinase Inhibitors , Protein Processing, Post-Translational/drug effects , Quercetin/pharmacology , Signal Transduction/drug effects , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/ultrastructureABSTRACT
In order to investigate signal transduction pathways and related changes of actin cytoskeleton organization in cellular senescence, H-ras double mutants--V12S35, V12G37, and V12C40--were constitutively expressed in human foreskin fibroblast (HDF). Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells, V12S35 and V12G37 expressers, revealed a failure to export actin fiber from nucleus to cytoplasm and also to form stress fibers. Perinuclear expression of Rac1 was prominent in the HDF cells and V12C40 expresser; however, in the V12S35 expresser, translocation of Rac1 from perinucleus to nucleus and strong expression of RhoA were obvious. In summary, the H-ras double mutant expressers induced premature senescence through the MEK pathway, accompanied by nuclear accumulation of actin and Rac1 proteins, cytoplasmic retention of p-Erk1/2, and marked induction of RhoA expression, suggesting the translocational inefficiency of the intracellular proteins in the senescent HDF cells.
Subject(s)
Active Transport, Cell Nucleus , Cellular Senescence/physiology , Cytoskeleton/physiology , Fibroblasts/cytology , Genes, ras , MAP Kinase Kinase Kinase 1 , MAP Kinase Signaling System , 3T3 Cells/drug effects , 3T3 Cells/ultrastructure , Actins/metabolism , Animals , Blood Proteins/metabolism , Cell Nucleus/metabolism , Cell Surface Extensions , Cyclin-Dependent Kinase Inhibitor p16/physiology , Cytoplasm/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/ultrastructure , Diploidy , Fibroblasts/metabolism , Genes, p16 , Genes, p53 , Humans , Male , Membrane Proteins/metabolism , Mice , Microfilament Proteins/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins p21(ras)/physiology , Stress Fibers/metabolism , Tumor Suppressor Protein p53/physiology , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.
Subject(s)
Integrin beta1/physiology , Oncogene Proteins/physiology , Receptors, Growth Factor/physiology , Signal Transduction/physiology , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , 3T3 Cells/drug effects , 3T3 Cells/metabolism , 3T3 Cells/ultrastructure , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Actins/metabolism , Amino Acid Substitution , Animals , Cell Membrane/ultrastructure , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cells, Cultured/ultrastructure , Cytoskeleton/physiology , Cytoskeleton/ultrastructure , Epidermal Growth Factor/pharmacology , Extracellular Matrix/physiology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Guanosine Diphosphate/physiology , Guanosine Triphosphate/physiology , Humans , Mice , Oncogene Proteins/chemistry , Oncogene Proteins/genetics , Phenotype , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Protein Processing, Post-Translational , Protein Structure, Tertiary , Proto-Oncogene Proteins c-vav , Rabbits , Receptors, Growth Factor/drug effects , Recombinant Fusion Proteins/physiology , Signal Transduction/drug effects , Transfection , cdc42 GTP-Binding Protein/chemistry , cdc42 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/chemistry , rac1 GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/chemistry , rhoA GTP-Binding Protein/geneticsABSTRACT
The study was designed to characterize the surface properties (including water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility) of a biological patch fixed with genipin, a naturally occurring crosslinking agent. Fresh and glutaraldehyde-fixed counterparts were used as controls. It was found that both glutaraldehyde and genipin are effective crosslinking agents for biological tissue fixation. Fixation of biological tissue with glutaraldehyde or genipin significantly increased its hydrophilicity and surface tension and reduced its mol ratio of adsorbed fibrinogen to adsorbed albumin as well as the amount of adhered platelet. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues. However, the cellular compatibilities of fresh and the genipin-fixed tissues were significantly superior to the glutaraldehyde-fixed tissue.