The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover: pH sensitivity of F-actin binding by human ADF, but not of Acanthamoeba actophorin.

Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G-actin)-ATP, and both bind co-operatively to F-actin. ADF decorates filaments below pH 7.3 and induces substantial depolymerization at higher pH values [Hawkins, M., Pope, B., Maciver, S. K. & Weeds, A. G. (1993) Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments, Biochemistry 32, 9985-9993], but, at all pH values tested, actophorin binds to filaments in a similar manner to ADF at pH 6.5. Both proteins increase the depolymerization rate at the pointed ends of gelsolin-capped filaments, but the effect of ADF is more marked at pH 8.0. Both proteins accelerate the nucleating activity when mixed with filamentous actin (F-actin), but not with gelsolin-capped filaments, and they rapidly decrease the lengths of filaments as evidenced by electron microscopy. Both of these effects are best explained by a weak severing activity. Our results are discussed in relation to earlier models and to the structural changes observed when ADF binds F-actin [McGough, A., Pope, B., Chiu, W. & Weeds, A. (1997) Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function, J. Cell Biol. 138, 771-781]. We also discuss the relevance of these observations to their possible roles in facilitating actin turnover in cells, thereby regulating filament dynamics in cell motility.

Fine-structural investigation of rat brain microvascular endothelial cells: tight junctions and vesicular structures in freshly isolated and cultured preparations.

A comparison was made between endothelial cells in freshly-isolated rat brain microvessels, and following culture of the cells for 1-10 days during growth to confluence. Attention focused on tight junctions and vesicular structures, as seen in thin sections and freeze-fracture replicas. Freshly-isolated vessels had an abnormal appearance, with a profusion of luminal microvillar processes, and extensive cytoplasmic vacuolation. There were numerous vesicular profiles, reaching a density of approximately 60 microns-2, and with a large proportion open to the surface, as shown by labelling with cationized ferritin at 4 degrees C for 5 min. Junctional zones were relatively loosely organized, with evidence for some cell:cell separation, as well as some residual tight junctional sites within zonula adhaerens junctions. In freeze-fracture replicas, junctional strands showed segments of tightly packed intramembrane particles, generally on the P face. After 1 day in culture, the cells appeared more normal, with no vacuolation or luminal processes. Vesicles were still numerous, some associated with junctional zones, while tight junctions were relatively sparse; freeze-fracture showed some incomplete tight junctional strands, with some of the intramembrane particles fracturing onto the E face. The double offset fibrillar nature of the strands could occasionally be seen. Cells cultured for 4 and 10 days showed a progressive increase in the completeness of the junctional zone, with more tight junctional contacts within the length of the adhaerens junction, and an aggregation of microfilaments in the underlying cytoplasm. The number of vesicular profiles declined, and they were progressively excluded from the junctional zone. These observations have relevance for studies on the physiology of the brain endothelium in vitro, and for comparisons with the in vivo condition.

Nuclear envelope dynamics and nucleocytoplasmic transport.

We have combined structural, biochemical and recombinant DNA methods to explore molecular interactions involved in nuclear envelope assembly dynamics and nucleocytoplasmic transport. Electron microscopy has established the overall architecture of the envelope and the relationship between nuclear pores, lamina fibres and pore-connecting fibrils. The lamin proteins that constitute the lamina resemble intermediate filament proteins, and assemble and disassemble during mitosis in response to phosphorylation. Lamins have been expressed in E. coli to facilitate structural investigations and the exploration of interaction sites with other envelope components. Disruption of envelopes has shown that nuclear pores are constructed from a central cylinder with cytoplasmic and nucleoplasmic rings. Examination of envelopes transporting gold-labelled nucleoplasmin has indicated that the transport pathway is complex and probably involves ring components in addition to the central cylinder. Molecular motors may be involved in changes in pore shape to enable transport and in the translocation mechanism.

Selective digestion of nuclear envelopes from Xenopus oocyte germinal vesicles: possible structural role for the nuclear lamina.

We have used enzymic digestion as a structural probe to investigate components of the nuclear envelope of germinal vesicles from Xenopus oocytes. Previous studies have shown that these envelopes are composed of a double membrane in which nuclear pore complexes are embedded. The nuclear pore complexes are linked to a fibrous lamina that underlies the nucleoplasmic face of the envelope. The pores are also linked by pore-connecting fibrils that attach near their cytoplasmic face. Xenopus oocyte nuclear envelopes were remarkably resistant to extraction with salt solutions and, even after treatment with 1 M NaCl or 3 M MgCl2, pores, lamina and pore-connecting fibrils remained intact. However, mild proteolysis with trypsin selectively removed the lamina fibres from Triton-extracted nuclear envelopes to leave only the pore complexes and connecting fibrils. This observation confirmed that the pore-connecting fibrils were different from the lamina fibres and were probably constructed from different proteins. Trypsin digestion followed by Triton treatment resulted in the complete disintegration of the nuclear envelope, providing direct evidence for a structural role for the lamina in maintaining envelope integrity. Digestion with ribonuclease did not produce any marked change in the structure of Triton-extracted nuclear envelopes, indicating that probably neither the pore-connecting fibrils nor the cytoplasmic granules on the pore complexes contained a substantial proportion of RNA that was vital for their structural integrity.

Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes.

We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoplasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60%), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.

Preparation of shadowed nuclear envelopes from Xenopus oocyte germinal vesicles for electron microscopy.

Methods for examining the structure of the nuclear envelope of oocytes of Xenopus laevis by electron microscopy using metal shadowing have been developed and evaluated. Minor modifications were made to existing methods for preparing specimens by freeze drying, mainly to eliminate unnecessary steps and a rapid method for examining the structure and arrangement of nuclear envelope components, based on dehydration in an ethanol series followed by amyl acetate and then air drying, was also developed. The preservation of the lamina and connections between the nuclear pore complexes using the rapid air drying method was satisfactory for observing the fibrous components of the envelope and their attachment to the pores. Furthermore, air drying required only simple laboratory apparatus and, moreover, offered several advantages compared to freeze drying when assessing the effect of various disruptive treatments on the nuclear envelope or examining the connections between its components. In specimens prepared by either the more rapid air drying method or by freeze drying, the lamina meshwork beneath the nuclear face of the envelope was clear, but the fine structure of the nuclear pore complexes was superior in freeze dried preparations. In views of the nucleoplasmic face of the envelope, the lamina meshwork was suspended above the support film in freeze dried preparations, but collapsed in most air dried specimens. This collapse was not without its advantages, however, as it facilitated observation of the connections between nuclear pore complexes and lamina fibres, which were often masked in freeze dried preparations.

Chromatin-like artifacts from nuclear sap.

Nuclear sap, on fixation in glutaraldehyde, forms a fibrous network that resembles chromatin in its dimensions and staining properties. This artifactual network is easily confused with true chromatin fibres in sections of nuclei. With formaldehyde a homogeneous array of beads is produced. These are approximately 10 nm in diameter - the size of nucleosomes - and are interconnected by exceedingly fine fibrils. Each fixative and buffer imposes its own distinctive, reproducible pattern on nuclear sap. The structure of nuclear sap in life cannot be deduced from this range of patterns.

The fixation of nuclei in glutaraldehyde.

The production of an artifactual network in the nuclear sap of the salivary glands of Drosophila has been investigated. Mechanical stress to the cells, 3% glutaraldehyde containing more than 4 mM calcium, tannins, or cacodylate buffer or whose temperature is above 10 degrees C all enhance this artifactual effect. Over the range pH 6.65-7.4 there is no significant effect of pH. The inclusion of sucrose, and adenine nucleotides or other 'chelating' agents in the fixative reduces the effect, especially at low temperature (6-10 degrees C). These influences are additive and can abolish the artifactual effect. So too can 8% glutaraldehyde, but this is irrespective of temperature.

Intranuclear electrophoresis of the chromatin of living cells.

Intranuclear electrophoresis of living cells under appropriate conditions causes the chromatin and nucleoli to move rapidly into the anodal side of the nucleus. In pig kidney cells, chromatin lengths attached to the nuclear envelope are oriented by the current and freed from surrounding non-oriented chromatin. Individual chromatin strands isolated in this way are often long and have not been subjected to the trauma of isolation from the nucleus. This has allowed us to demonstrate oriented lines of up to 8 chromomeres in a strand, linked by fine single fibres. These chromomeres of chromatin have the same linear dimensions as the bands and interbands of polytene chromosomes. A very wide range of morphology of chromatin is revealed - from lines of nucleosomes in open array, to strands uniformly 25 nm thick. Doublet strands and multiple strands - often embedded in darkly staining material - are also seen. All morphological types may be seen in the same nucleus. Many of the oriented threads appear to be transcriptionally active. The variable morphology of these sites and their relation to peripheral heterochromatin is discussed. Histone nucleosomes are present in these apparently transcriptionally active regions. The method is useful for investigating the relationships between chromatin and the nuclear envelope. Approximately 1500 attachment sites per nucleus are found in these cells. Some nucleoli are attached to the nuclear envelope.

The fine structure of human chromosomes isolated by shearing-sieving.

Chromosomes isolated by the new technique of shearing-sieving, even if unstained, show a less degraded organisation than those prepared for the electron microscope by other techniques. The chromosomes are banded, may show more bands if stretched, and the centromere is a precisely defined structure. Appearances resulting from this technique are compared with those from other techniques.

The fixation of nuclei and chromosomes.

Chromosomes in the nuclei of living salivary glands of Simulium have been observed with Nomarski optics and polarized light before and during irrigation with various fixatives. All cause loss of chromosomal organization to some degree. The best fixative is redistilled glutaraldehyde; even so, although it leaves the nucleus visually unaltered, a network forms in the nuclear sap and birefringence is lost. Calcium ions in the fixative cause chromosomal movements. The fixatives may alter nucleoli and may dissolve them completely. New fixatives are needed for reliable fine-structural studies of chromosomal organization.

Interpretation of the three-dimensional structure of living nuclei by specimen tilt.

A mechanism is described that enables a specimen to be tilted through a known angle and continuously observed under the highest power of the optical microscope. Objects can thus be localized accurately in space. This facility in conjunction with serial optical sectioning by Nomarski optics has been used to construct models of the arrangement of polytene chromosomes in nuclei of Drosophila, Simulium and Chironomus. Telomeres and chromocentre lie on the nuclear envelope. In Simulium they lie close to an equator of the nucleus. Despite these constraints, different nuclei in the same gland do not resemble each other closely.