Conception Ferraz

Immunocytochemical localisation of Gelsolin in Oligodendroglia of the developing rabbit central nervous system

Antibodies raised against gelsolin of rabbit lung macrophages were used in an immunocytochemical study during development of the rabbit central nervous system. With the exception of a transient staining of the cerebellar Purkinje cells during the first postnatal week, gelsolin was found a new marker for oligodendrocytes, especially in developing animals. In the macrophages, gelsolin is involved in the control of locomotion, secretion and endocytosis. In the oligodendrocytes, which produce the myelin sheaths in the central nervous system, gelsolin could therefore be involved in the control of the complex motile events leading to myelin wrapping.

Effect of thyroid deficiency on actin mRNA content in the developing rat cerebellum

The actin mRNA content of the cerebellum was determined in normal and hypothyroid developing rats using RNA dot hybridization with a β‐actin cDNA probe. The decline in actin mRNA content occurring during the second postnatal week in normal development was delayed by about 1 week in hypothyroid rats. Since this effect coincides exactly with the delay in actin filament formation recently reported in thyroid‐deficient rats, it strengthens the hypothesis of an inverse relationship in the developing brain between the polymerization state of actin and the production of actin mRNA.

High gelsolin content of developing oligodendrocytes

The actin‐binding protein gelsolin that severs and caps the actin microfilaments under the control of the cytoplasmic free calcium and the membranous phosphatidylinositol 4,5‐bisphosphate, is essentially restricted to the oligodendroglia in the central nervous system. Immunocytochemistry showed that gelsolin is an early marker of oligodendrocytes, both in vivo, in the rat cerebellum, and in vitro, in oligodendrocyte culture. We report the early appearance of gelsolin in A2B5‐positive precursor oligodendrocyte cells and the specific expression of gelsolin in OL‐1‐, GC‐, and MBP‐positive oligodendrocytes in culture. The protein was distributed throughout the cell body and in the branched cell processes of cultured oligodendrocytes, but not in the MBP‐positive membrane sheets. Gelsolin is thus cytosolic and not a myelin component. The quantitative study demonstrated that the cerebellar gelsolin content changes significantly with age, with the maximal value at the age of 21 days, confirming that large amounts of gelsolin are transiently synthesized during development, especially from the first events of myelinogenesis. The results are consistent with gelsolin being involved, through its effects on the actin cytoskeleton, in the motile events occurring during the growth of the oligodendroglial processes towards the axons and the wrapping of the myelin sheaths around the axons.

Cloning and deletion mutagenesis using direct protein-protein interaction on an expression vector identification of the calmodulin binding domain of α-fodrin☆

We have screened a lambda gt11 library, constructed with mouse macrophage cDNA, in order to isolate clones that code for calmodulin binding proteins. We have developed a new approach for this purpose using radioactive calmodulin (produced by genetic engineering) to detect fusion proteins that interact with this protein with high affinity. A cDNA clone that codes for mouse macrophage fodrin was isolated, sequenced and identified. By deleting part of the sequence the calmodulin binding domain was located on the fodrin sequence. The site is situated on repeat 11 of fodrin and probably on the extra arm of this repeat. The method we developed is widely applicable to site-directed mutagenesis of interacting proteins.

Evolution of tropomyosin functional domains: Differential splicing and genomic constraints

SummaryWe have cloned and determined the nucleotide sequence of a complementary DNA (cDNA) encoded by a newly isolated human tropomyosin gene and expressed in liver. Using the leastsquare method of Fitch and Margoliash, we investigated the nucleotide divergences of this sequence and those published in the literature, which allowed us to clarify the classification and evolution of the tropomyosin genes expressed in vertebrates. Tropomyosin undergoes alternative splicing on three of its nine exons. Analysis of the exons not involved in differential splicing showed that the four human tropomyosin genes resulted from a duplication that probably occurred early, at the time of the amphibian radiation. The study of the sequences obtained from rat and chicken allowed a classification of these genes as one of the types identified for humans.The divergence of exons 6 and 9 indicates that functional pressure was exerted on these sequences, probably by an interaction with proteins in skeletal muscle and perhaps also in smooth muscle; such a constraint was not detected in the sequences obtained from nonmuscle cells. These results have led us to postulate the existence of a protein in smooth muscle that may be the counterpart of skeletal muscle troponin.We show that different kinds of functional pressure were exerted on a single gene, resulting in different evolutionary rates and different convergences in some regions of the same molecule.Codon usage analysis indicates that there is no strict relationship between tissue types (and hence the tRNA precursor pool) and codon usage. G+C content is characteristic of a gene and does not change significantly during evolution. These results are in good agreement with an isochore composition of the genome, and thus suggest a similar chromosomal environment in chicken, rat, and human.

Expression of tropomyosin genes during the development of the rat cerebellum.

Abstract: The expression of the tropomyosin genes in the rat nervous system was examined during the postnatal development of the cerebellum, using human‐specific α‐, β‐, γ‐, and d‐tropomyosin cDNA probes and rat‐specific α‐, β‐, and d‐tropomyosin oligonucleotide probes. The β‐ and γ‐genes do not seem to be expressed in the rat brain. The δ‐tropomyosin gene produces two mRNAs: a major one of 2.4 kb, which is highly concentrated during the first postnatal week and then decreases fourfold in level until the age of 35 days, and a minor one of 2 kb, with the same developmental profile as the 2.4‐kb mRNA. A 3‐kb mRNA is expressed by the α‐tropomyosin gene and is characteristic of the mature rat. The expression of the tropomyosin genes during the development of the rat cerebellum does not seem to be regulated through alternative splicing but rather implies the differential expression of two different isogenes. The multiple isoforms of tropomyosin produced during neuronal differentiation may be intimately involved in the regulation of the organization and function of actin microfilaments.

Distribution of gelsolin in the retina of the developing rabbit

SummaryThe distribution of gelsolin, a calcium-dependent actin-severing and capping protein, in the retina of the developing and adult rabbit was studied. Gelsolin immunoreactivity was found in the photoreceptors and ganglion cells, where it may have a role in neuronal morphogenesis. Only the inner segment of the photoreceptors retained a high gelsolin content in the adult retina, perhaps because the attached outer segment is continuously renewed throughout life. Gelsolin, which is a major component of the rabbit brain oligodendrocytes, was also found in the myelin of the medullary ray region of the rabbit retina. Müller cells in all regions of the rabbit retina also contain gelsolin from early in development to adulthood. Since one of the functions of these cells is to ensheath neuronal elements in the inner plexiform and optic fiber layers, we suggest that gelsolin may play the same role in Müller cells as it does in oligodendrocytes, i.e., sheath formation via its calcium-dependent action on the actin microfilament networks.

Gelsolin immunoreactivity and development of the tectorial membrane in the cochlea of normal and hypothyroid rats

SummaryGelsolin was localized by immunocytochemistry in the developing cochlea of the rat. In normal animals, the protein appeared at 18 th day in utero in cells of the Köllikers organ, which are involved in the secretion of the tectorial membrane. The Köllikers organ cells were not immunoreactive after the first postnatal week, which is when they cease their secretory activity. Gelsolin immunoreactivity was similar in thyroid-deficient rats until the second postnatal week but, at this age, Köllikers organ did not transform and its gelsolin immunoreactivity persisted, together with its secretory activity. As a result, the tectorial membrane was greatly distorted and out of contact with the hair cells, which dramatically impaired the mechanical properties of the organ of Corti. The developing cochlea thus provides an example of the involvement of gelsolin in a secretory process that is of importance in the development of hearing.

Structural analysis of human skeletal β-tropomyosin produced in E. coli

We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside. We show that a new protein was synthesized by bacteria containing the engineered plasmid. This protein was heat stable and reacted with antibodies against tropomyosin. We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal. Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine. The circular dichroism spectrum is compatible with 100% alpha-helices. These results show that the protein synthesized in E. coli possesses a native structure.

Profilin and profilin mRNA in the cerebellum of the developing rat.

Profilin and profilin mRNA concentrations were determined in the cerebellum of the developing rat. Profilin represented nearly 0.1% of total Triton-soluble protein both at birth and at the end of the developmental period. During brain maturation, the profilin concentration was regulated at the transcriptional level. A transient increase of profilin mRNA and profilin was observed at the end of the first postnatal week, a period of intense cell proliferation, neuritic outgrowth and synaptogenesis. We propose that profilin may act by forming, during the critical period of cerebellar development, a reserve pool of monomeric actin that can easily be mobilized in cell regions such as growth cones or synaptic junctions where filamentous actin is highly concentrated.