Objetivo
Studies focussed on 2 major areas:
examining the fine structure of actin crosslinking proteins that modulate the structure and assembly of F-actin filaments into gels and networks that closely resemble those found in vivo;
correlating in vitro structural and biochemical studies of these crosslinking proteins with their function in vivo.
The investigation concentrated on 2 actin crosslinking proteins, alpha-actinin and gelation factor, that are involved in motility and cytoskeletal dynamics in the motile amoeba Dictyostelium discoideum.
In alpha-actinin distinctive EF-hand calcium ion binding motifs are located at carboxyl termini (residues 697-861) and regulate the interaction with actin. The importance of the EF-hands for calcium ion binding and crosslinking was investigated by generating point mutations in the alpha-actinin complementary deoxyribonucleic acid (cDNA) in such a way that the calcium ion binding residues were replaced by alanine residues. 3 mutant alpha-acinins were obtained, with EF-hand I, EF-hand II and EF-hands I and II mutated. The mutated cDNAs were expressed in Dictyostelium discoideum and the corresponding proteins purified. They were investigated with regard to calcium binding and activitity in a crosslinking assay. The affinities for calcium ion differed for the 2 EF-hands with EF-hands II exhibiting the higher affinity. In contrast, for F-actin crosslinking EF-hand I seems to be important since only molecules with this calcium ion binding site intact could increase the viscosity of an actin solution.
Alpha-actinin was purified as a dimer by gel filtration chromatography under nondenaturing conditions. Isoelectric focussing showed 1 isoelectric species at pH 5.2. The protein was studied using biophysical methods. X-ray scattering experiments were performed using the small angle instrument D24 at the Synchrotron radiation laboratory LURE (Orsay). Electron microscopy was performed at the Centre National de la Recherche Scientifique (C NRS) in Gif-sur-Yvette by Dr J Lepault. By electron microscopy of rotary shadowed samples, alpha-actinin appeared as a rod shaped particle of 38 nm in length and 3 to 4 nm wide which is in agreement with previous studies.
The actin-binding domain of alpha-actinin is located at the amino terminus. Residues 28 to 257 and residues 98 to 174 of Dictyostelium alpha-actinin was expressed in Escherichia coli (E Coli) and its actin-binding properties investigated. Whereas the larger of the recombinant proteins bound to F-actin under routine conditions, the smaller was only able to do so under more acidic conditions.
The rod domain of alpha-actinin is a module spacer which places the actin-binding domain in the correct position to perform its function. It is thought to be essential in the formation of a stable dimer. To further investigate the rod domain, a 1.4 Kilobase deoxyribonucleic acid (DNA) (kb) fragment comprising this domain was expressed in E coli K38 using the bacteriophase T7 ribonucleic acid (RNA) polymerase promoter system. The 55 kD recombinant protein was purified from the soluble fraction by fractionation on a diethylaminoethyl (DEAE) cellulose column and size exclusion chromatography and used for electron microscopy (performed at CNRS by Dr Jean Lepault). The length of the rod-shaped molecules was determined as between 27 and 35 um.
By analyzing its sequence, 2 domains were defined in the gelation factor: an actin-binding domain analogous to that in alpha-actinin; and a rod domain containing 6 100-residue repeating motifs. To obtain structural information each domain was expressed separately in E coli.
The actin-binding domain islocated at the amino terminus. A construct corresponding to residues 1 to 250 was expressed and large quantities of pure material obtained. Sedimentation assays verified that this fragment possessed actin-binding activity.
The repeating motifs of the rod domain of gelation factor are very different from those found in a lpha-actinin. Analysis of the sequence indicated a cross-beta structure and circular dichroism (CD) spectroscopy was to confirm the presence of a high content of beta-structure.
Construct corresponding to the entire rod (domains 1 to 6; 600 residues) were expressed in E Coli as well as domains 1 to 5 (500 residues) and 3 to 4 (200 residues). All protein were soluble and substantial quantities could be purified without great difficulty. CD spectroscopy showed that constructs 1 to 6 and 1 to 5 had retained their beta-conformation, whereas construct 3 to 4 appeared not to have folded correctly and so was not investigated further. Sedimentation equilibrium in an analytical ultracentrifuge indicated a molecular weight of 110 kD for construct 1 to 6 and 87 kD for 1 to 5, indicating that both had dimerized. Dimerization was confirmed using chemical crosslinking with both glutaraldehyde and 1-ethyl-3-dimethyl-aminopropyl-carbodiimide EDC).
Structural examinations of the rod constructs were performed by Dr Stewart at the Medical Research Council (MRC) Cambridge. Electron microscopy of shadowed specimens confirmed they were rod-shaped. Construct 1 to 6 was approximately 30 nm long whereas 1 to 5 was 25 nm long. Since the entire gelation factor is 35 nm long these results were consistent with a 5 nm actin-binding domain and were also consistent with the proposed size of the 100-residue domains. Dictyostelium mutants lacking alpha-actinin and gelation factor are impaired in developed. Nitrosoguanidine was used to introduce mutations into Dictyostelium DNA and mutants carrying defects only in the alpha-actinin and gelation factor genes isolated. To achieve this, these genes were destroyed by gene replacement and homologous recombination and 2 independent mutant cell lines were isolated. Both of them exhibited defects in development and are now being used as recipients in gene transfer experiments with various gelation factor constructs. In a first attempt the defect in dev elopment could be restored by introducing an intact gelation factor gene.
We intend employ the complementary structural, biochemical and molecular biology skills possessed by our three laboratories to explore the structural basis for interactions involving actin crosslinking proteins that are important for cytoskeletal structure and its modulation during cell motility. Initially we will
concentrate on the DICTYOSTELIUM gelation factor. Because this protein shares homologies with a range of other actin-binding and crosslinking proteins, such as alpha-actinin and dystrophin, the results we obtain should be of broad general significance. We will characterise the molecular basis for interactions between chains in the protein and also those with actin together with their control. This information will be then used to construct mutants to investigate the function of these proteins in vivo.
Ámbito científico (EuroSciVoc)
CORDIS clasifica los proyectos con EuroSciVoc, una taxonomía plurilingüe de ámbitos científicos, mediante un proceso semiautomático basado en técnicas de procesamiento del lenguaje natural. Véas: El vocabulario científico europeo..
CORDIS clasifica los proyectos con EuroSciVoc, una taxonomía plurilingüe de ámbitos científicos, mediante un proceso semiautomático basado en técnicas de procesamiento del lenguaje natural. Véas: El vocabulario científico europeo..
- ciencias naturales ciencias de la tierra y ciencias ambientales conexas geología sedimentología
- ciencias naturales ciencias biológicas genética ADN
- ciencias naturales ciencias químicas química inorgánica metales alcalinotérreos
- ciencias naturales ciencias biológicas bioquímica biomoléculas proteínas
- ciencias naturales ciencias físicas óptica microscopía electron microscopy
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82152 MARTINSRIED
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