Search PubMed⌕ Search

Biomedical subjects

M Schliwa

Publications and source records attributed to M Schliwa.

At least 91 records · Page 5Linked to original sources
PubMed1986

Motility in the siphonous green alga Bryopsis. II. Chloroplast movement requires organized arrays of both microtubules and actin filaments.

The cortical cytoplasm of the giant cells of Bryopsis contains hundreds of interconnected microtubule (MT) bundles aligned along the cell's long axis. Actin fibers show an extensive but not exclusive superposition with these MT bundles. Chloroplasts move parallel to the bundles. Colchicine (0.5 mM), vinblastine (0.1 mM), and the herbicide ami-prophosmethyl (APM, 1-5 microM) strongly inhibit chloroplast movement and severely disrupt both the MT and the actin network. Additionally, APM leads to the appearance of large actin bundles up to 5 microns in diameter and several tens of micron in length. Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA, 1 mM) does not block chloroplast movement, but affects chloroplast behavior by causing transient aggregations. The MT network is not significantly changed by EHNA, but actin fibers converge in large, radially symmetric complexes in regions of chloroplast aggregates. Cytochalasin D (CD, 1-10 micrograms/ml) leads to a significant but transient reduction of chloroplast speed within the first 60 min, as the actin network breaks down into small foci. Within the next 1 to 3 h of treatment, these foci segregate into massive clusters where chloroplasts remain immobilized. At the same time, chloroplast movement recovers in other areas of the cell. This recovery coincides with the reappearance of actin filament bundles in these cell regions. The MT cytoskeleton is not significantly affected by CD. These data are inconsistent with a mechanism of chloroplast movement in Bryopsis based solely on either MTs or actin, but instead they suggest an intimate interaction of both cytoskeletal networks in maintaining the spatial organization of the cytoplasm and in supporting chloroplast movement.

Actin Cytoskeleton↗
PubMed1985

Electrical and ionic controls of tissue cell locomotion in DC electric fields.

The motility of fish epidermal cells (keratocytes) was examined in the presence and absence of DC electric fields. In fields of 0.5-15 V/cm, single epidermal cells, cell clusters, and cell sheets migrate toward the cathode. Cell clusters and sheets break apart into single migratory cells in the upper range of these field strengths. During locomotion, keratocytes extend broad lamellipodia, which contain a pervasive actomyosin network. The lamellipodial extension and locomotion of keratocytes are reversibly inhibited by a variety of calcium channel antagonists, whereas their motility is unaffected by hyperpolarizing and depolarizing (low and high K+) media. Microtubule disassembly has no effect on cell morphology, motility or the ability of the cells to be guided by a DC electric field. Using these results, the role that membrane-regulated Ca2+ influx may play in generating cytoskeletal and protrusive activity in keratocytes and other cells is discussed in some detail. Mechanisms by which an external electric field may bias transmembrane ion fluxes and thereby control cell locomotion are also examined.

Animals↗
PubMed1985

Bidirectional organelle transport can occur in cell processes that contain single microtubules.

Intracellular organelle transport was studied in a new model system, the giant freshwater ameba Reticulomyxa. The ameba extends a large reticulate network of cytoplasmic strands in which various phase-dense organelles can be seen to move at a rate of up to 25 microns/s. This combined light and high voltage electron microscopic study shows that organelles move bidirectionally in even the finest network strands that contain only a single microtubule. In terms of microtubule-associated intracellular transport, this observation defines a minimum set of conditions necessary for such movement. The implications of this finding for possible models of force generation are discussed.

Amoeba↗
PubMed1985

Evidence for an involvement of actin in the positioning and motility of centrosomes.

Cultured human polymorphonuclear leukocytes exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) spread on the substratum and undergo centrosome splitting. The two centrioles may separate by a distance of several micrometers, each being surrounded by an aster of microtubules. Here we show that the centriole/aster complexes are in constant, rapid motion through the cytoplasm, carrying with them some of the cytoplasmic granules while pushing aside others, or deforming and displacing the nucleus. An analysis of this unique motility phenomenon was undertaken. We show that intact microtubules are required for TPA-induced centrosome splitting and aster motility, but not for cell spreading. More importantly, disruption of the actin network inhibits both centrosome splitting and cell spreading, and even reverses splitting (induces convergence and fusion of asters) in polymorphonuclear leukocytes pretreated with TPA alone. These observations indicate the existence of a dynamic relationship between microtubules and actin networks and provide evidence for a role of actin in determining the position of the centrosome by way of interaction with the microtubules radiating from it.

Actins↗
PubMed1985

Microtubule organization of unstimulated and stimulated adherent human neutrophils in Chediak-Higashi syndrome.

The numbers and length of centriole-associated microtubules of two patients with Chediak-Higashi syndrome (CHS) were examined. Detergent-extracted whole-mount preparations of adherent cells were studied by stereo high-voltage electron microscopy. Under conditions of random migration, neutrophils from both patients had a microtubule organization similar to that of the control; microtubule numbers (28 +/- 3) and length (7.0 +/- 2.8 micron) were within normal range. When cells were treated with phorbol myristate acetate (PMA), differences in the response of the two patients were noted. Neutrophils from patient No. 2 and the control showed a significant rise in numbers (38 +/- 5) and length (9.5 +/- 3.6 micron) of microtubules. In contrast, neutrophils from patient No. 1 were unresponsive to PMA treatment. Because vitamin C is used therapeutically in CHS patients and has been shown to correct microtubule-related cell function, neutrophils were exposed to ascorbic acid. A significant increase in microtubule numbers (35 +/- 6) was observed in cells from the control and patient No. 2 after ascorbate treatment; neutrophils from patient No. 1 showed no increase in microtubule numbers. While ascorbic acid did not affect microtubule length in the control cells, it caused a significant increase in microtubule length in neutrophils from both patients. Results suggest that adherent CHS neutrophils contain centriole-associated microtubules which are normal in number and length. However, differences between patients are observed regarding neutrophil responsiveness to stimuli which induce microtubule polymerization.

Ascorbic Acid↗
PubMed1984

erythro-9-[3-(2-Hydroxynonyl)]adenine is an effective inhibitor of cell motility and actin assembly.

erythro-9-[3-(2-Hydroxynonyl)]adenine (EHNA) has been reported previously to be an agent that arrests sperm motility by inhibiting the axonemal dynein ATPase activity and has been used to probe the involvement of putative cytoplasmic dyneins in mitosis and intracellular organelle transport. We report here that EHNA profoundly and reversibly affects several actin-dependent processes, both in vivo and in vitro. It induces dramatic changes in actin organization in cultured cells, inhibits cell translocation, blocks actin-dependent cytoplasmic streaming, interferes with actin-dependent gelation of cytoplasmic extracts, and inhibits actin assembly. Just as the cytochalasins, EHNA appears to be a highly effective inhibitor of actin-based motility, whose effects in complex biological systems should be interpreted with caution.

Actins↗
PubMed1984

A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells.

Treatment of epithelial African green monkey kidney (BSC-1) cells with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid and reversible redistribution of actin and vinculin that is detectable after only 2 min of treatment. Within 20-40 min, stress fibers disappear, while at the same time large actin-containing ribbons resembling ruffles develop both at the cell periphery and in more central regions. Vinculin is associated with these actin ribbons or bands in a punctate or patchy staining pattern. Adhesion to the substratum is changed from predominantly focal contacts associated with stress fiber ends in untreated cells to broad zones of close contact after TPA treatment. High voltage electron microscopic observations disclose the ribbons to consist of highly cross-linked actin filament networks. Thus, association of vinculin with filament networks, rather than (the ends of) filament bundles, is demonstrated. The integrity of microtubules and vimentin filaments is not affected by TPA treatment, but their distribution is altered to conform with the highly distorted cell shape. The response to TPA is neither prevented nor modified by nocodazole-induced depolymerization or taxol-induced stabilization of microtubules. An intact intermediate filament network seems not required either since colcemid-induced collapse of vimentin filaments towards the nucleus does not affect the cell's response to TPA. Rapid redistribution of actin and vinculin also takes place in enucleated cells and in the presence of cycloheximide, but is prevented by dinitrophenol or oligomycin. TPA-induced cytoskeletal alterations are independent of fibronectin expression and not mimicked, modified, or prevented by calmodulin inhibitors or experimentally elevated levels of calcium and cyclic AMP. Thus the morphological response to TPA involves rapid redistribution of actin and vinculin independent of transcription and translation, fluctuations in the levels of calcium or cyclic AMP, or changes in the organization of microtubules, intermediate filaments, and fibronectin.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses.

A cellular protein of 36,000 daltons becomes phosphorylated at tyrosine in chicken embryo fibroblasts transformed with avian sarcoma viruses. We have used cellular fractionation and immunofluorescence to locate the 36-kdalton protein in virus-transformed and uninfected chicken fibroblasts. The 36-kdalton protein in transformed cells fractionated mainly with high-speed particulate material, and in density gradient separations, the 36-kdalton protein was found in association with light density membranes together with most of the plasma membrane marker. Increasing the concentration of salt or adding ion chelators solubilized some of the 36-kdalton protein that otherwise was pelletable with high g forces. Based on these data, we conclude that this protein is peripherally or indirectly attached to light density membranes, including plasma membranes. Indirect immunofluorescent staining of the 36-kdalton protein in fixed cells revealed that it was located inside the cell in an extensive reticulum apposed to surface membranes. The same pattern of staining was found in both uninfected and virus-transformed cells. Pretreatment of cells with nonionic detergents before fixation altered or abolished 36-kdalton staining. The 36-kdalton protein appeared to be excluded from regions of the cells where actin cables were present. The pattern of staining observed with the anti-36-kdalton antibody was similar, but not identical, to that observed with antiserum against nonerythroid spectrin. Thus, the data obtained by biochemical fractionation and by immunofluorescent staining indicate that the 36-kdalton protein is found in a reticulum at the inner surface of the plasma membrane, possibly in association with cytoskeletal proteins.

Animals↗
PubMed1983

Tumor promoter-induced centrosome splitting in human polymorphonuclear leukocytes.

Human neutrophilic polymorphonuclear leukocytes (neutrophils) adhering to a substratum undergo a dramatic change in cellular morphology when treated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). Within a few minutes of TPA treatment, the cells cease locomotion and spread symmetrically on the substratum. Concomitantly, TPA initiates centrosome splitting in a manner similar to that induced by treatment of randomly locomoting cells with a chemotactic factor. The two centrioles of a centrosome separate by a distance of several micrometers, and each of the solitary centrioles is surrounded by an aster of microtubules. Some cells also establish a third, centriole-free aster of microtubules. TPA treatment increases the total number of microtubules associated with the centrosome(s) and also increases overall polymer length. The frequency of centrosome splitting is enhanced transiently by treatment with the synthetic chemotactic peptide f-Met-Leu-Phe. Centrosome splitting is interpreted in terms of an interaction between the cell periphery and the microtubule system. Possible cellular mechanisms of this unusual phenomenon are considered.

Carcinogens↗
PubMed1983

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes.

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.

Actins↗
PubMed1982

Implications of cytoskeletal interactions for cellular architecture and behavior.

The three major filamentous components of the cytoskeleton (microfilaments, microtubules and intermediate filaments) do not just coexist in the cell, but interact with each other in various ways. This paper discusses some examples of structural interactions visualized in critical-point-dried cells by stereoscopic high-voltage electron microscopy. The relative contribution of two classes of interactions to the consolidation of different cytoskeletal domains is considered. One class is represented by T-junctions (end-to-side contacts) of actin filaments with other filaments, and the other by 3 nm links. Attention is then turned to what may be called the behavioural consequences of cytoskeletal interactions. As an example of a coordinated interplay between events at the cell membrane and the cytoskeleton, we discuss changes in cytoskeletal organization of polymorphonuclear leucocytes upon stimulation with a chemotactic factor. These changes culminate in some cells in centriole separation and the establishment of two microtubule asters, each centred around a single centriole.

Actins↗
PubMed1982

Centrosome splitting in neutrophils: an unusual phenomenon related to cell activation and motility.

Detergent-extracted whole mount preparations of human neutrophilic polymorphonuclear leukocytes (neutrophils) were used to study centrosome position and microtubule organization during random, chemokinetic and chemotactic locomotion. Chemotaxing neutrophils have a polarized external and internal morphology, with centrosomes between the anterior lamellipod and the nucleus. In randomly locomoting cells centrosome position is highly variable with respect to these two cell organelles. Stimulation of randomly locomoting cells with a chemoattractant (chemokinesis) rapidly (within 1 min) induces pronounced spreading and ruffling and concomitantly initiates, in up to 55% of the cells, transient separation of the centrosome into two solitary centrioles, each surrounded by an aster of microtubules. In addition, about 10% of the cells with separated centrosomes have a third, centriole-free aster of microtubules radiating from an accumulation of dense material. Neutrophil activation also leads to an increase in the number of microtubules and an approximately twofold increase in polymer length. This unusual response of the centrosome supports a correlation between cell movement and microtubule organization.

Adult↗
PubMed1982

Action of cytochalasin D on cytoskeletal networks.

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end-to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.

Actins↗
PubMed1981

Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins.

Detergent-extracted BSC-1 monkey cells have been used as a model system to study the Ca(2+) sensitivity of in vivo polymerized microtubules under in vitro conditions. The effects of various experimental treatments were observed by immunofluorescence microscopy. Whereas microtubules are completely stable at Ca(2+) concentrations below 1 muM, Ca(2+) at greater than 1-4 muM induces microtubule disassembly that begins in the cell periphery and proceeds towards the cell center. At concentrations of up to 500 muM, both the pattern and time course of disassembly are not markedly altered, suggesting that, within this concentration range, Ca(2+) effects are catalytic rather than stoichiometric. Higher (millimolar) Ca(2+) concentration results in rapid destruction of microtubules. Of other divalent cations, only Sr(2+) has a slight depolymerizing effect, whereas millimolar Ba(2+), Mg(2+), or Mn(2+) is ineffective. Disassembly induced by micromolar Ca(2+) is inhibited by pharmacological agents known to bind to calmodulin and inhibit its function, suggesting that calmodulin mediates Ca(2+) effects. Both the addition of exogenous brain microtubule-associated proteins (MAPs) after lysis and the retention of endogenous cellular MAPs normally extracted during the lysis step stabilize microtubules against the depolymerizing effect of micromolar Ca(2+). The results indicate that, in this model system, microtubules are sensitive to physiological Ca(2+) concentrations and that this sensitivity may be conferred by calmodulin associated with the microtubules. MAPs appear to have a modulating effect on microtubular Ca(2+) sensitivity and thus may function as a discriminating factor in cellular functions performed by calmodulin. It is hypothesized that Ca(2+)-stimulated microtubule disassembly depends on the relative amount of MAPs.

Animals↗