Urs-Peter Roos

Chapter 15 Probing the Mechanisms of Mitosis with Dictyostelium discoideum

Publisher Summary Mitosis in D. discoideum is studied in live amoebae, by immunofluorescence and by electron microscopy. From the live observation, it is learned that the spindle elongates by a factor of three or more during anaphase and telophase at a velocity of 4 μm/min, which is faster than the rate of chromosome movement in many cells. The immunofluorescence studies provide insight into the overall mode of formation of the microtubules (MT) spindle as the interphase complex of MTs breaks down, and the reverse process at the end of nuclear division. Electron microscopy has revealed that the spindle consists of the ensemble of kinetochore MTs that link chromosomes directly to the spindle pole bodies (SPBs), plus the central spindle, which is made up essentially of two sets of interdigitating pole MTs. As the spindle elongates, the number of MTs decreases while the remaining MTs elongate by addition of subunits at their distal, overlapping ends. Concomitantly, the order between interdigitating MTs from opposite poles increases. These results are compatible with the idea that the spindle elongates by MT sliding and elongation.

Germ tube growth and the microtubule cytoskeleton in Phytophthora infestans: Effects of antagonists of hyphal growth, microtubule inhibitors, and ionophores

We carried out a set of experiments designed to test hypotheses on the relationship between the microtubule (MT) cytoskeleton and tip growth in fungal hyphae. Using a growth assay with 1·5 h-old germ tubes of Phytophthora infestans we tested various chemicals that inhibit growth or disrupt MTs, or that affect both. Germ tubes were measured after 2 h exposure to the test compounds and MTs were revealed by immunofluorescence. The phytoalexin glyceollin had a dual effect as it stimulated germ tube growth at concentrations of 10−5 m and 10−6 m , but inhibited it at 10−4 m . It affected the MT cytoskeleton at neither of these concentrations. Metalaxyl, the active ingredient of the fungicide Ridomil, inhibited germ tube growth slightly at 10−4 m and 10−6 m , but it had no effect on MTs. Benomyl, the active ingredient of the fungicide Benlate, had no effect on MTs at a concentration of 10−5 m , although it partially inhibited germ tube growth. Nocodazole, a benzimidazole inhibitor of MTs, disrupted the subapical MT cytoskeleton in germ tubes of P. infestans at 10−4 m , at which growth was still 79% of the control. The potassium ionophore nigericin and the calcium ionophore A23187 stimulated germ tube growth at concentrations between 10−6 m and 10−9 m , but inhibited it at 10−4 m . Nigericin, but not A23187, disrupted MTs at this concentration. These results point out that no simple relationship exists between an intact MT cytoskeleton and germ tube growth in P. infestans.

Microtubules in Interphase and Mitosis of Cellular Slime Molds

Cellular slime molds are mycetozoan protists characterized by a trophic stage during which they exist as solitary cells that phagocytize bacteria or yeasts, and by the formation of stalked sporocarps that bear one or many walled spores (Bonner, 1967; Olive, 1975; Raper, 1973). The cells of most species are non-flagellated amoebae, but several taxa have amoeboflagellate cells.

Nucleic acid cytochemistry of the nucleus and microtubule-organizing centers in dictyostelium discoideum.

We used fluorescence microscopy with DAPI, Hoechst 33258, acridine orange, and ethidium bromide, as well as ultracytochemical regressive staining, the Feulgen-type reaction with osmium-ammine, and the enzyme-gold method to investigate the presence and distribution of DNA and RNA in the nucleoplasm, nucleolus, nucleus-associated body, and spindle pole bodies. We found that the nucleoplasm of interphase nuclei contains mostly DNA dispersed in a fibrillar meshwork, with which some RNA is probably associated as perichromatin granules or fibers. With DNase-gold and RNase-gold the nucleolus, which consists of interspersed fibrillar and granular components, was the most heavily labelled of five cellular compartments analyzed. Accordingly, its fluorescence with acridine orange and ethidium bromide was brightest. In mitotic nuclei the nucleolus was dispersed, filling most of the nuclear volume. Chromosomes were brightly stained by DNA-specific fluorochromes and the osmium-ammine reaction revealed that only the innermost layer of the trilaminar kinetochores contains DNA. Neither the nucleus-associated body of interphase cells nor the spindle pole bodies of mitotic cells contain DNA or RNA.

Morphology of the nucleolus in undifferentiated amoebae of Dictyostelium discoideum

We investigated the morphology and behavior of phase-opaque and electron-opaque intranuclear structures in undifferentiated amoebae of the cellular slime mold Dictyostelium discoideum by video microscopy of live amoebae and 3-D reconstruction from serial ultrathin sections. We conclude from our observations and results that these structures, with the possible exception of some small intranuclear granules, represent the nucleolus, which occupies 31 % to 39% of the nuclear volume. The nucleolus thus is a very complex body whose morphology varies from cell to cell. It consists of a single element that is extensively or punctually associated with the nuclear envelope, or of several larger and smaller elements, some of which may be joined. The number of the nucleolar elements in a given nucleus is fixed, i.e. no fusions between elements and no splitting of elements occur. The position of these elements is also fixed, for as the nucleus rotates and changes shape in response to changes in cell shape during amoeboid movement the nucleolar elements maintain their relative position in the nucleus. The morphological variation between nucleoli of different amoebae contrasts remarkably with the constant morphology within any one cell. These features distinguish the nucleolus of D. discoideum from that of higher eukaryotes and pose many questions regarding genetic control over nucleolar morphogenesis and maintenance of its shape.

Mitosis in Amoebae of the cellular slime mold (Mycetozoan) Protostelium mycophaga

Summary We investigated mitosis in amoebae of Protostelium mycophaga by video microscopy of live cells, by indirect immunofluorescence with antibodies against tubulins, and by transmission electron microscopy of ultrathin sections. Amoebae in interphase usually contain two microtubule centers (MCs) on opposite sides of the nucleus, from which microtubules (MTs) radiate into the cytoplasm. During prophase these MTs shorten to form two asters between which the mitotic spindle develops during prometaphase. Concomitantly, the nucleolus fragments, the numerous small chromosomes orient amphitelically in the spindle and congress to the spindle equator, and the asters diminish further until metaphase. The spindle is open and acentric, but with complex spindle pole bodies. Each sister-chromatid is attached to a single MT by a tiny, layered kinetochore. During anaphase and telophase, asters develop anew and enlarge to become the elaborate MT cytoskeletons of the daughter cells. Anaphase lasted 2 min on average (s.d. = 0.6 min, n = 4), during which the chromosomes moved poleward with a mean velocity of 4.0 μm/min (s.d. = 0.8 μm/min, n = 5). The intermingling of kinetochore MTs and the numerous non-kinetochore MTs allows for a sliding interaction between them, but depolymerisation-driven chromosome movement is also possible. The spindle elongated at a mean rate of 5.9 μm/min (s.d. = 2.2 μm/min, n = 5), and the mean elongation factor was 2.4 in live cells. In immunofluorescence preparations the longest spindles were 3.5 times longer than the average metaphase spindle. Spindle elongation thus requires the growth of interzonal MTs that assemble as several bundles from an ample pool of tubulin. At the end of telophase the nuclear envelope is reconstructed from membrane vesicles and flattened cisternae that appose to the masses of decondensed chromosomes and nucleolar material.

Cell shape, motility and distribution of F-actin in amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea. A comparison with Dictyostelium discoideum

Summary We investigated, by a computer-assisted video analysis, by F-actin cytochemistry, and by scanning electron microscopy (SEM), the dynamic morphology, the locomotory behavior, and the actin cytoskeleton of trophic amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea, and compared them with amoebae of the cellular slime mold Dictyostelium discoideum. The three species represent protist taxa marked by different reproductive systems and by differences of amoeboid motility in the non-reproductive phase of the life cycle. Live observations and the numerical analysis of video recordings revealed characteristic traits of cell shape changes and motility in each species. The leading edge of Protostelium amoebae is dominated by a large lamellipodium that is studded with pseudodigits. We computed a mean cell size (outline area) of 498 μm2 and a mean velocity of 26.5 μm/min (centroid displacement) for this species (N = 30, as for the other organisms). Most amoebae followed a sinuous path during the observation period, which resulted in a mean standard deviation of vector direction (SDV) of 0.64, but some hardly moved at all. Amoebae of Acrasis are essentially monopodial, with lobose, smooth pseudopodia. Their mean cell size was 759 μm2 and their mean velocity 71.6 um/min. They are thus among the fastest “crawling” eucaryotic cells, which may be related to the fact that they contain no cytoplasmic microtubules. Most amoebae followed a straight path, but some made one or two abrupt turns during the observation period (SDV = 0.58). The corresponding values for Dictyostelium amoebae with filose or lobose pseudopodia were 220 μm2, 10.3 μm/min, and SDV = 0.80. In amoebae of all the three species F-actin was localized in the cell cortex, especially in hyaline pseudopodia. As seen by SEM, Protostelium amoebae had the largest area of contact with the substratum, whereas Acrasis amoebae adhered with a foot-like structure, the leading edge and the uropod being detached from the support.

A novel type of unorthodox mitosis in amoebae of the cellular slime mold (Mycetozoan) Acrasis rosea

Summary We investigated mitosis in trophic amoebae of Acrasis rosea (Olive and Stoianovitch) by video microscopy of live cells, by immunofluorescence with an antibody against tubulin, and by transmission electron microscopy. As interphase cells contain neither microtubules (MTs) nor microtubule-organizing centers (MTOCs) the mitotic spindle forms entirely de novo from presumptive diffuse intranuclear MTOCs. Mitosis is closed and devoid of any visible pole organelles during all its stages. The spindle axis is probably established by parallel alignment of MTs due to spatial constraints or lateral interaction. Chromosomes condense only when spindle formation is well advanced; their kinetochores presumably acquire MTs (kMTs) by capture. The two main elements of the nucleolus, viz. granular body and dense granules, disperse, but do not disintegrate. They later arrange in the center of the spindle along its axis and eventually segregate as two granular bodies and two groups of granules. Reconstruction of the nucleolus during telophase involves the compaction of the granular bodies and coaggregation of the dense granules. At metaphase the chromosomes are aligned at the equator of a spindle that tapers towards the blunt spindle poles. Chromosomes are small, but they have distinct layered kinetochores with two MTs each that terminate in their outer layer. Cytokinesis is accomplished ca. eight minutes after the first signs of prophase. Chromosome segregation during anaphase is effectuated almost solely by spindle elongation, which begins when chromosomes are still undivided and aligned at the equator. The velocity of spindle elongation and chromosome segregation was 6 μm/min. Chromosome congression and segregation are most likely driven by interactions, static or dynamic, between kMTs and non-kinetochore MTs (nkMTs), whereas the mechanism of spindle elongation is probably based on movements between staggeredly overlapping nkMTs. At the end of telophase the closure of the nuclear envelope around the daughter nuclei pinches off a membrane tube containing remnant nkMTs. Mitosis in A. rosea differs markedly from that in other cellular slime molds and has much in common with that in several protozoa.

Mitosis in amoebae of the cellular slime mold (mycetozoan) Acytostelium leptosomum

Summary We investigated mitosis in amoebae of Acytostelium leptosomum, grown in liquid culture, by video microscopy of live cells, by indirect immunofluorescence with antibodies against tubulins, and by transmission electron microscopy of ultrathin sections. Amoebae in interphase contain a single microtubule-organizing center (MTOC, [30]) at each nucleus, from which microtubules (MTs) radiate into the cytoplasm. These disappear as the intranuclear spindle forms. Concomitantly, the nucleolus disperses, and the chromosomes that are visible in phase contrast congress to the spindle equator. The spindle is closed except for polar fenestrae occupied by broad, amorphous spindle pole bodies (SPBs). The chromosomes at metaphase are joined to form several blocks, each attached to several kinetochore MTs. Anaphase was accomplished within 2.2 min (s.d. = 0.5 min, n = 11). Anaphase A was virtually absent, but anaphase B contributed substantially to chromosome segregation. The mean velocity of pole separation was 3.2 μm/min (s.d. = 0.8 μm/min) and the mean elongation factor was 2.8 (range 1.9 to 3.4). The telophase spindle was a shaft consisting of a few MTs traversing each incipient daughter nucleus and joining in the interzone. The amorphous SPBs were reconverted to compact interphase MTOCs as the chromosomes decondensed and the nucleolus re-formed during cytokinesis. Duration of mitosis and velocities of its movements are within values typical for lower eucaryotes. In most aspects of mitosis A. leptosomum is very similar to two other dictyostelid cellular slime molds, Dictyostelium discoideum and Polysphondylium violaceum, and the three lower eucaryotes are clearly distinct from other mycetozoans.