Manfred Hauser

Demonstration of tubulin, actin and α-actinin by immunofluorescence in the microtubule-microfilament complex of the cytopharyngeal basket of the ciliate Pseudomicrothorax dubius

Abstract The feeding organelle (cytopharyngeal basket) of the ciliate Pseudomicrothorax dubius contains a highly complex pattern of microtubular lattices closely associated with microfilamentous web-like material. Indirect immunofluorescence microscopy on cell ‘ghosts’ and isolated cytopharyngeal baskets with antibodies against brain tubulin and mammalian muscle proteins show the presence of not only tubulin but also actin and α-actinin within this organelle. These findings demonstrate a special microtubule-microfilament complex and suggest the participation of contractile elements in conjunction with microtubules in the complex movement phenomena observed during the feeding process.

ULTRASOUND ENHANCED ENDOCYTOTIC ACTIVITY OF HUMAN FIBROBLASTS

Although various in vitro studies have shown that low-intensity pulsed ultrasound influences cytoskeletal components and biochemical pathways, the exact biologic mechanisms are still not fully understood. In this study, we analysed the effect of therapeutic ultrasound on the endocytotic activity of human foreskin fibroblasts. Fibroblasts were incubated with two different endocytotic markers (transferrin Alexa 488 and Lucifer yellow; Sigma Bioprobes, Eugene, OR, USA). To evaluate the amount of internalized markers in sonicated and nonsonicated control cells, confocal microscopy and plate reader experiments were performed. Additionally, the structural integrity of the cell membrane was monitored by electron-microscopy. After ultrasound treatment a clear increase (1.6-fold/Lucifer yellow and 1.4-fold/transferrin Alexa 488) of fluorescent marker uptake was detected. Confocal microscopy and plate reader experiments revealed that whole populations of sonicated fibroblasts showed a significant higher fluorescence compared with cells not sonicated (p<0.05; t-test for unpaired samples). The electron microscopic analysis of the cells showed no signs of structural membrane damage or a loosening of the membrane integrity. However, an exceedingly high amount of endocytotic vesicles and clathrin coated pits were observed in the sonicated group.

Occurrence and non-detectability of maytansinoids in individual plants of the genera Maytenus and Putterlickia.

Individual plants belonging to different species of the family Celastraceae collected from their natural habitats in South Africa (Putterlickia verrucosa (E. Meyer ex Sonder) Szyszyl., Putterlickia pyracantha (L.) Szyszyl., Putterlickia retrospinosa van Wyk and Mostert) and Brazil (Maytenus ilicifolia Mart. ex Reiss., Maytenus evonymoides Reiss., Maytenus aquifolia Mart.) were investigated for the presence of maytansinoids and of maytansine, an ansamycin of high cytotoxic activity. Maytansinoids were not detectable in plants grown in Brazil. Analysis of plants growing in South Africa, however, showed clearly that maytansinoids were present in some individual plants but were not detectable in others. Molecular biological analysis of a Putterlickia verrucosa cell culture gave no evidence for the presence of the aminohydroxybenzoate synthase gene which is unique to the biosynthesis of aminohydroxybenzoate, a precursor of the ansamycins including maytansinoids. Moreover, this gene was not detectable in DNA extracted from the aerial parts of Putterlickia plants. In contrast, observations indicate that this gene may be present in microbes of the rhizosphere of Putterlickia plants. Our observations are discussed with respect to the possibility that the roots of Putterlickia plants may be associated with microorganisms which are responsible for the biosynthesis of maytansine or maytansinoids.

Mitosis-like macronuclear division in a ciliate

The macronucleus of a small marine ciliate of the genus Protocrucia consists of a cluster of ten vesicles which give rise to 20 distinct chromosomal elements in the course of prophase-like condensation stages. Size differences of vesicles and chromosomes are cytological indications of their genetic individuality. In an anaphase-like stage, the chromosomal elements are separated in two daughter groups which re-form 10 vesicles each. The micronucleus divides simultaneously. The existence of a precisely functioning mode of chromosome distribution is also indicated by DNA measurements. Since the macronucleus contains much more DNA than the micronucleus, the macronuclear chromosomes are thought to be oligotenic. This hypothesis is supported by the much larger size of the macronuclear chromosomes. In contrast to other modes of macronuclear division known so far, this ciliate has retained some essential features of mitosis.

Aktomyosinartige Filamente im Teilungsmakronukleus des Ciliaten Ichthyophthirius Multifiliis

Just before nuclear division, the chromosomal elements within the large, highly polyploid macronucleus of I. multifiliis carry out rotational movements. Electron micrographs of cells fixed during the rotational movements show islets filled with microfilaments in various states of aggregation. Both thick (80–200 Å) and thin (30–80 Å) filaments occur, either as a highly dense network or as straight, in part parallel, filaments embedded in a filamentous network of lower density. Other “islets” of the macronucleus contain large and dense aggregates of filaments, sometimes with globular particles measuring 50–60 Å arranged along the thick filaments, occasionally forming cross-bridges with the thinner ones. — After incubation of the cells before fixation in a contractionsolution containing 0.002 M ATP, all nuclear islets show a nearly uniform appear ance of filamentous aggregates: numerous long and thick filaments are arranged in parallel with thin filaments with which they are in some parts connected by bridges. The probable myosinoid and actinoid nature of thick and thin filaments is discussed. It is suggested that the pre-divisional intranuclear rotational movement is a mechanism to avoid aneuploidy by producing a random arrangement of replicated hereditary units prior to division.

The intranuclear mitosis of the ciliates Paracineta limbata and Ichthyophtirius multifiliis

Electron microscope studies on the premetaphase stages of micronuclear divisions of Paracineta limbata and Ichthyophtirius multifiliis showed that spindle material also exists during interphase. In the case of I. multifiliis scattered microtubule fragments persist in the nuclear space; in P. limbata the micronuclei contain a small paracrystalloid which is suggested to be microtubular protein. Wide microtubules, varying in diameter from 300 to 400 Å develop during intranuclear prophase near the nuclear envelope in both cases. There are good reasons to assume that they function as a kind of stem body during the enlargement of the surface area of the nuclear envelope. Later micronuclear prophase stages of both species show a some-what different development. In I. multifiliis, there are scattered groups of short microtubular segments, partly in parallel array, whereas in P. limbata the wide tubules are transformed into normal microtubules of 180–200 Å diameter. The nuclei of both species are similar at late prophase and prometaphase stages. Bundles of interpolar microtubules run between the chromosomes, and single microtubules, presumably induced by the chromosomes, cross them at different angles. The chromosome-induced microtubules appear a short time after the interpolars. At prometaphase stage all microtubules show a highly parallel arrangement and therefore it is suggested that chromosomal tubules reach their final polar orientation by the action of cross-bridges.

Ultrasound-induced modifications of cytoskeletal components in osteoblast-like SAOS-2 cells.

In clinical and experimental studies an acceleration of fracture healing and increased callus formation induced by low‐intensity pulsed ultrasound (LIPUS) has been demonstrated. The exact molecular mechanisms of ultrasound treatment are still unclear. In this study ultrasound transmitted cytoskeletal and growth rate changes of SAOS‐2 cells were examined. Osteoblast‐like cell lines (SAOS‐2) were treated using low‐intensity pulsed ultrasound. Cytoskeletal changes were analyzed using rhodamine phalloidine for f‐actin staining and indirect immunofluorescence techniques with different monoclonal antibodies against several tubulin modifications. To examine changes of cell number after ultrasound treatment cell counts were done. Significant changes in cytoskeleton structure were detected compared to controls, including an enhancement of stress fiber formation combined with a loss of cell migration after ultrasound application. We further observed that sonication altered the proportion of the more stable microtubules to the more labile microtubule subclass. The labile tyrosinated microtubules appeared highly enhanced, whereas the amount of the more stable acetylated microtubules was remarkably diminished. All these observations were quantified by fluorometric measurements. The centrosomal γ‐tubulin was frequently scattered throughout the cells cytoplasm, giving rise to additional polyglu‐positive microtubular asters, which induced multipolar spindles, leading either to aneuploid mini‐or giant cells. Moreover, a significant increase of cell number was noticed in the sonicated group. These experiments demonstrate that ultrasound treatment increases cell number and leads to significant changes of the cytoskeletal structure and composition in vitro.

In vivo effects of ortho-vanadate on spindle structure and dynamics of locust spermatocytes I

After external application of vanadate, a potent inhibitor of several ATPases including dynein, the following effects on living spermatocytes I are detectable: spherical metaphase cells change to a lemon shape due to a concentration dependent elongation of the spindle, apparently achieved by pulling the plasma membrane-inserted poles apart, presumably through the assistance of cytoskeletal filaments. The observed dismembering of the spindle seems to be due to the separation of the half-spindle fibres, composed of usually interdigitating kinetochore microtubules (kMTs), free MTs (fMTs) and polar MTs (pMTs). As revealed by microcinematographic recordings, the lengthening of the half-spindles is accompanied by counter-clockwise twisting movements of the polar regions which, after prolonged vanadate treatment, lead to the formation of filiform appendices. Bundles of 5 nm microfilaments, which could be identified by indirect immunofluorescence microscopy (IIF) as actin, are concentrated within these appendices. In spite of a certain derangement of spindle architecture, half of the metaphases in 1 mM vanadate are capable of entering anaphase, but the rates of chromosome-to-pole movement have changed depending on the incubation time and the cell shape developed, respectively. Thus, chromosomes move with the highest speed in lemon-shaped cells but lag in cells with filiform appendices. However, it remains an open question whether the acceleration of chromosome migration is the result of spindle dismemberment or whether the slowing of anaphase motion is the consequence of a far-reaching displacement of the filamentous component from the spindle framework.

Primary Cilia in Locust Spermatocytes: Formation, Fate, and Possible Function

During locust spermatocyte development, rudimentary cilia originate from all centrioles of the doubled prophasic centrosome, located in the centrosphere and specifically linked to the nuclear envelope. Membranous vesicles at the bases of the centrioles fuse to form an intracellular ampulla that contributes the ciliary membranes. Later in prophase the ampulla’s membrane is integrated into the plasma membrane so that all primary cilia project into the extracellular medium and lose contact with the nucleus. As the centrioles remain ciliated on their way to their polar positions, w e propose a mechanism for this migration which is based on this intimate association between centrosomes and plasma membrane, on membrane fluidity and on a contractile cell cortex. We noted a translocation of the ciliated centrioles below the cell surface at the metaphase/-anaphase transition which may be regarded as “deciliation” by light microscopy. Some possible explanations for the primary cilium’s role in cell cycle regulation are suggested.

Nuclear phenomena during conjugation of the suctorian Heliophrya erhardi: I. EM-Observations of micronuclear meiosis

Summary We investigated micronuclear meiosis in the conjugating suctorian Heliophrya erhardi by time lapse cinemicrography of living cells, by indirect immunofluorescence (IIF) with monoclonal antibodies against modified tubulins, and by transmission electron microscopy, respectively high voltage electron microscopy (HVEM) of ultrathin and semithin sections. The fine structure of mid meiotic prophase reveals moderately condensed chromatin threads forming a “parachute” arrangement. Since only extremely short and also somewhat atypically structured synaptonemal complexes (SCs) are first discernible as early as diplotene, when partial chromosomal disjunction is seen, it is suggested that we are dealing here with achiasmatic meiosis. The spindle axis is established during prometaphase I by parallel alignment of polar MTs (pMTs) and the congression of chromosomes towards the equator is brought about by outgrowing kinetochore MTs (kMTs) becoming increasingly parallel. Metaphase I is indicated by a complete depolymerization of pMTs, revealing a truncated spindle type consisting exclusively of kMTs which never reach the presumptive poles before the metaphase/anaphase transition, and, as also seen by IIF, several of the small chromosomes converge with their kMTs forming numerous “subspindles”. These are focused on electron dense bodies located at half the distance to the presumptive poles. At late metaphase stages spindles become stabilized by their anchorage at the lamin-like sheets of the inner membrane of the nuclear envelope. Anaphase I is clearly subdivided into anaphase A and into anaphase B. While chromosome segregation in anaphase A I is not conspicuous an enormous interzonal spindle elongation that contributes substantially to chromosomal segregation is formed at anaphase B I. The mean velocity of pole separation of 0.5 μm/min corresponds rather well with the anaphase AI poleward migration. The telophase spindle develops from a centrally located torsional zone of the stembody in anaphase B by separation into two twisted areas which push the chromosomal fronts into opposite directions. During the dumbbell-shaped telophase stage, the daughter nuclei are pinched off by rotational movements of the terminalized contorted regions of the spindle. The nuclear events leading to meiosis II are highly similar to meiosis I.