A 48 kilodalton intermediate filament associated protein (IFAP) in reactive-like astrocytes induced by dibutyryl cyclic AMP in culture and in reactive astrocytes in situ.

In this paper we demonstrate that the 48 kilodalton (kDa) intermediate filament associated protein (IFAP), previously reported to be present in normal astrocytes, is also present in reactive astrocytes in situ and in reactive-like astrocytes induced by dibutyryl cyclic AMP in vitro. This IFAP is detectable by antibodies in normal rabbit serum (F2N) and is closely associated with glial fibrillary acidic protein-containing intermediate filaments (IF). The expression of 48 kDa IFAP is related to the acquisition of stellate shape by normal and reactive-like astrocytes in vitro. It is proposed that 48 kDa IFAP may be responsible for cross-linking IF into bundles and is thereby associated with cell process formation.

Contractile units in stress fibers of fetal human astroglia in tissue culture.

The interaction between myosin and F-actin requires the enzyme, myosin light chain kinase (MLCK), as well as Ca(2+)-calmodulin and the calmodulin binding protein, caldesmon, which also binds to F-actin. Using immunofluorescence staining, we have demonstrated that in human fetal astroglia as in mouse astroglia (Abd-El-Basset et al., 1991) the stress fibers contain these contractile elements: F-actin, myosin, tropomyosin and caldesmon. F-actin extends continuously along the stress fibers, whereas myosin, tropomyosin and caldesmon are localized discontinuously in a periodic pattern. In addition, we have demonstrated that fetal human astroglia have the enzyme MLCK and calmodulin. The association of the contractile elements listed above together with calmodulin and MLCK constitutes what may be termed 'contractile units', suggesting that the stress fibers in astroglia may be contractile. Contractile stress fibers would enable astroglia to exert tension on the matrix surrounding them, thus facilitating rapid changes in cell shape.

Immunolocalization of the alpha isoform of smooth muscle actin in mouse astroglia in cultures.

In mouse astroglia colony cultures, cells display dramatic changes in their 'social behavior' during differentiation. The non-motile glioblasts progress through highly motile pro-astroblast and astroblast stages before becoming non-motile fibrous astrocytes. These changes in the behavior of astroglia are paralleled by changes in the organization of their microfilaments. Immunofluorescence staining of the astroglia colony cultures with rhodamine phalloidin, which binds to actin filaments (F-actin) formed by any of the 6 actin isoforms and with monoclonal antibody to alpha-smooth muscle (alpha-sm) actin (an actin isoform typical of smooth muscle cells), shows that the majority of the cells are positive for both alpha-sm actin and F-actin. Approximately 15% of the cells, however, were negative for alpha-sm actin but stained positively for F-actin, indicating that the actin of these cells belongs to isoforms other than the alpha-sm isoform.

Effect of interleukin-1ß on the expression of actin isoforms in cultured mouse astroglia.

Cytokines are soluble mediators that are thought to act as communication signals between astroglia and neighboring neural cells. They are both released by, and act on, astroglia. It is hypothesized that it is this effect on astroglia that may be important in widespread phenomena including traumatic brain injury, inflammation, and scar formation. In this article, we examine the effect of mouse recombinant interleukin-1ß (IL-1ß) on the morphology, organization, and expression of glial fibrillary acidic protein (GFAP) and actin isoforms in cultured mouse astroglia. This study shows that the majority of the astroglia treated with IL-1ß acquire long processes. Immunofluorescence staining shows that there are no remarkable changes in the organization of GFAP, F-actin, α-smooth muscle (α-sm) actin, and ß-actin isoforms. In fluorescent microplate assay, the short-term treated astroglia (range, 1-2 days) show an increase in the intensity of GFAP and ß-actin isoform over the level observed in untreated control, whereas no remarkable changes are observed in the intensity of α-sm actin isoform. In the case of long-term treatment (range, 4-8 days), the intensity of GFAP and α-sm actin isoform progressively decreases below the level of untreated control. In addition, the intensity of ß-actin isoform increases above the control level. These results have been confirmed by immunoblotting experiments. The upregulation of ß-actin isoform may be important in limiting the noxious effects of an inflammatory reaction. This gives credence to the hypothesis that it might be possible to modulate astroglial effects on neuronal inflammation and scar formation with appropriate therapies.

The effect of dibutyryl cyclic AMP on the expression of actin isoforms in astroglia.

Mammalian cells contain at least 8 actin isoforms. The functional significance and the mechanisms that regulate the expression of each actin isoform are not yet known. Using immunofluorescence staining, it was found that all astroglia in tissue culture express beta-actin isoform and about 86% of astroglia express alpha-smooth muscle actin isoform. When astroglia were treated with dibutyryl cyclic AMP for 4, 7, 14 and 21 days, it was found that the number of the cells expressing alpha-smooth muscle actin isoform progressively decreased, whereas, the number of the cells expressing beta-actin isoform remained constant. The western blot experiment showed that the amount of total alpha-smooth muscle actin isoform (soluble and insoluble) and of the insoluble isoform expressed by astroglia treated with dibutyryl cAMP decreased whereas, the amount of total and insoluble beta-actin isoform expressed by the same cells did not show any significant changes. The cells treated with the cAMP failed to migrate and to close the area created by the scratch wound in monolayer culture. However, the non-treated cells migrated and closed the area created by the scratch after 3 days. This study shows that the astroglia have different mechanisms in regulating the expression of different actin isoforms and that the alpha-sm actin isoform is important in migration of astroglia.

Actin and actin-binding proteins in differentiating astroglia in tissue culture.

In this paper we have described the organization of F-actin and actin-binding proteins (ABP): alpha-actinin, myosin, tropomyosin, caldesmon, vinculin, talin, and spectrin, in differentiating astroglia in colony cultures. We observed that the microfilament (MF) network arrangements differ at various stages of astroglia development, but the composition of MF bundles and stress fibers is the same at all developmental stages. F-actin is closely colocalized with myosin, tropomyosin, caldesmon, and alpha-actinin. The striated pattern of myosin, tropomyosin, and caldesmon are superimposable. Tropomyosin and caldesmon extend along F-actin but are interrupted for short periods, whereas myosin is interrupted for longer periods. alpha-actinin colocalizes with tropomyosin and caldesmon but not with myosin. In astroglia at different stages of development spectrin is arranged in the form of fine networks spreading through the cell and does not follow the arrangement of MF bundles. Only F-actin, alpha-actinin, and vinculin can be detected at cell-cell junctions. In the areas of the focal contacts, F-actin, alpha-actinin, vinculin, and talin are present. They overlap each other, although talin and vinculin extend toward the cell membrane beyond F-actin and alpha-actinin. Astroglia undergo well-defined states of nonmotility, motility, and nonmotility again during differentiation. The changes in motility are paralleled by changes in the organization of F-actin and ABP: as GFAP-containing intermediate filaments increase in differentiating astroglia, the F-actin and ABP are down-regulated, leading to non motility.

Dynamics of actin filaments in microglia during Fc receptor-mediated phagocytosis.

The phagocytic ability of mouse microglia during their differentiation in culture and after stimulation with bacterial wall lipopolysaccharide (LPS) has been investigated using Fc receptor-mediated phagocytosis of immunoglobulin (IgG)-coated sheep erythrocytes (SRBCs). We observed that in 10-14 day-confluent neopallial cell cultures some immature microglia are not phagocytic but, on further culturing, they do become phagocytic. LPS-stimulated microglia are able to phagocytose larger numbers of IgG-coated SRBCs and at a faster rate than non-stimulated microglia. Within 5-10 min of phagocytosis the actin filaments of the LPS-stimulated microglia become depolymerized, leaving only bundles of actin filaments around the phagocytosed SRBCs (phagosome cups). At 30 min after the start of phagocytosis the actin filaments of the LPS-stimulated microglia begin to polymerize, and within 2 h the original pre-phagocytosis pattern of the actin filament network is re-established. The non-LPS-stimulated microglia exhibit actin filament depolymerization in only a few lamellipodia and polymerization of actin filaments around engulfed particles, but much later during phagocytosis.

Upregulation of F-actin and alpha-actinin in reactive astrocytes.

We have shown previously that in tissue culture stellate astrocytes downregulate F-actin and actin binding proteins (ABPs) (Abd-El-Basset et al.: J Neurosci Res 30:1-17, 1991), whereas the reactive-like astrocytes upregulate their F-actin (Fedoroff et al.: Neuroscience 22:255-266, 1987). In the present study we report that in normal brain, as in tissue culture, neither F-actin nor alpha-actinin (an ABP) could be detected in stellate astrocytes. When a stab wound was made in brain, F-actin and alpha-actinin were upregulated in reactive astrocytes. We also demonstrated that reactive-like astrocytes in tissue culture express alpha-actinin, which has a "dotted" appearance when immunostained, and is colocalized with F-actin in a specific arrangement.

Expression of 48-kilodalton intermediate filament-associated protein in differentiating and in mature astrocytes in various regions of the central nervous system.

In this paper we present evidence that the 48-kD intermediate filament-associated protein (IFAP) is expressed relatively late in maturation of astrocytes, after they have acquired the glial fibrillary acidic protein (GFAP). In the astrocytes of white matter in the cerebellum the GFAP is detected at P3, whereas the 48-kD IFAP is detected only at P11. In the periventricular region and the hippocampus the 48-kD IFAP was detected at P6, long after the appearance of GFAP. In adult mice the 48-kD IFAP was observed in GFAP-positive astrocytes in the white matter of cerebellum, spinal cord, brainstem, and corpus callosum as well as in GFAP-positive cells in the grey matter of cerebral cortex and spinal cord. The 48-kD IFAP was not, however, detected in radial glia and their derivatives, in Bergmann glia or in Müller glia. Thus, not all the GFAP-positive astroglia express the 48-kD IFAP. Similarly, 48-kD IFAP was not detected in cells which were GFAP-negative.

48-Kilodalton intermediate-filament-associated protein in astrocytes.

We provide evidence that a protein of 48 kilodaltons (KD), recognized by a normal rabbit serum (F2N), is associated with intermediate filaments (IF) of astrocytes both in cell cultures and in situ. Immunofluorescence staining shows that the F2N serum gives a fibrous staining pattern similar to that seen with anti-serum to glial filament protein (GFP), a protein specific for IF of astrocytes, and that both proteins are present in the perinuclear fibrous aggregates of IF produced by treating the cells with colchicine. At the ultrastructural level the gold particles decorating the 48-KD protein are localized in clusters along the IF, whereas the gold particles decorating the GFP are localized on the IF in a linear pattern. This difference in distribution and the fact that the two proteins have different electrophoretic mobilities on SDS gels indicates that the 48-KD protein although associated with IF is different from GFP. The 48-KD protein appears to be a distinct, developmentally regulated intermediate-filament-associated protein (IFAP), different from other IFAPs reported to date and the first IFAP described in astrocytes. Its appearance in late developmental stages when motile astroblasts are changing into nonmotile stellate cells suggests that the 48-KD protein may be involved in cross-linking the GFP-containing IF to provide more tensile strength to the cytoplasm at the expense of flexibility.

Up-regulation of cytoskeletal proteins in activated microglia.

OBJECTIVES: This study investigates how the tumor necrosis factor (TNF-alpha) and interleukin-1beta (IL-1beta) affect the morphology, organization, and expression of actin, beta-actin and tubulin in microglia. MATERIALS AND METHODS: Microglia cultures were prepared from neopallia of newborn mice. Immunofluorescence, immunoblotting, and ELISA studies were used. RESULTS: When microglia are treated with TNF-alpha, IL-1beta or a combination of both for 1-5 days, the majority change from an ameboid to a large, round and flat shape. F-actin and beta-actin isoform, which are diffusely arranged throughout the cytoplasm before stimulation, are reorganized into filamentous bundles underneath and parallel to the cell membrane, which projects into many ruffles. This organization is maintained even after withdrawal of the cytokines. The dense microtubule network of tubulin in nontreated microglia becomes less dense and extends to occupy the cytoplasm of the treated microglia. Immunoblotting shows that the amount of total actin, beta-actin isoform and tubulin increases in treated microglia. In addition, IL-1beta and a combination of both TNF-alpha and IL-1beta stimulate the release of IL-6 by microglia. CONCLUSION: This study suggests that TNF-alpha and IL-1beta have an effect on the expression of cytoskeletal proteins similar to some extent to that of LPS. The up-regulation of actin, beta-actin and tubulin may play a key role in the motility and recruitment of microglia to the area of central nervous system inflammation.

Immuno-electron microscopical localization of vimentin and glial fibrillary acidic protein in mouse astrocytes and their precursor cells in culture.

Immuno-electron microscopy was used to localize the distribution of vimentin and glial fibrillary acidic protein (GFAP) in mouse astrocytes and their precursor cells in primary cultures. In astroblasts and astrocytes, vimentin and GFAP form intermediate filaments (IF), which are heteropolymers, as previously observed in gliomas. Astrocytes and their precursor cells may have IF composed of GFAP-vimentin heteropolymer or vimentin alone, but IF composed of GFAP only were not seen. It seems that the formation of IF that are GFAP-vimentin heteropolymers is a feature of normal astroglia development and that the ratio of GFAP to vimentin in these IF reflects the degree of differentiation and functional state of the cell.