Yoshio Fukui

Nuclear actin bundles in Amoeba, dictyostelium and human HeLa cells induced by dimethyl sulfoxide

In a previous study we demonstrated that dimethyl sulfoxide (DMSO) induces the formation of microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium mucoroides [12], in which the microfilaments bound rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ structure, and that this binding could be reversed by Mg2+ and ATP. In the present study, we show electron microscopic data demonstrating the occurrence of such microfilament bundles in the nucleus of Amoeba proteus and human HeLa cells, as well as in D. mucoroides. The similarities in the morphology and dimension of the microfilanets, as well as the specific conditions by which they are induced, suggested that these microfilaments are actin. We present evidence that actin is involved in interphase nucleus of a variety of organisms, and that DMSO acts on the molecules to induce microfilament bundles specifically in the nucleus.

Dictyostelium MTOC: Structure and Linkage to the Nucleus

SummaryThe microtubule organizing center (MTOC) was isolated fromDictyostelium discoideum to investigate the fine structure of the components as the first step in clarifying its molecular organization and function. The isolation protocol was designed to preserve microtubules bound to the MTOC by using indirect immunofluorescence employing anti-α-tubulin. After cell lysis with Triton X-100, the MTOCs were isolated in association with the nucleus by centrifugation in a microtubule-stabilizing buffer. The MTOC was found to be bound to the nucleus via an electron-dense fibrous structure, and this linkage could not be destroyed by KI, KCl, or sonication. We named this complex composed of microtubules, MTOC, and the anchor the MTOC-complex. Negative staining of the isolated MTOC-complex revealed that distinct vesicles decorated with 11-nm tacks were associated with microtubules radiating from the MTOC. Fine filaments, 4–5 nm wide, were also present close to the MTOC, aligned parallel to the microtubules. The three-dimensional profile of the central core of the MTOC, examined by transmission electron microscopy of serial thin sections of the isolated MTOC fraction supplemented by a microcomputer analysis, was concluded to be a matchbox-like cuboid (180 × 210 × 370 nm) of 15 layers.We propose that theDictyostelium MTOC is the structural domain of a more complicated unit composed of 1. MTOC, 2. microtubules, and 3. a firm fibrous linkage connecting the MTOC to the nucleus, with the MTOC core being a multilayered cuboid, associated with nodules and surrounded by amorphous electron-dense material including peculiar vesicles with 11 nm-tacks. The possible functions of these domains are discussed.

Disruption of microtubules and retardation of development ofDictyostelium with ethylN-phenylcarbamate and thiabendazole

SummaryThe effects of ethylN-phenylcarbamate (EPC) and thiabendazole (TB) onDictyostelium discoideum andD. mucoroides cells were examined as a step toward purifying tubulin and clarifying the function of microtubules in cellular slime molds. EPC (1.5 × 10−3M) or TB (5 × 10−5M) inhibited the development ofDictyostelium, inducing the formation of aberrant fruiting bodies with stalks irregular in shape and sori containing spores of various sizes and shapes.EPC and TB inhibited cell division but not cell growth, resulting in the production of giant cells up to ten times larger than untreated cells. The giant cells either had a single huge nucleus of irregular shape or contained multiple nuclei. The effects of the inhibitors were reversible. After the removal of the inhibitors, the giant cells underwent successive cell divisions producing many daughter cells. Interestingly, most of the giant cells induced by EPC treatment contained gigantic secondary lysosomes probably produced by extensive lysosomophagy.Light microscopy using Nomarski optics revealed that these inhibitors caused the round-up of the cells resulting in the inhibition of cell locomotion, whereas non-Brownian movement of the cytoplasmic granules was not affected. Indirect immunofluorescence using anti-α-tubulin revealed that networks of microtubules were apparently destroyed by the EPC or TB treatment.These results show both EPC and TB are potent inhibitors of microtubules inDictyostelium and are effective tools for studying the function of microtubules either in cellular or multicellular organization throughout its life cycle.

Role of the MT-MTOC complex in determination of the cellular locomotory unit inDictyostelium

SummaryThe microtubule inhibitors, ethyl-N-phenylcarbamate (EPC) and thiabendazole (TB), which disrupt cytoplasmic microtubules and induce giant cells inDictyostelium (Kitanishiet al. 1984), were found to induce the occurrence of multiple microtubule organizing centers (MTOCs) in these giant cells. Probing was done by indirect immunofluorescence using monoclonal anti-α-tubulin. The nuclear DNA content of the giant cells increased in parallel with an increase in the number of MTOCs, as shown by microspectrophotometory of cells stained with the fluorescent DNA stain DAPI (4′,6-diamidino-2-phenylindole).Shortly after the inhibitors were removed, the MTOCs of the giant cell formed multiple mitotic spindles or synchronously reconstituted numerous cytoplasmic MT-networks. These events apparently reflected the cell-cycle dependent activities of the MTOCs at the time the inhibitors were removed. When multiple spindles were formed, numerous cytoplasmic MT-networks became organized subsequent to the breakdown of the spindles. In either case, reconstitution of the cytoplasmic MT-networks was followed by apparently normal cytokinesis resulting in the production of many daughter cells each containing a single MT-MTOC complex. The evidence suggested the possible mechanism of the induction of multiple MTOCs, and implied that the MT-MTOC complex is significant in the cytokinesis ofDictyostelium by determining the cell locomotory unit.

In vivo identification of Tetrahymena actin probed by DMSO induction of nuclear bundles

Abstract We report the first successful identification of actin, an ubiquitous contractile protein, in Tetrahymena pyriformis (strain W). We employed dimethyl sulfoxide (DMSO) as a probe to induce the formation of actin bundles in the cell nucleus [1, 2] through disruption of cytoplasmic microfilament organization [3, 4]. The cells were incubated for 30 min at 22 °C in the inorganic medium of Prescott & James [5] containing 10% DMSO, and observed under a transmission electron microscope (TEM). Microfilarment bundles were formed in interphase macronuclei, and these microfilaments, approx. 6 nm in diameter, could be decorated by rabbit skeletal muscle heavy meromyosin (HMM) in the glycerinated model. In many cases, the bundles formed closely parallel to natively existing bundles of microtubules. Interestingly, these microtubules had prominent striation with 15–16 nm periodicity. SDS-polyacrylamide gel electrophoresis was designed to show the low actin content of Tetrahymena cells in comparison with that of Dictyostelium . Actin was suggested to comprise less than 1.7% of the total protein in Tetrahymena , whereas as much as 6% was actin in Dictyostelium cells. In assessing the physiological significance of the bundle formation, we further performed HMM and myosin subfragment-1 (S1)-binding studies to clarify the organization process and the polarity of the DMSO-induced nuclear actin filaments by using the tannic acid staining technique [6]. Randomly oriented short filaments appeared in the nucleus treated with 10% DMSO for 10 min. These filaments became elongated and associated with each other to form loose bundles in the following 10 min. With 30-min treatment, the filaments were organized and large bundles with single axes developed. With these well-developed bundles, the Students t -test was performed on 172 pairs of neighboring filaments and the probability ( p ) of the deviation from random polarity was 0.08, suggesting that the filaments were organized in an anti-parallel manner. The results show that the DMSO induction of nuclear actin is a powerful tool to demonstrate the existence of cellular actin in vivo and to study the mechanism of microfilament organization in relation to cell physiological activities.

ENZYMATIC DISSOCIATION OF NASCENT MACROCYSTS AND PARTITION OF THE LIBERATED CYTOPHAGIC GIANT CELLS IN DICTYOSTELIUM MUCOROIDES

Nascent macrocysts of the cellular slime mold Dictyostelium mucoroides were dissociated enzymatically and the liberated cytophagic giant cells were partitioned by dextrin density gradient centrifugation. Enzymatic and cytochemical studies revealed that the primary wall is composed mainly of cellulose (β‐1,4‐glucan) associated with polysaccharides including hemicellulose, pectic substances and á‐1,4‐glucan. The buoyant density of the liberated cytophagic giant cells and peripheral cells was determined by density gradient centrifugation, and partitioning of the cells was possible due to the difference in this property. The process of macrocyst reconstitution was investigated using dissociated cells. The isolated cytophagic giant cell has a specific affinity for other cytophagic giant cells and predominantly ingests them by phagocytosis, while it retains the ability to ingest peripheral cells. The present study provides a clue for investigating the differentiation and development of sexual cells, since only the cytophagic giant cell gives rise to a zygote in macrocyst formation.

A cortical cytoskeleton associated with phagosomes disclosed by a monoclonal antibody against capped membrane ofDictyostelium

SummaryThe formation of the ligand-receptor complex on cell surface causes a tight association of microfilaments with the plasma membrane. Since capping is an energy-dependent process which involves contractile proteins, it seemed reasonable to look for physiologically important cytoskeletal elements in regions of the capped membrane. We isolated the capped membrane ofDictyostelium induced with lentil lectin, separated the proteins by electrophoresis, and prepared monoclonal antibodies against proteins extracted from the gel. These antibodies were reactive to distinct antigens involved in the cytoskeleton-membrane complex, as observed by improved immunofluorescence (“agar-overlay” technique). We further characterized one of these antibodies (DCC-32) (DCC:Dictyostelium cortical cytoskeleton), and found that it was reactive to a new cytoskeletal element of 41.5 K (pI 10.65) according to a two-dimensional Western blotting. Immunoelectron microscopy showed this antigen to be localized in the cortical cytoskeleton associated with the plasma membrane and at the cytoplasmic side of the phagosome membrane. The evidence suggests that monoclonal antibodies against capped membrane are useful probes for analysing functional cytoskeleton domains; and the 41.5K-dalton component is probably involved in the phagocytosis.