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At least 19 recordsLinked to original sources
PubMed1992

Correlation of axenic linkage groups with the position of the microtubule-organizing center in aggregating Dictyostelium.

Positioning of the microtubule-organizing center (MTOC) in Dictyostelium discoideum was found to be genetically regulated. We examined the wild-type strain NC-4 cells independently maintained in different laboratories, freshly recovered cells from spores stocked for over 20 years, the temperature-sensitive growth mutant HU49 isolated from NC-4, as well as strain V-12 which is the opposite mating-type to NC-4. During aggregation on nonnutrient agar plates, all these strains showed similar cell polarity, as defined by the alignment of the nucleus ahead of the MTOC. By contrast, in Ax2 and Ax3, axenic strains carrying axenic mutations on linkage groups II and III, the MTOC was usually positioned ahead of the nucleus. Cells containing axenic linkage group II but not III positioned the MTOC ahead of the nucleus. Conversely cell polarity of strains including axenic linkage group III but not II was similar to that of wild-type cells. Thus axenic linkage group II, probably axeC or other linked gene(s) not yet identified, is responsible for the location of the MTOC anterior to the nucleus during aggregation. The anterior positioning of the MTOCs was prevented by growth on bacteria in cells carrying both axenic linkage groups, but not in those carrying only axenic linkage group II.

Animals↗
PubMed1979

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements.

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

Animals↗
PubMed1991

Intracellular organization of hippocampal neurons during the development of neuronal polarity.

In culture, hippocampal neurons initially establish several short, apparently identical processes; of these, only one acquires axonal characteristics, the remainder becoming dendrites. We examined the organization of cytoplasmic constituents that might influence which of the initial processes becomes the axon. The Golgi complex was visualized using either fluorescent wheat germ agglutinin or a specific antibody. Presumptive microtubule-organizing centers were identified by depolymerizing microtubules with nocodazole, then allowing them to repolymerize for brief periods. As judged by light microscopy, hippocampal neurons contained a single Golgi region and a single microtubule-organizing center, which were frequently localized together adjacent to a shallow indentation in the nucleus. In cells fixed shortly after the axons had emerged, there was no correlation between the position of the Golgi complex or the microtubule organizing center and the site of origin of the axon. Based on nuclear shape, the position of the Golgi complex and microtubule-organizing center could also be inferred in living cells. When axonal outgrowth was followed in individual cells by time-lapse microscopy, so that the location of the Golgi complex and microtubule-organizing center could be determined at the exact moment when the axon emerged, no correlation was apparent. Antibodies that recognize specific posttranslational modifications of alpha-tubulin--acetylation and de-tyrosination--were used to assess the distribution of arrays of stable microtubules. Stable microtubules were present in all processes, both before and after the emergence of the axon. They were not confined to the axon. Thus the localization of these cellular constituents does not play a major role in determining which of the processes initially extended by hippocampal neurons becomes the definitive axon.

Animals↗
PubMed1992

Cell cycle-dependent assembly and disassembly of cytoplasmic microtubules in the plasmodium of the myxomycete Physarum polycephalum.

The giant syncytium of Physarum plasmodia possesses a complex cytoplasmic microtubule network except during the occurrence of the intranuclear mitosis. In early prophase stages, intranuclear spindles assemble concomitantly as the cytoplasmic microtubule network disassembles. No cytoplasmic microtubules are present in metaphase. They begin to reassemble in telophase. The complex cytoplasmic microtubule network reappears in early reconstruction stages. The assembly of cytoplasmic microtubules occurs on cytoplasmic foci, both in telophase stage and during rewarming after cold microtubule disassembly. These foci, independent of the nuclei, correspond to the foci observed in the cytoplasm during interphase, both by immunofluorescence and electron microscopy. As cytoplasmic and intranuclear microtubule-organizing centers are spatially distinct, plasmodial syncytia offer the possibility to study the effects of cell regulatory pathways on two types of microtubule-organizing centers that differ in their nucleating activity during the cell cycle.

Animals↗
PubMed2025

Diversity of microtubule arrays in animal cells at a glance.

Microtubules are cytoskeletal filaments important for various cellular processes such as intracellular transport, cell division, polarization and migration. Microtubule organization goes hand in hand with cellular function. Motile cells, such as immune cells or fibroblasts, contain microtubule asters attached to the centrosome and the Golgi complex, whereas in many other differentiated cells, microtubules form linear arrays or meshworks anchored at membrane-bound organelles or the cell cortex. Over the past decade, new developments in cell culture, genome editing and microscopy have greatly advanced our understanding of complex microtubule arrays. In this Cell Science at a Glance article and the accompanying poster, we review the diversity of microtubule arrays in interphase animal cells. We describe microtubule network geometries present in various differentiated cells, explore the variety in microtubule-organizing centers responsible for these geometries, and discuss examples of microtubule reorganization in response to functional changes and their interplay with cell motility and tissue development.

Microtubules↗
PubMed1992

Heterogeneity of microtubule organizing center components as revealed by monoclonal antibodies to mammalian centrosomes and to nucleus-associated bodies from dictyostelium.

The molecular composition of two morphologically distinct microtubule-organizing centers (MTOCs) was compared by probing with monoclonal antibodies raised against (i) nucleus-associated bodies (NABs) isolated in a complex with nuclei from the cellular slime mold Dictyostelium discoideum and (ii) mammalian mitotic spindles isolated from Chinese hamster ovary (CHO) cells. The staining patterns observed by immunofluorescence microscopy in whole CHO cells and Dictyostelium amoebae showed that the distribution of thirteen MTOC antigens is heterogeneous. Not all antibodies recognized the MTOC in both interphase and mitosis. Most of the anti-MTOC antibodies cross-reacted with other cellular organelles such as nuclei, Golgi apparatus-like aggregates and cytoskeletal elements. Two antibodies, CHO3 and AX3, recognized phosphorylated epitopes present in both mammalian centrosomes and Dictyostelium NABs. On immunoblots, most of the antibodies showed multiple bands, often of high molecular weight, indicating that the antigenic determinants are shared among different molecules. One antibody inhibited the regrowth of microtubules onto centrosomes in vitro after addition of exogenous tubulin to detergent-lysed CHO cells on coverslips; this antibody binds to an antigen(s) that might be essential for the microtubule-nucleating activity of centrosomes. These observations demonstrate that molecular components in different MTOCs exhibit a variety of distinct subcellular localizations and functional properties, and that some antigenic molecules have been conserved among morphologically distinct MTOCs.

Animals↗
PubMed1992

Activation of maternal centrosomes in unfertilized sea urchin eggs.

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.

Ammonia↗
PubMed1992

A high molecular weight centrosomal protein of mammalian cells is antigenically related to myosin II.

Available data on the molecular composition of the centrosome, the typical microtubule-organizing center of animal cells, are still fragmentary. To address this important issue we have taken advantage of centrosome isolation from a human lymphoblastic cell line (KE37) to generate a monoclonal antibody (mAb) library. Here we present the characterization of one of these mAbs (CTR56). On the basis of both its immunofluorescence staining pattern and its reactivity with a major 200 kD antigen on immunoblots, CTR56 has been tentatively classified as an anticellular myosin heavy chain. In light of cytological and biochemical data obtained in parallel with two other well-characterized myosin antibodies, it appears that myosin cannot be considered as a genuine centrosomal protein. We have resolved the paradoxical results with CTR56 by showing that in addition to the cellular myosin heavy chain, this antibody also recognizes a high molecular weight protein specifically enriched in centrosomal fractions. The possible biological significance of this finding is discussed in structural and functional terms.

Animals↗
PubMed1990

Oogenesis: chromatin and microtubule dynamics during meiotic prophase.

Changes in the organization of germinal vesicle chromatin in mouse oocytes have been analyzed by fluorescence microscopy with respect to progressive stages of follicular development and the disposition of oocyte cytoplasmic microtubules. Four discrete patterns of chromatin organization exist in germinal vesicle (GV)-stage oocytes isolated from the ovaries of 21-25-day-old gonadotropin-primed mice. Analysis of ovarian cryosections stained with the DNA-binding fluorochrome Hoechst 33258 indicates that sequential changes in GV chromatin occur during folliculogenesis that result in the formation of a continuous perinucleolar chromatin sheath at the time of antrum formation. Specific alterations in the cytoplasmic microtubule complex of GV-stage oocytes were observed that correlate with chromatin patterns. The extensive cytoplasmic microtubule complex seen in oocytes of preantral follicles initially localizes to perinuclear areas of the ooplasm. This is followed by a progressive reduction in cytoplasmic microtubules and the appearance of prominent microtubule-organizing centers at the nuclear periphery. Coordinated nuclear and microtubular alterations also occur under in vitro conditions prior to progression of meiosis to prometaphase-1. The results are discussed with respect to the ongoing differentiation of the oocyte nucleus and the microtubule cytoskeleton during folliculogenesis in preparation for the resumption of meiosis.

Animals↗
PubMed1991

Maternal inheritance of centrosomes in mammals? Studies on parthenogenesis and polyspermy in mice.

The centrosome, the microtubule-organizing center of the cell, is introduced typically by the sperm at fertilization. In some mammals, however, this paternal pattern of inheritance appears to be violated. The hypothesis that the centrosome is maternally inherited was tested during parthenogenesis, polyspermy, and polygyny as well as after recovery from microtubule inhibition at first mitosis. During parthenogenesis the paternal contribution was absent, and in polyspermy the paternal contribution was multiplied. Haploid and diploid parthenogenotes as well as polyspermic and digynic fertilized eggs each segregated their centrosomes to organize a bipolar mitotic apparatus. Oocytes recovering from a nocodazole block formed two normal bipolar mitotic apparatus; the paternal chromosomes aligned at one spindle equator, while the maternal chromosomes were found at the other. These results show that the centrosome is maternally inherited from cytoplasmic sites in the mouse. The evolutionary switch from paternal to maternal inheritance in mammals might be related to the additional dangers that parthenogenesis represents: a threat to the life of the mother as well as to the life of the fetus.

Animals↗
PubMed1992

Identification of ubiquitous high-molecular-mass, heat-stable microtubule-associated proteins (MAPs) that are related to the Drosophila 205-kDa MAP but are not related to the mammalian MAP-4.

AX3, a monoclonal antibody raised against isolated microtubule-organizing centers of Dictyostelium discoideum, stains microtubule-containing structures in species ranging from Dictyostelium to human. On immunoblots, the AX3 antibody recognizes heat-stable proteins in the 260- to 280-kDa molecular-mass range in a number of different species. The AX3 antigens from HeLa and embryonic mouse fibroblast cells coprecipitate with microtubules in vitro, indicating that these antigens are, indeed, MAPs. The AX3 antigens are not immunologically related to the mammalian MAP-2 or MAP-4 but are related to the 205-kDa MAP of Drosophila. This report describes a structural-type MAP in Dictyostelium and a MAP that is detected in a wide variety of species. The Drosophila 205-kDa MAP had previously been proposed to represent a member of the MAP-4 class of proteins. From the results reported here, however, it is suggested that proteins recognized by AX3 monoclonal antibody, including the Drosophila 205-kDa MAP, represent a distinct class of MAPs that has been widely conserved through evolution.

Animals↗
PubMed1992

Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex.

The localization of the Golgi complex depends upon the integrity of the microtubule apparatus. At interphase, the Golgi has a restricted pericentriolar localization. During mitosis, it fragments into small vesicles that are dispersed throughout the cytoplasm until telophase, when they again coalesce near the centrosome. These observations have suggested that the Golgi complex utilizes a dynein-like motor to mediate its transport from the cell periphery towards the minus ends of microtubules, located at the centrosome. We utilized semi-intact cells to study the interaction of the Golgi complex with the microtubule apparatus. We show here that Golgi complexes can enter semi-intact cells and associate stably with cytoplasmic constituents. Stable association, termed here "Golgi capture," requires ATP hydrolysis and intact microtubules, and occurs maximally at physiological temperature in the presence of added cytosolic proteins. Once translocated into the semi-intact cell cytoplasm, exogenous Golgi complexes display a distribution similar to endogenous Golgi complexes, near the microtubule-organizing center. The process of Golgi capture requires cytoplasmic tubulin, and is abolished if cytoplasmic dynein is immunodepleted from the cytosol. Cytoplasmic dynein, prepared from CHO cell cytosol, restores Golgi capture activity to reactions carried out with dynein immuno-depleted cytosol. These results indicate that cytoplasmic dynein can interact with isolated Golgi complexes, and participate in their accumulation near the centrosomes of semi-intact, recipient cells. Thus, cytoplasmic dynein appears to play a role in determining the subcellular localization of the Golgi complex.

Adenosine Triphosphate↗
PubMed1979

Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells.

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.

Cell Cycle↗
PubMed1992

CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae.

A genetic screen was devised to identify genes important for spindle pole body (SPB) and/or microtubule functions. Four mutants defective in both nuclear fusion (karyogamy) and chromosome maintenance were isolated; these mutants termed cik (for chromosome instability and karyogamy) define three complementation groups. The CIK1 gene was cloned and characterized. Sequence analysis of the CIK1 gene predicts that the CIK1 protein is 594 amino acids in length and possesses a central 300-amino-acid coiled-coil domain. Two different CIK1-beta-galactosidase fusions localize to the SPB region in vegetative cells, and antibodies against the authentic protein detect CIK1 in the SPB region of alpha-factor-treated cells. Evaluation of cells deleted for CIK1 (cik1-delta) indicates that CIK1 is important for the formation or maintenance of a spindle apparatus. Longer and slightly more microtubule bundles are visible in cik1-delta strains than in wild type. Thus, CIK1 encodes a SPB-associated component that is important for proper organization of microtubule arrays and the establishment of a spindle during vegetative growth. Furthermore, the CIK1 gene is essential for karyogamy, and the level of the CIK1 protein at the SPB appears to be dramatically induced by alpha-factor treatment. These results indicate that molecular changes occur at the microtubule-organizing center (MTOC) as the yeast cell prepares for karyogamy and imply that specialization of the MTOC or its associated microtubules occurs in preparation for particular microtubule functions in the yeast life cycle.

Amino Acid Sequence↗
PubMed1992

Establishment and expression of cellular polarity in fucoid zygotes.

Zygotes of fucoid algae have long been studied as a paradigm for cell polarity. Polarity is established early in the first cell cycle and is then expressed as localized growth and invariant cell division. The fertilized egg is a spherical cell and, by all accounts, bears little or no asymmetry. Polarity is acquired epigenetically a few hours later in the form of a rhizoid/thallus axis. The initial stage of polarization is axis selection, during which zygotes monitor environment gradients to determine the appropriate direction for rhizoid formation. In their natural setting in the intertidal zone, sunlight is probably the most important polarizing vector; rhizoids form away from the light. The mechanism by which zygotes perceive environmental gradients and transduce that information into an intracellular signal is unknown but may involve a phosphatidylinositol cycle. Once positional information has been recorded, the cytoplasm and membrane are reorganized in accordance with the vectorial information. The earliest detectable asymmetries in the polarizing zygote are localized secretion and generation of a transcellular electric current. Vesicle secretion and the inward limb of the current are localized at the presumptive rhizoid. The transcellular current may establish a cytoplasmic Ca2+ gradient constituting a morphogenetic field, but this remains controversial. Localized secretion and establishment of transcellular current are sensitive to treatment with cytochalasins, indicating that cytoplasmic reorganization is dependent on the actin cytoskeleton. The nascent axis at first is labile and susceptible to reorientation by subsequent environmental vectors but soon becomes irreversibly fixed in its orientation. Locking the axis in place requires both cell wall and F-actin and is postulated to involve an indirect transmembrane bridge linking cortical actin to cell wall. This bridge anchors relevant structures at the presumptive rhizoid and thereby stabilizes the axis. Approximately halfway through the first cell cycle, the latent polarity is expressed morphologically in the form of rhizoid growth. Elongation is by tip growth and does not appear to be fundamentally different from tip growth in other organisms. The zygote always divides perpendicular to the growth axis, and this is controlled by the microtubule cytoskeleton. Two microtubule-organizing centers on the nuclear envelope rotate such that they align with the growth axis. They then serve as spindle poles during mitosis. Cytokinesis bisects the axial spindle, resulting in a transverse crosswall. Although the chronology of cellular events associated with polarity is by now rather detailed, causal mechanisms remain obscure.

Cell Cycle↗
PubMed1976

Ultrastructure of Pharyngostomoides procyonis Harkema 1942 (Diplostomatidae). I. Observations on the male reproductive system.

Spermatogonia, nutritive cells, and developmental stages of spermatids were observed with the electron microscope. Spermatogonia are near the surface of the testis and contain large nuclei and comparatively little cytoplasm. Nutritive or supporting cells are associated with the spermatogonia. Early spermatids are characterized by a circle of mitochondria around the nucleus. Late spermatids have 2 parallel free flagella separated by a cytoplasmic process, and a nucleus containing electron-dense strands of chromatin arranged in coils or concentric layers. Mature sperm have 2 flagella enclosed by cytoplasm. Their nuclei contain dense, fibrillar chromatin. A microtubule-organizing center (MTOC) found between basal bodies of spermatids is described. Descriptions are presented of the seminal reservoir, seminal vesicle, and the sperm found in those organs.

Animals↗