Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes

The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.     

Dissociation and sorting out of Drosophila imaginal disc cells

Previous attempts to study sorting out of Drosophila imaginal disc cells have been hampered by an inability to thoroughly dissociate these cells and the need to use cuticular markers which require several days of in vivo culture. This study overcomes these limitations by using a new dissociation procedure and a genetic marker for undifferentiated cells, the succinate dehydrogenase8 (sdh8) mutation. Dissociated and reaggregated cells from wing and leg imaginal discs segregated or "sorted out" from one another after only 24 hr of in vivo culture. It was also found that leg cells from different body segments may sort out, but to a lesser degree than wing and leg cells. Mixtures of wing and haltere cells did not sort out, in contrast to previous reports. These results constitute the first unambiguous study of sorting out with Drosophila imaginal disc cells and indicate that dorsally situated imaginal cells share a recognition specificity which is different from that of ventral imaginal cells.     

Telolysomes in cultured iris epithelial cells and in the TVI cell-line.

Iris epithelial cells of adult newts, which are fully differntiated melanocytes and non-dividing, become dedifferentiated and converted into lens cells when put in culture. A recent study shows that this dedifferentiation is based on an autophagic process which is associated with proliferation and mainly affects melanosomes. The present report shows that in primary culture of iris epithelial cells after the majority of melanosomes have disappeared, myelinoid bodies, which are interpreted to be telolysosomes of autophagic nature, appear in high frequencies. This suggest that in these cells autophagy persists after the loss of melanosomes. A possible connection of this type of autophagy with the differentation of lens fiber which occurs in this culture is discussed. In the TVI cell line which is believed to be derived from the same cell type, but devoid of melanosomes, similar myelinoid bodies are a characteristic cell component, suggesting that the tendency for autophagy is inherited in theis cell line.     

BDNF increases the phagocytic activity in cultured iris pigment epithelial cells

To investigate the effect of brain derived neurotrophic factor (BDNF) on the phagocytic activity in iris pigment epithelial (IPE) cells, purified porcine photoreceptor outer segments (POS) were applied to cultured IPE cells for three hours. To measure phagocytic activities, bound and total POS were differentially stained using a double immunofluorescence staining method. BDNF increased the binding of POS in IPE cells in a dose-dependent manner. Ingestion of POS, however, was not affected throughout the concentrations used in this study. To investigate the signal transduction pathways of BDNF, a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and MAPK/ERK kinase (MEK) inhibitor, PD98059, were used for this study. LY294002 had no effect on the binding and ingestion of POS in BDNF-treated IPE cells. On the other hand, PD98059 completely inhibited the increase of POS binding in BDNF-treated cells and also decreased the ingestion of POS. These results indicate that increased POS binding activity by BDNF and the decreased ingestion of POS were mediated through the MAPK pathway.     

Purification of mitotic spindles from cultured human cells

In eukaryotes, both chromosome segregation and the determination of the cell division cleavage plane depend on the mitotic spindle apparatus. Spindle malfunctioning can lead to chromosome mis-segregation and cytokinesis defects and hence result in aneuploidy. Thus, the understanding of the structure and function of mitotic spindles is of interest not only from the perspective of basic science, but has implications also for human health and disease. Until recently, this complex microtubule-based structure was studied mainly by cell biological techniques in mammalian cells, by biochemical assays in Xenopus egg extracts, and by genetic approaches in genetically tractable organisms such as yeast, flies, and nematodes. With the rapid development of mass spectrometry and its increasing application to biological problems, it has become possible to subject highly complex structures, such as the mitotic spindle apparatus, to proteomics approaches. Such studies require the isolation of the mitotic spindle, or its substructures, in sufficient amounts and free of excessive contaminants. A number of methods for the isolation of mitotic spindles from mammalian tissue culture cells have been developed in the past. We have compared these methods and found that protocols based on the stabilization of microtubules by taxol were most efficient and reproducible. Here, we describe the further optimization of a taxol-based method, originally developed by Zieve and Solomon [Cell 28 (1982) 233-242], and its application to the isolation of human mitotic spindles at a scale suitable for mass spectrometric analysis [G. Sauer, R. Korner, A. Hanisch, A. Ries, E.A. Nigg, H.H.W. Sillje, Mol. Cell. Proteomics 4 (2005) 35-43].     

Diphenylhydantoin and mitotic spindle abnormalities in cultured mouse and human cells

The present study demonstrates that the antiepileptic drug diphenylhydantoin (DPH) is capable of inducing aneuploidy but not structural aberrations in cultured mouse embryonic fibroblasts. A high concentration of 200 micrograms/ml was found to increase the percentage of hyperdiploidy from 4.8 (control) to 16.0. The treatment was found to increase mitotic indices as a consequence of a mitotic-arresting action of the drug. These effects are probably due to the effect of the drug on the structure of the mitotic apparatus. Abnormal cell divisions and mitotic disturbance were found to increase in a dose-dependent manner after DPH treatment. In a parallel study, human amnion cells were found to show similar response to DPH treatment.     

Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.     

Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells

The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits. siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology. Simultaneous depletion of both complexes causes the severest defect. In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes. Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro. We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.     

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.     

Action of ribonuclease upon the polysomes of cultured mouse cells

Summary Pancreatic ribonuclease (RNase) and³H-uridine were used to study certain compositional and ontogenetic features of the polysomes of strain L mouse cells. Growing cells were exposed to the radioactive nucleoside,³H-uridine, for brief defined periods, and the sensitivity of the polysomes to digestion by RNase was determined. The RNase-resistant RNA of polysomes is shown to be primarily ribosomal, and the RNase-sensitive material formed during brief pulse labeling studies is largely messenger RNA. Actinomycin D inhibition of RNA synthesis was used to confirm this identification. The technique described here was used to investigate the effects of hydrocortisone on polysome formation. The hormone (10⁻⁶ m) lessens the extent of the nucleoside incorporation into polysomal and total RNA and delays the appearance of newly synthesized 18 S and 28 S rRNA into cytoplasmic polysomes. This work was partially supported by grants from the United States Public Health Service (GM 10866), from the National Science Foundation (GB 13924), and from The University of Kansas General Research Fund.     

Separating centrosomes interact in the absence of associated chromosomes during mitosis in cultured vertebrate cells

We detail here how "free" centrosomes, lacking associated chromosomes, behave during mitosis in PtK(2) homokaryons stably expressing GFP-alpha-tubulin. As free centrosomes separate during prometaphase, their associated astral microtubules (Mts) interact to form a spindle-shaped array that is enriched for cytoplasmic dynein and Eg5. Over the next 30 min, these arrays become progressively depleted of Mts until the two centrosomes are linked by a single bundle, containing 10-20 Mts, that persists for > 60 min. The overlapping astral Mts within this bundle are loosely organized, and their plus ends terminate near its midzone, which is enriched for an ill-defined matrix material. At this time, the distance between the centrosomes is not defined by external forces because these organelles remain stationary when the bundle connecting them is severed by laser microsurgery. However, since the centrosomes move towards one another in response to monastrol treatment, the kinesin-like motor protein Eg5 is involved. From these results, we conclude that separating asters interact during prometaphase of mitosis to form a spindle-shaped Mt array, but that in the absence of chromosomes this array is unstable. An analysis of the existing data suggests that the stabilization of spindle Mts during mitosis in vertebrates does not involve the chromatin (i.e., the RCC1/RanGTP pathway), but instead some other chromosomal component, e.g., kinetochores.     

In situ analysis of normal and abnormal patterns of the mitotic apparatus in cultured rat-kangaroo cells

Dividing cells in monolayers of the rat-kangaroo (Potorous tridactylis) cell line Pt-K₁ have large spindles and are flat, thus making possible studies of interactions between the achromatic and chromatic parts of the mitotic apparatus during the cell cycle. At prophase, asters and centrioles seem to exert pressure on the nuclear membrane leading to its rupture and penetrance of the centrioles. Apparently, the long axis of the spindle is shorter than the nuclear diameter. What appears as persistent, large portions of the nuclear membrane were observed in some metaphase and anaphase cells. Such a condition might also indicate an arrested mitosis. The midbody, which was often bipartite, was found to be of a ribonucleoprotein nature. — Three-group metaphases were of common occurrence and might represent early stages of chromosome orientation preceding the final alignment of the chromosomes on the equatorial plate. They could also be an expression of an anomalous condition as a result of mitotic arrest during prometaphase owing to spindle inactivation or breakage, errors in centromere-spindle attachments, interference with chromosome movement, or a duplicated centriolar constitution. Most of these aberrations could be attributed to the flatness of dividing cells, which might also bring about the failure of centriole separation and spindle organization in prometaphase stages, as well as multipolar mitosis.De novo organization of half spindles might take place in cells with ruptured spindles. Anaphase cells showing signs of a previous three-group orientation were rare. — Multipolar mitoses were prevalent mainly in cells with high chromosome numbers. They were often star-shaped with the chromosomes oriented between opposite and adjacent poles, and rarely as end-to-end associations of spindles. Apparently, one or more centrioles might share a common polar region. Multipolar configurations have either a mono- or multinuclear origin. Nuclei usually enter division synchronously in binucleate cells and the spindles become organized between centrioles associated with individual or different nuclei.