Molecular genetic approaches to understanding the actin cytoskeleton.

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.

Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis.

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.

Studies on the transcription, translation, and structure of alpha-actinin in Dictyostelium discoideum.

A clone coding for the F-actin cross-linking protein alpha-actinin was obtained by screening a genomic library of Dictyostelium discoideum DNA in lambda gt11 with monoclonal antibodies specific for Dictyostelium alpha-actinin. The 1.2-kilobase (kb) genomic clone was confirmed as containing part of the alpha-actinin gene by comparing its nucleotide sequence with the amino acid sequence of tryptic peptides from purified alpha-actinin. The clone recognized a 3.0-kb message in a Northern blot. Hybridization to RNA isolated from different developmental stages of several D. discoideum strains indicated that the mRNA content increased during early development. A similar result was obtained when the alpha-actinin content of the cells was followed by Western blot analysis. Hybridization of the clone to DNA from different wild-type strains of D. discoideum indicated a polymorphism on the DNA level that coincided with a polymorphism on the protein level. The data suggest continuous transcription of the alpha-actinin gene throughout the development of D. discoideum, up- and down-regulation of the levels of alpha-actinin mRNA and protein with maximum levels at the onset of aggregation, and a high diversity of alpha-actinin at the DNA and protein level among different D. discoideum strains. The structural data make it conceivable that the highly conserved nature of alpha-actinin resides only at the functional sites, whereas the helical portions of the alpha-actinin molecule allow a higher level of diversity throughout evolution.

Comparisons of CapG and gelsolin-null macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing.

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.

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein.

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.

Delayed retraction of filopodia in gelsolin null mice.

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.

The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis.

Dictyostelium alpha-actinin is a Ca(2+)-regulated F-actin cross-linking protein. To test the inhibitory function of the two EF hands, point mutations were introduced into either one or both Ca(2+)-binding sites. After mutations, the two EF hands were distinguishable with respect to their regulatory activities. Inactivation of EF hand I abolished completely the F-actin cross-linking activity of Dictyostelium discoideum alpha-actinin but Ca2+ binding by EF hand II was still observed in a 45Ca2+ overlay assay. In contrast, after mutation of EF hand II the molecule was still active and inhibited by Ca2+; however, approximately 500-fold more Ca2+ was necessary for inhibition and 45Ca2+ binding could not be detected in the overlay assay. These data indicate that EF hand I has a low affinity for Ca2+ and EF hand II a high affinity, implying a regulatory function of EF hand I in the inhibition of F-actin cross-linking activity. Biochemical data is presented which allows us to distinguish two functions of the EF hand domains in D. discoideum alpha-actinin: (a) at the level of the EF-hands, the Ca(2+)-binding affinity of EF hand I was increased by EF hand II in a cooperative manner, and (b) at the level of the two subunits, the EF hands acted as an on/off switch for actin-binding in the neighboring subunit. To corroborate in vitro observations in an in vivo system we tried to rescue the abnormal phenotype of a mutant (Witke, W., M. Schleicher, A. A. Noegel. 1992. Cell. 68:53-62) by introducing the mutated alpha-actinin cDNAs. In agreement with the biochemical data, only the molecule modified in EF hand II could rescue the abnormal phenotype. Considering the fact that the active construct is "always on" because it requires nonphysiological, high Ca2+ concentrations for inactivation, it is interesting to note that an unregulated alpha-actinin was able to rescue the mutant phenotype.

Mena is required for neurulation and commissure formation.

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.

Gelsolin and diseases.

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.

Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I.

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.

Calcium-sensitive non-muscle alpha-actinin contains EF-hand structures and highly conserved regions.

The F-actin crosslinking molecule alpha-actinin from the slime mould Dictyostelium discoideum carries two characteristic EF-hand structures at the C-terminus. The calcium-binding loops contain all necessary liganding oxygens and most likely form the structural basis for the calcium sensitivity of strictly calcium-regulated non-muscle alpha-actinins. Furthermore, the sequence exhibits at the N-terminal site of the molecule a high degree of homology to chicken fibroblast alpha-actinin. This stretch of amino acids appears to have remained essentially constant during evolution and might represent the actin-binding site. The findings have led us to propose a model for the inhibitory action of Ca2+ on non-muscle alpha-actinins.

Purkinje cell loss and motor coordination defects in profilin1 mutant mice.

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.

Electron microscopical localization of the alpha 2-macroglobulin thiol ester sites.

The active thiol ester groups of alpha 2-macroglobulin (alpha 2M) were reacted with a biotin derivative and the sites labelled with avidin-ferritin complexes. Electron micrographs show a strong preference of attachment of the ferritins to the ends of the rods of the H-shaped molecules. A mutual "cross-labelling" was observed in an alpha 2M preparation which yielded dimers of the molecules which must have been formed during purification. The molecules were mostly attached to each other at the ends of the rods of the H-shaped molecules. It is concluded that the thiol esters responsible for the covalent attachment of the proteinases (and other molecules) may be located more in the distal parts of the alpha 2M molecules, while the proteinase molecules are finally trapped near to the centre of the alpha 2M molecules.

Inactivation of the alpha-actinin gene in Dictyostelium.

alpha-Actinin-negative transformants of Dictyostelium have been obtained by transforming cells with a transformation vector carrying part of the alpha-actinin gene in either sense or antisense orientation. The transformants did not produce detectable alpha-actinin anymore and contained an altered RNA lacking the 3' part of the coding sequences. The deficiency in alpha-actinin was due to an integration of the transformation vector into the gene, since it could be detected by Southern blot analysis in the endogenous gene.

A single base exchange in an intron of the Dictyostelium discoideum alpha-actinin gene inhibits correct splicing of the RNA but allows transport to the cytoplasm and translation.

The genomic structure of the alpha-actinin gene from Dictyostelium discoideum has been determined. The coding region consists of three exons separated by two short introns located at either end of the gene. In an alpha-actinin mutant, HG1130, about 1% of alpha-actinin protein products are found compared to the parent strain AX2. The size of the mutant proteins are 88 and 95 kDa. In HG1130, the second intron is improperly spliced from the primary alpha-actinin transcript resulting from a mutation at the 5' splice site from a GT to an AT. The premature RNA is less efficiently cleaved at the mutated 5' splice site and the following step of the splicing reaction, ligation of exons 2 and 3, is prevented. Thus, two RNA species are generated, one in which intron 2 is not removed and one in which exon 2 is not ligated to exon 3. The two RNA species are stable, transported to the cytoplasm, bound to polysomes and translated. In vitro and in vivo, the partially spliced RNAs are translated into proteins of 88 and 95 kDa which can be immunoprecipitated with an alpha-actinin-specific monoclonal antibody. The in vivo stability of the mutant proteins is comparable to the wild type alpha-actinin.

Homologous recombination in the Dictyostelium alpha-actinin gene leads to an altered mRNA and lack of the protein.

Mutation of the alpha-actinin gene in Dictyostelium has been achieved by transforming cells with the Dictyostelium transformation vector pDNeoII containing a 1.2 kb fragment of the alpha-actinin gene. Transformants deficient in alpha-actinin, an actin-binding protein, produced an altered mRNA that lacked the 3' portion of the coding region. The defect in alpha-actinin production was not due to integration of the vector within the gene, but was apparently caused by errors produced during homologous recombination between the introduced alpha-actinin sequence and its complementary sequence in the coding region of the endogenous gene.

Quinones from archaebacteria, II. Different types of quinones from sulphur-dependent archaebacteria.

From the sulphur-dependent, anaerobically grown archaebacterium Sulfolobus ambivalens Caldariella quinone, CQ-6(12H) and the new Sulfolobus quinone SQ-6(12H), 6-(3,7,11,15,19,23-hexamethyltetracosyl)-5-methyl-benz[b]thioph en-4, 7-quinone have been isolated as main components. Lower homologues SQ-5-(10H), SQ-4(8H), SQ-3(6H), phylloquinone-like species CQ-6(10H), SQ-6(10H) and the menaquinone MK-6(12H) are present as minor components. The results are compared with those from Sulfolobus acidocaldarius. Thermococcus celer, Desulfurococcus mucosus and Desulfurococcus mobilis do not contain quinones in comparable amounts.

Redundancy in the microfilament system: abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins.

We generated by gene disruption Dictyostelium cells that lacked both the F-actin cross-linking proteins, alpha-actinin and gelation factor. Several major cell functions, such as growth, chemotaxis, phagocytosis, and pinocytosis, were apparently unaltered. However, in all double mutants, development was greatly impaired. After formation of aggregates, cells were very rarely able to form fruiting bodies. This ability was rescued when mutant and wild-type strains were mixed in a ratio of 70 to 30. The developmental program in the mutant was not arrested, since the expression pattern of early and late genes remained unchanged. Development of the mutant was rendered normal when a functional alpha-actinin gene was introduced and expressed, showing the morphogenetic defect to be due to the absence of the two F-actin cross-linking proteins. These findings suggest the existence of a functional network allowing mutual complementation of certain actin-binding proteins.

Alternative splicing of the mouse profilin II gene generates functionally different profilin isoforms.

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.