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1.
Neuroscience ; 223: 355-64, 2012 Oct 25.
Article in English | MEDLINE | ID: mdl-22864186

ABSTRACT

Profilin1 is an actin monomer-binding protein, essential for cytoskeletal dynamics. Based on its broad expression in the brain and the localization at excitatory synapses (hippocampal CA3-CA1 synapse, cerebellar parallel fiber (PF)-Purkinje cell (PC) synapse), an important role for profilin1 in brain development and synapse physiology has been postulated. We recently showed normal physiology of hippocampal CA3-CA1 synapses in the absence of profilin1, but impaired glial cell binding and radial migration of cerebellar granule neurons (CGNs). Consequently, brain-specific inactivation of profilin1 by exploiting conditional mutants and Nestin-mediated cre expression resulted in a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers, and ectopic CGNs. Apart from these findings we noted a loss of PCs and an irregularly shaped PC layer in adult mutants. In this study, we show that PC migration and development are not affected in profilin1 mutants, suggesting cell type-specific functions for profilin1 in PCs and CGNs. PC loss begins during the second postnatal week and progresses until adulthood with no further impairment in aged mutants. In Nestin-cre profilin1 mutants, defects in cerebellar cortex cytoarchitecture are associated with impaired motor coordination. However, in L7-cre mutants, lacking profilin1 specifically in PCs, the cerebellar cortex cytoarchitecture is unchanged. Thereby, our results demonstrate that the loss of PCs is not caused by cell-autonomous defects, but presumably by impaired CGN migration. Finally, we show normal functionality of PF-PC synapses in the absence of profilin1. In summary, we conclude that profilin1 is crucially important for brain development, but dispensable for the physiology of excitatory synapses.


Subject(s)
Brain/pathology , Mutation/genetics , Profilins/genetics , Psychomotor Disorders/genetics , Psychomotor Disorders/pathology , Purkinje Cells/physiology , Action Potentials/genetics , Age Factors , Animals , Animals, Newborn , Biophysics , Brain/growth & development , Disease Models, Animal , Disease Progression , Electric Stimulation , Gene Expression Regulation, Developmental/genetics , In Vitro Techniques , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Mice , Mice, Transgenic , Motor Activity/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin , Patch-Clamp Techniques , Purkinje Cells/pathology
2.
Thorax ; 64(6): 467-75, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19213772

ABSTRACT

BACKGROUND: Despite intense research efforts, the aetiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. Gelsolin, an actin-binding protein that modulates cytoskeletal dynamics, was recently highlighted as a likely disease modifier through comparative expression profiling and target prioritisation. METHODS: To decipher the possible role of gelsolin in pulmonary inflammation and fibrosis, immunocytochemistry on tissue microarrays of human patient samples was performed followed by computerised image analysis. The results were validated in the bleomycin-induced animal model of pulmonary inflammation and fibrosis using genetically-modified mice lacking gelsolin expression. Moreover, to gain mechanistic insights into the mode of gelsolin activity, a series of biochemical analyses was performed ex vivo in mouse embryonic fibroblasts. RESULTS: Increased gelsolin expression was detected in lung samples of patients with idiopathic interstitial pneumonia as well as in modelled pulmonary inflammation and fibrosis. Genetic ablation of gelsolin protected mice from the development of modelled pulmonary inflammation and fibrosis attributed to attenuated epithelial apoptosis. CONCLUSIONS: Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis, while the caspase-3-mediated gelsolin fragmentation was shown to be an apoptotic effector mechanism in disease pathogenesis and a marker of lung injury.


Subject(s)
Gelsolin/metabolism , Pneumonia/metabolism , Pulmonary Fibrosis/metabolism , Adult , Aged , Animals , Apoptosis , Bleomycin , Disease Models, Animal , Epithelial Cells/pathology , Female , Gelsolin/deficiency , Gelsolin/physiology , Humans , Lung/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Neutrophil Infiltration , Pneumonia/chemically induced , Pneumonia/pathology , Pneumonia/prevention & control , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/prevention & control , Respiratory Mucosa/pathology
3.
Subcell Biochem ; 45: 55-69, 2007.
Article in English | MEDLINE | ID: mdl-18193634

ABSTRACT

Gelsolin is a calcium-activated actin filament severing and capping protein found in many cell types and as a secreted form in the plasma of vertebrates. Mutant mice for gelsolin as well as clinical studies have shown that gelsolin is linked to a number of pathological conditions such as inflammation, cancer and amyloidosis. The tight regulation of gelsolin by calcium is crucial for its physiological role and constitutive activation leads to apoptosis. In the following we will give an overview on how gelsolin is regulated by calcium, and which clinical conditions have been linked to lack or misregulation of gelsolin.


Subject(s)
Calcium/physiology , Gelsolin/physiology , Actins/physiology , Amyloidosis/genetics , Animals , Gelsolin/genetics , Genes, Tumor Suppressor/physiology , Humans , Inflammation/physiopathology , Phosphatidylinositols/physiology
4.
J Cell Biol ; 154(4): 775-84, 2001 Aug 20.
Article in English | MEDLINE | ID: mdl-11514591

ABSTRACT

Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a null Capg allele engineered into mice. Both CapG-null mice and CapG/gelsolin double-null mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG-null macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin-null macrophages and no additive effects are observed in CapG/gelsolin double-null macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.


Subject(s)
Actins/metabolism , Cell Movement/physiology , Gelsolin/genetics , Macrophages/physiology , Microfilament Proteins/genetics , Nuclear Proteins/genetics , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Intracellular Membranes/physiology , Macrophages/cytology , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Neutrophils/cytology , Neutrophils/physiology , Phagocytosis/physiology
5.
Proc Natl Acad Sci U S A ; 98(7): 3832-6, 2001 Mar 27.
Article in English | MEDLINE | ID: mdl-11274401

ABSTRACT

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1(ko)) allele in mice. Homozygous pfn1(ko/ko) mice are not viable. Pfn1(ko/ko) embryos died as early as the two-cell stage, and no pfn1(ko/ko) blastocysts were detectable. Adult pfn1(ko/wt) mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1(ko/wt) embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.


Subject(s)
Blood Platelets/metabolism , Contractile Proteins , Embryo, Mammalian/metabolism , Microfilament Proteins/physiology , Animals , Cell Division/physiology , Cell Survival/physiology , Mice , Mice, Inbred BALB C , Mice, Knockout , Profilins
6.
J Cell Sci ; 113 Pt 21: 3795-803, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11034907

ABSTRACT

Profilins are a conserved family of proteins participating in actin dynamics and cell motility. In the mouse, two profilin genes are known. Profilin I is expressed universally at high levels, while profilin II is expressed mainly in the brain. Here we describe the occurrence of two mouse profilin II isoforms, A and B, which are derived by alternative splicing. They are identical through residue 107 of the protein, but then have distinct C-terminal sequences. Profilin IIA binds to poly-L-proline and actin with high affinity similar to profilin I. Profilin IIB on the other hand does not bind to actin and the affinity for poly-L-proline is greatly diminished. However, tubulin was found to bind to GST-profilin IIB, and in vivo GFP-profilin IIB was recruited to spindles and asters during mitosis in HeLa cells. Our results indicate unexpected diversity in the functions of the profilin family of proteins, and suggest that in mouse profilin IIA is intimately involved in actin dynamics, while profilin IIB associates with other cytoskeletal components.


Subject(s)
Alternative Splicing , Contractile Proteins , Microfilament Proteins/metabolism , Protein Isoforms/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , Microfilament Proteins/genetics , Molecular Sequence Data , Profilins , Protein Isoforms/genetics , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism
7.
J Immunol ; 165(5): 2451-7, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10946270

ABSTRACT

Phagocytosis and the microbicidal functions of neutrophils require dynamic changes of the actin cytoskeleton. We have investigated the role of gelsolin, a calcium-dependent actin severing and capping protein, in peripheral blood neutrophils from gelsolin-null (Gsn-) mice. The phagocytosis of complement opsonized yeast was only minimally affected. In contrast, phagocytosis of IgG-opsonized yeast was reduced close to background level in Gsn- neutrophils. Thus, gelsolin is essential for efficient IgG- but not complement-mediated phagocytosis. Furthermore, attachment of IgG-opsonized yeast to Gsn- neutrophils was reduced ( approximately 50%) but not to the same extent as ingestion ( approximately 73%). This was not due to reduced surface expression of the Fcgamma-receptor or its lateral mobility. This suggests that attachment and ingestion of IgG-opsonized yeast by murine neutrophils are actin-dependent and gelsolin is important for both steps in phagocytosis. We also investigated granule exocytosis and several steps in phagosome processing, namely the formation of actin around the phagosome, translocation of granules, and activation of the NADPH-oxidase. All these functions were normal in Gsn- neutrophils. Thus, the role of gelsolin is specific for IgG-mediated phagocytosis. Our data suggest that gelsolin is part of the molecular machinery that distinguishes complement and IgG-mediated phagocytosis. The latter requires a more dynamic reorganization of the cytoskeleton.


Subject(s)
Gelsolin/deficiency , Gelsolin/genetics , Immune Tolerance/genetics , Immunoglobulin G/physiology , Neutrophils/immunology , Neutrophils/metabolism , Phagocytosis/genetics , Actins/metabolism , Animals , Binding Sites/immunology , Biological Transport/immunology , Calcium/physiology , Cell Membrane/immunology , Cell Membrane/metabolism , Cytoplasmic Granules/metabolism , Gelsolin/physiology , Guanosine 5'-O-(3-Thiotriphosphate)/physiology , Immunoglobulin G/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nitroblue Tetrazolium/metabolism , Opsonin Proteins/metabolism , Oxidation-Reduction , Phagosomes/metabolism , Receptors, Complement/biosynthesis , Receptors, Complement/metabolism , Receptors, Complement/physiology , Receptors, IgG/biosynthesis , Receptors, IgG/metabolism , Receptors, IgG/physiology
8.
Nature ; 401(6749): 133-41, 1999 Sep 09.
Article in English | MEDLINE | ID: mdl-10490020

ABSTRACT

Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.


Subject(s)
Adaptor Proteins, Signal Transducing , Arachidonic Acid/metabolism , Carrier Proteins/physiology , Cell Membrane/physiology , Lysophospholipids/metabolism , Membrane Lipids/physiology , Synaptic Vesicles/physiology , Acyl Coenzyme A/metabolism , Acyltransferases/metabolism , Amino Acid Sequence , Animals , Arachidonic Acids/pharmacology , Carrier Proteins/metabolism , Cattle , Cell Membrane/chemistry , Cell Membrane/metabolism , Enzyme Inhibitors/pharmacology , Humans , Membrane Fusion , Membrane Lipids/chemistry , Mice , Models, Biological , Molecular Conformation , Molecular Sequence Data , Organophosphonates , PC12 Cells , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Phosphorylation , Protein Binding , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , src Homology Domains/physiology
9.
Neuron ; 22(2): 313-25, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10069337

ABSTRACT

Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton.


Subject(s)
Brain/embryology , Carrier Proteins/physiology , Contractile Proteins , Cytoskeletal Proteins , Nervous System/embryology , Animals , Animals, Newborn/physiology , Axons/physiology , Carrier Proteins/genetics , Embryo, Mammalian/physiology , Embryonic and Fetal Development/physiology , Gene Deletion , Growth Cones/physiology , Mice/embryology , Microfilament Proteins/genetics , Mutation/physiology , Profilins , Tissue Distribution
10.
Curr Opin Cell Biol ; 11(1): 142-51, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10047521

ABSTRACT

New tools in molecular genetics, such as genetic interaction screens and conditional gene targeting, have advanced the study of actin dynamics in a number of model systems. Yeast, Dictyostelium, Caenorhabditis elegans, Drosophila, and mice have contributed much in recent years to a better understanding of both the numerous functions and modes of regulation of the actin cytoskeleton.


Subject(s)
Actins/physiology , Contractile Proteins , Cytoskeleton/physiology , Genetic Techniques , Animals , Caenorhabditis elegans , Cell Division/physiology , Dictyostelium , Drosophila Proteins , Drosophila melanogaster , Endocytosis/physiology , Gelsolin/metabolism , Mice , Microfilament Proteins/metabolism , Myosins/metabolism , Profilins , Signal Transduction
11.
J Mol Evol ; 46(5): 552-61, 1998 May.
Article in English | MEDLINE | ID: mdl-9545466

ABSTRACT

The serum albumin gene family is composed of four members that have arisen by a series of duplications from a common ancestor. From sequence differences between members of the gene family, we infer that a gene duplication some 580 Myr ago gave rise to the vitamin D-binding protein (DBP) gene and a second lineage, which reduplicated about 295 Myr ago to give the albumin (ALB) gene and a common precursor to alpha-fetoprotein (AFP) and alpha-albumin (ALF). This precursor itself duplicated about 250 Myr ago, giving rise to the youngest family members, AFP and ALF. It should be possible to correlate these dates with the phylogenetic distribution of members of the gene family among different species. All four genes are found in mammals, but AFP and ALF are not found in amphibia, which diverged from reptiles about 360 Myr ago, before the divergence of the AFP-ALF progenitor from albumin. Although individual family members display an approximate clock-like evolution, there are significant deviations-the rates of divergence for AFP differ by a factor of 7, the rates for ALB differ by a factor of 2.1. Since the progenitor of this gene family itself arose by triplication of a smaller gene, the rates of evolution of individual domains were also calculated and were shown to vary within and between family members. The great variation in the rates of the molecular clock raises questions concerning whether it can be used to infer evolutionary time from contemporary sequence differences.


Subject(s)
Albumins/genetics , Evolution, Molecular , Vitamin D-Binding Protein/genetics , alpha-Fetoproteins/genetics , Animals , Genetics, Population , Humans , Models, Genetic , Multigene Family , Serum Albumin , Time Factors
12.
J Cell Sci ; 111 ( Pt 5): 615-24, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9454735

ABSTRACT

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.


Subject(s)
ErbB Receptors/physiology , Isoenzymes/physiology , Signal Transduction/physiology , Tissue Adhesions/physiopathology , Type C Phospholipases/physiology , Animals , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Adhesion/physiology , Cell Line , Cell Movement/physiology , Fibroblasts/physiology , Gelsolin/analysis , Heterozygote , Homozygote , Mice , Phospholipase C gamma
13.
Chem Biol ; 5(5): 273-81, 1998 May.
Article in English | MEDLINE | ID: mdl-9578635

ABSTRACT

BACKGROUND: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyphosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility. We sought to investigate the site of interaction of profilin with phosphoinositides. RESULTS: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The P-1-tethered D-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of PtdIns(4,5)P2 but not with Ins(1,4,5)P3. The acyl-modified PtdIns(4,5)P2 analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P2 phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [3H]BZDC-Ins(1,4,5)P3-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix. CONCLUSIONS: The PtdIns(4,5)P2-binding site of profilin I includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of PtdIns(4,5)P2 and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.


Subject(s)
Contractile Proteins , Microfilament Proteins/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Binding Sites , Humans , Microfilament Proteins/chemistry , Molecular Probes , Phosphatidylinositol 4,5-Diphosphate/chemistry , Profilins , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
14.
EMBO J ; 17(5): 1362-70, 1998 Mar 02.
Article in English | MEDLINE | ID: mdl-9482733

ABSTRACT

Rac, a member of the rho family of GTPases, when activated transmits signals leading to actin-based membrane ruffling in fibroblasts. Compared with wild-type fibroblasts, gelsolin null (Gsn-) dermal fibroblasts have a markedly reduced ruffling response to serum or EGF stimulation, which signal through rac. Bradykinin-induced filopodial formation, attributable to activation of cdc42, is similar in both cell types. Wild-type fibroblasts exhibit typical lamellipodial extension during translational locomotion, whereas Gsn- cells move 50% slower using structures resembling filopodia. Multiple Gsn- tissues as well as Gsn- fibroblasts overexpress rac, but not cdc42 or rho, 5-fold. Re-expression of gelsolin in Gsn- fibroblasts by stable transfection or adenovirus reverts the ruffling response, translational motility and rac expression to normal. Rac migrates to the cell membrane following EGF stimulation in both cell types. Gelsolin is an essential effector of rac-mediated actin dynamics, acting downstream of rac recruitment to the membrane.


Subject(s)
Cell Movement/physiology , Fibroblasts/cytology , GTP-Binding Proteins/physiology , Gelsolin/physiology , Actins/analysis , Actins/biosynthesis , Actins/metabolism , Animals , Bradykinin/pharmacology , Cell Cycle Proteins/physiology , Cell Membrane , Cells, Cultured , Coculture Techniques , Epidermal Growth Factor/pharmacology , GTP-Binding Proteins/analysis , Gene Expression Regulation/physiology , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Pseudopodia , Signal Transduction/physiology , Skin/cytology , cdc42 GTP-Binding Protein , rac GTP-Binding Proteins
15.
EMBO J ; 17(4): 967-76, 1998 Feb 16.
Article in English | MEDLINE | ID: mdl-9463375

ABSTRACT

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.


Subject(s)
Actins/metabolism , Brain/metabolism , Contractile Proteins , Endocytosis , Microfilament Proteins/metabolism , Animals , Brain Chemistry , Cells, Cultured , Chromatography, Affinity , Dynamin I , Dynamins , GTP Phosphohydrolases/metabolism , Hippocampus/metabolism , Mice , Microfilament Proteins/biosynthesis , Microfilament Proteins/isolation & purification , Models, Molecular , Neurons/metabolism , Profilins , Protein Binding , Rats , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
16.
J Neurosci ; 17(21): 8178-86, 1997 Nov 01.
Article in English | MEDLINE | ID: mdl-9334393

ABSTRACT

Calcium influx through NMDA receptors and voltage-dependent calcium channels (VDCC) mediates an array of physiological processes in neurons and may also contribute to neuronal degeneration and death in neurodegenerative conditions such as stroke and severe epileptic seizures. Gelsolin is a Ca2+-activated actin-severing protein that is expressed in neurons, wherein it may mediate motility responses to Ca2+ influx. Primary hippocampal neurons cultured from mice lacking gelsolin exhibited decreased actin filament depolymerization and enhanced Ca2+ influx after exposure to glutamate. Whole-cell patch-clamp analyses showed that currents through NMDA receptors and VDCC were enhanced in hippocampal neurons lacking gelsolin, as a result of decreased current rundown; kainate-induced currents were similar in neurons containing and lacking gelsolin. Vulnerability of cultured hippocampal neurons to glutamate toxicity was greater in cells lacking gelsolin. Seizure-induced damage to hippocampal pyramidal neurons was exacerbated in adult gelsolin-deficient mice. These findings identify novel roles for gelsolin in controlling actin-mediated feedback regulation of Ca2+ influx and in neuronal injury responses. The data further suggest roles for gelsolin and the actin cytoskeleton in both physiological and pathophysiological events that involve activation of NMDA receptors and VDCC.


Subject(s)
Actins/metabolism , Calcium Channels/drug effects , Calcium/metabolism , Gelsolin/physiology , Glutamic Acid/toxicity , Hippocampus/drug effects , Kainic Acid/toxicity , Nerve Tissue Proteins/physiology , Neurons/drug effects , Neurotoxins/toxicity , Receptors, N-Methyl-D-Aspartate/drug effects , Signal Transduction/drug effects , Animals , Cells, Cultured , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Drug Resistance , Feedback , Gelsolin/deficiency , Gelsolin/genetics , Glutamic Acid/pharmacology , Hippocampus/cytology , Hippocampus/embryology , Injections , Ion Transport/drug effects , Kainic Acid/pharmacology , Mice , Mice, Knockout , Neurotoxins/pharmacology , Patch-Clamp Techniques , Receptors, N-Methyl-D-Aspartate/physiology , Seizures/chemically induced , Seizures/pathology , Stimulation, Chemical
17.
J Cell Biol ; 138(6): 1279-87, 1997 Sep 22.
Article in English | MEDLINE | ID: mdl-9298983

ABSTRACT

Growth cones extend dynamic protrusions called filopodia and lamellipodia as exploratory probes that signal the direction of neurite growth. Gelsolin, as an actin filament-severing protein, may serve an important role in the rapid shape changes associated with growth cone structures. In wild-type (wt) hippocampal neurons, antibodies against gelsolin labeled the neurite shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt and gelsolin null (Gsn-) mice (Witke, W., A.H. Sharpe, J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41-51.) was recorded with time-lapse video microscopy. The number of filopodia along the neurites was significantly greater in Gsn- mice and gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed from the region of the growth cone and remained as protrusions from the newly consolidated shaft after the growth cone advanced. Histories of individual filopodia in Gsn- mice revealed elongation rates that did not differ from controls but an impaired retraction phase that probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function in the initiation of filopodial retraction and in its smooth progression.


Subject(s)
Gelsolin/genetics , Mice, Knockout/physiology , Neurites/physiology , Animals , Cell Size/physiology , Cells, Cultured , Hippocampus/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Video , Neurites/ultrastructure , Neurons/chemistry , Neurons/physiology , Neurons/ultrastructure , Pseudopodia/physiology
18.
Circulation ; 94(6): 1394-401, 1996 Sep 15.
Article in English | MEDLINE | ID: mdl-8822998

ABSTRACT

BACKGROUND: Gene delivery is a rapidly expanding field with potential applications to every human organ system. Recently, adenoviruses have been used as efficient vectors for in vivo gene transfer into the myocardium. These methods, however, have shown a sharp decline of gene expression after 1 week. To test the hypothesis that an immune-effector mechanism is involved in this decline, we compared the results after injection of adenovirus-5 carrying the beta-galactosidase gene (Ad beta-gal) into the left ventricular myocardium of athymic nude rats (NDRs) versus immunocompetent Sprague-Dawley rats (SDRs). METHODS AND RESULTS: Ad beta-gal (5.0 x 10(9) PFU/mL) was injected into the left ventricle of NDRs (n = 16) and SDRs (n = 22). Hearts were harvested, embedded in paraffin, and sectioned and stained for beta-gal activity, hematoxylin and eosin and picrosirius red at 4, 21, 35, 85, and 120 days. Representative samples were immunostained with antibodies directed at inflammatory markers. beta-gal activity was quantified by digital planimetry and expressed as area of staining (% +/- SEM). Peak beta-gal activity was highest at 4 days, with NDRs displaying significantly greater staining (83 +/- 3.0% versus 54 +/- 8.0%; P = .03). SDRs sustained a rapid drop in activity, such that at 35 (1 +/- 0.19%) and 85 (1 +/- 0.4%) days, only occasional cells stained positive and by 120 days (0.3 +/- 0.0%), activity had been extinguished. NDRs continued to show transgene expression at all time periods (35 and 85 days, 25 +/- 7.1% and 7.4 +/- 2.7%, respectively) and was still readily detected at 120 days. An inflammatory response was limited in NDRs compared with SDRs, in which there was intense mononuclear cell infiltration, with collagen deposition and scar formation. Immunostaining identified the majority of these inflammatory cells as not being of lymphocyte lineage, although small numbers of lymphocytes and phagocytic and activated plasma cells were identified. CONCLUSIONS: Our data suggest that immune-effector mechanisms can severely affect the expression of genes delivered by adenovirus. The present model provides efficient gene expression for at least 120 days without significant inflammatory reaction.


Subject(s)
Adenoviridae/physiology , Gene Expression , Gene Transfer Techniques , Heart/physiology , Myocardium/immunology , Virus Replication , Animals , Antibody Formation , Collagen/metabolism , Female , Immunohistochemistry , Myocarditis/genetics , Myocarditis/pathology , Myocardium/metabolism , Myocardium/pathology , Rats , Rats, Nude , Rats, Sprague-Dawley , Time Factors
19.
J Cell Biol ; 134(2): 389-99, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8707824

ABSTRACT

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F-actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.


Subject(s)
Actins/metabolism , Blood Platelets/metabolism , Gelsolin/metabolism , Microfilament Proteins/metabolism , Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors , Animals , Blood Platelets/drug effects , Blood Platelets/ultrastructure , CapZ Actin Capping Protein , Cell Membrane Permeability , Chickens , Contractile Proteins/metabolism , Cytoskeleton/metabolism , Destrin , Filamins , Glucosides/pharmacology , Humans , Hydrogen-Ion Concentration , Mice , Muscle Proteins , Phosphatidylinositol 4,5-Diphosphate , Phosphatidylinositol Phosphates/pharmacology , Platelet Activation , Receptors, Thrombin/metabolism
20.
Biophys J ; 70(2): 1054-60, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8789124

ABSTRACT

To determine the specific contribution of cytoskeletal proteins to cellular viscoelasticity we performed rheological experiments with Dictyostelium discoideum wild-type cells (AX2) and mutant cells altered by homologous recombination to lack alpha-actinin (AHR), the ABP120 gelation factor (GHR), or both of these F-actin cross-linking proteins (AGHR). Oscillatory and steady flow measurements of Dictyostelium wild-type cells in a torsion pendulum showed that there is a large elastic component to the viscoelasticity of the cell pellet. Quantitative rheological measurements were performed with an electronic plate-and-cone rheometer, which allowed determination of G', the storage shear modulus, and G", the viscous loss modulus, as a function of time, frequency, and strain, respectively. Whole cell viscoelasticity depends strongly on all three parameters, and comparison of wild-type and mutant strains under identical conditions generally produced significant differences. Especially stress relaxation experiments consistently revealed a clear difference between cells that lacked alpha-actinin as compared with wild-type cells or transformants without ABP120 gelation factor, indicating that alpha-actinin plays an important role in cell elasticity. Direct observation of cells undergoing shear deformation was done by incorporating a small number of AX2 cells expressing the green fluorescent protein of Aequorea victoria and visualizing the strained cell pellet by fluorescence and phase contrast microscopy. These observations confirmed that the shear strain imposed by the rheometer does not injure the cells and that the viscoelastic response of the cell pellet is due to deformation of individual cells.


Subject(s)
Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Dictyostelium/genetics , Dictyostelium/metabolism , Mutation , Actinin/genetics , Actinin/metabolism , Animals , Biomechanical Phenomena , Biophysical Phenomena , Biophysics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Elasticity , Fungal Proteins/genetics , Fungal Proteins/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Phenotype , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Rheology , Stress, Mechanical , Viscosity
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