Centrosome behavior in the exposure of tissue culture cells to energy metabolism inhibitors

The organization of the centrosome in PK cells has been analysed according to several parameters: the presence of primary cilium, the number of pericentriolar satellites, the presence of striated rootlets, the distance between two centrioles and their orientation as regards the substrate plane. 2,4-Dinitrophenol (DNP), DNP with deoxyglucose (DOG), sodium azide cause the increase of frequency of occurrence of primary cilia and the growth of mean number of satellites per active centriole. The distribution of active and inactive centrioles in control cells is described by a histogram corresponding to a histogram of accidental distribution. Under the action of DNP or DNP with DOG, but not of sodium azide a part of active centrioles settled down perpendicularly to the substrate plane increases. The orientation of inactive centrioles under all the treatments used doesn't practically change.     

The ultrastructure of the centrosome in cytoplasts and its alteration under the action of ouabain]

It has been shown that after enucleation of the PE cells with cytochalasin D the centrioles remain in approximately 80% of cytoplasts. Some cytoplasts contain only single centriole, either a mother (active) of a daughter (inactive) one. 20% cytoplasts have no centrioles. 2h after enucleation the centrosome structure in the cytoplasts did not differ from that in normal cells. 14-16 h after enucleation in many cytoplasts large secondary lysosomes and lipid droplets appeared around the centrosome. At this time in some cytoplasts in the centrosome we observed free microtubule convergence foci. 14-16 h after the enucleation, some cytoplasts have doubling centrioles. Under the influence of ouabain (30 min), the number of active centrioles oriented perpendicularly to the substrate plane in the cytoplasts increased. We suggest that the preferentially perpendicular orientation of centrioles to the substrate plane does not depend on the nuclear activity.     

A stereoscopic analysis of centrosome structure in the cells of a tissue culture under the action of energy metabolism inhibitors. I. 2,4-Dinitrophenol, deoxyglucose, sodium azide and the calcium ionophore A23187]

A 30-min action of energy transfer inhibitors (2,4-dinitrophenol, deoxyglucose, azide and calcium ionophore A23187) on tissue culture cells results in a significant increase in the quantity of microtubules around the centrosome. After the action of all the inhibitors, mostly increases the number of long microtubules with free proximal end oriented towards the centrosome. It is suggested that energy transfer inhibitors may stimulate foundation of microtubules on the centrosome and stabilize free microtubules, while they exert no effect on the frequency of detachment of microtubules from the centrosome.     

The centrosome reaction in tissue-culture cells to depolarization of the cell membranes

Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), ouabain and calcium ionophore A23187 caused a centrosome restructuring expressed in mean number increase of satellites on the active (mother) centriole, in an increase of the mean slope of centrioles to the substrate surface and in the more frequent occurrence of primary cilia. In the presence of FCCP the effect appeared only in 10 min and retained for 2 h. The ouabain caused the separation of active and inactive centrioles in more than in a half of the cells. The data obtained permit to conclude that depolarization of the plasmatic membrane only is needed for the initiation of centrosome restructuring. The authors propose that this reaction of centrosome is a component of the cell overall response to nonspecific lesions.     

A stereoscopic analysis of the centrosome structure in tissue culture cells under the action of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and ouabain]

The action of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and ouabain results in significant increase of the quantity of microtubules with attached and free proximal end around the centrosome. The majority of free microtubules are oriented with their proximal ends towards the heads of pericentriolar satellites or towards the walls of centriolar cylinders. The increasing of total number of microtubules is the result of the increasing of microtubules attached to or oriented towards the pericentriolar satellites. Comparing the action of FCCP and ouabain from one side and taxol from the other side it is possible to conclude that FCCP and ouabain promote the initiation of microtubule growth in the centrosome of they have an influence on the frequency of separation of the microtubules from microtubule nucleating centers.     

Behavior and function of paternally inherited centrioles in brown algal zygotes

In brown algal cells, the centrosome, consisting of a pair of centrioles and the pericentriolar material, is primarily involved in the organization of microtubules (MTs) throughout the cell cycle. In motile cells, the centrioles participate in the formation of flagellar axoneme as flagellar basal bodies, and in somatic cells they play a crucial role in many cellular activities as a part of the centrosome. With respect to the role of the centrosome as a microtubule organizing center (MTOC), brown algal cells resemble animal cells. In most animal fertilization processes, the sperm cell introduces centrioles, the core of the centrosome, into the egg cytoplasm. In this study, the behavior of centrioles from gametogenesis and fertilization to the first cell division of the zygote was examined in the three sexual reproduction patterns occurring in brown algae, i.e., oogamy, anisogamy and isogamy, by electron- and immunofluorescence-microscopy. The pair of centrioles contained in somatic cells was shown to be derived from the male gamete, irrespective of the sexual reproductive pattern. The paternally derived centrioles were duplicated before mitosis and were involved in spindle pole formation. Moreover, MTs from the centrosome play a crucial role in the process of cytokinesis, as the position of centrosomes accompanying daughter nuclei seems to determine the cytokinetic plane. A new approach to clarifying the mode of cytokinesis in brown algae is presented in this study.Chikako Nagasato was the recipient of the Botanical Society of Japan Award for Young Scientist, 2004.     

Centrosome Function: Sometimes Less Is More

Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles – a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.     

A non-canonical mode of microtubule organization operates throughout pre-implantation development in mouse

In dividing animal cells, the centrosome, comprising centrioles and surrounding pericentriolar-material (PCM), is the major interphase microtubule-organizing center (MTOC), arranging a polarized array of microtubules (MTs) that controls cellular architecture. The mouse embryo is a unique setting for investigating the role of centrosomes in MT organization, since the early embryo is acentrosomal, and centrosomes emerge de novo during early cleavages. Here we use embryos from a GFP::CETN2 transgenic mouse to observe the emergence of centrosomes and centrioles in embryos, and show that unfocused acentriolar centrosomes first form in morulae (~16–32-cell stage) and become focused at the blastocyst stage (~64–128 cells) concomitant with the emergence of centrioles. We then used high-resolution microscopy and dynamic tracking of MT growth events in live embryos to examine the impact of centrosome emergence upon interphase MT dynamics. We report that pre-implantation mouse embryos of all stages employ a non-canonical mode of MT organization that generates a complex array of randomly oriented MTs that are preferentially nucleated adjacent to nuclear and plasmalemmal membranes and cell-cell interfaces. Surprisingly, however, cells of the early embryo continue to employ this mode of interphase MT organization even after the emergence of centrosomes. Centrosomes are found at MT-sparse sites and have no detectable impact upon interphase MT dynamics. To our knowledge, the early embryo is unique among proliferating cells in adopting an acentrosomal mode of MT organization despite the presence of centrosomes, revealing that the transition to a canonical mode of interphase MT organization remains incomplete prior to implantation.     

A stereoscopic analysis of the centrosome structure in the cells of continuous and primary cell cultures]

A 3D reconstruction of the centrosome region was made based on series of semithick sections in tissue culture cells. It was shown that: 1) the total number of microtubules attached to the centrosome is about 30-50 of which only 20% or less run farther than 2 microns away from the centrosome; 2) a certain number of short microtubules (less than 1 micron length) is present in the vicinity of the centrosome, the majority of them are attached to the centrosome; 3) many microtubules around the centrosome have no direct contact with either centrioles, or other microtubule-convergent structures; 4) the majority of free microtubules are comparatively long (more than 1 micron length); 5) almost all the microtubules running closer than 2 microns to the centrosome are oriented towards it with their proximal ends. The radial distribution of free microtubules around the centrosome support the supposition that they may appear as a result of their detachment from the microtubule-nucleating centres.     

The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)

Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 μmol L^?1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome‐associated MT almost disappeared, and a massive assembly of cortical/subcortical, well‐organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome‐associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles.     

Origin and behavior of bicentriolar centrosomes in the bryophyteRiella americana

Summary Basal bodies in embryophyte spermatozoids develop from centrosomes which arisede novo in spermatid mother cells (SMC). The centrosomes at SMC spindle poles in those land plants producing biflagellated sperms comprise a coaxial pair of centrioles, a bicentriole (BC). Ultrastructural observations of antheridia of the aquatic liverwortRiella americana indicate that the centrosome is first evident as a dark staining body on the outer surface of the nucleus. Numerous short microtubules (MT) diverge from this body which next separates into two lobes, each with divergent MTs. Within each lobe, a BC differentiates-the cartwheel hub and spokes developing before the triplet MTs. Constituent centrioles of each BC are apposed by their proximal ends and connected only by the central hub. As the BCs migrate toward opposite spindle poles, they appear to be connected by MTs that terminate in granular material partially investing each BC. Each spermatid resulting from SMC division will inherit a bicentriole.     

Centrosomal and non-centrosomal microtubules.

While microtubule (MT) arrays in cells are often focused at the centrosome, a variety of cell types contain a substantial number of non-centrosomal MTs. Epithelial cells, neurons, and muscle cells all contain arrays of non-centrosomal MTs that are critical for these cells' specialized functions. There are several routes by which non-centrosomal MTs can arise, including release from the centrosome, cytoplasmic assembly, breakage or severing, and stabilization from non-centrosomal sites. Once formed, MTs that are not tethered to the centrosome must be organized, which can be accomplished by means of self-organization or by capture and nucleation of MTs where they are needed. The presence of free MTs requires stabilization of minus ends, either by MT-associated proteins or by an end-capping complex. Although some of the basic elements of free MT formation and organization are beginning to be understood, a great deal of work is still necessary before we have a complete picture of how non-centrosomal MT arrays are assembled in specific cell types.     

Microtubule dynamics in epithelial cells

Microtubules (MTs) are necessary components of all eukaryotic cells. They fulfill various functions being involved in cell division, ciliar and flagellar beating, cell shape maintaining, organelle distribution in the cell, organization of other cytoskeletal elements. Dynamic features of MTs have been commonly studied in vitro or on undiffirentiated cultured cells by means of molecular and ultrastructural methods. It is generally accepted that the phenomenon of dynamic instability is the major mechanism of MT turnover in the cell. MTs radiate from the centrosome and take part in the distribution of cell organelles. In addition, epithelial, nerve, and skeletal muscle cells contain non-centrosomal MTs. A few hypothesis of their origin have been so far put forward. According to the capture-release hypothesis, MTs are first nucleated on the a centrosome, then release to be driven in various parts of the cell by molecular motors. Some alternative mechanisms of non-centrosomal MT formation are also proposed in literature. For example, the nucleation sites were reported not only in centrosomes but also in other parts of cells, such as the apical membranes of epithelial cells, the nuclear membrane of muscle cells, pigment granule aggregates of melanophores. On studying frog urinary bladder and large intestine epithelial cells the authors observed in these cells numerous non-centrosomal MTs. This makes epithelial cells, good models for analysing structural and dynamic features of non-centrosomal MTs in differentiated cells. For the urinary bladder the pool of specific granules may serve as MT organizing centers. Non-cenrosomal MTs of these cells have big diameters (35-38 nm) and form bundles oriented in the apical-basal axis of the cell. In addition, non-centrosomal MTs of these cells may participate in the transport of specific granules and giant vacuoles that appear under stimulated water flows through the cell.     

Three-dimensional structural characterization of centrosomes from early Drosophila embryos

An understanding of the mechanism and structure of microtubule (MT)- nucleating sites within the pericentriolar material (PCM) of the centrosome has been elusive. This is partly due to the difficulty in obtaining large quantities of centrosomes for analysis, as well as to the problem of attaining interpretable structural data with conventional EM techniques. We describe a protocol for isolating a large quantity of functional centrosomes from early Drosophila embryos. Using automated electron tomography, we have begun a three-dimensional structural characterization of these intact centrosomes with and without regrown MTs. Reconstructions of the centrosomes to approximately 6-8 nm resolution revealed no large structures at the minus ends of MTs, suggesting that if MT-nucleating material physically contacts the MTs, it must conform closely to the shape of the minus end. While many MTs originate near the centrioles, MT minus ends were found throughout the PCM, and even close to its outer boundary. The MTs criss-crossed the PCM, suggesting that nucleating sites are oriented in many different directions. Reconstructions of centrosomes without MTs suggest that there is a reorganization of the PCM upon MT regrowth; moreover, ring-like structures that have a similar diameter as MTs are apparent in the PCM of centrosomes without MTs, and may be MT- nucleating sites.     

Autoregulation of the electrogenic sodium pump

The dependence of electrogenic sodium pump activity on changes in the cell volume of Helix pomatia neurons with different levels of intracellular sodium ion concentration was studied. Hypertonic solutions caused hyperpolarization of the membrane and increased membrane resistance in cells with a low sodium content (low-sodium cells; LSC). The activity of the electrogenic sodium pump in hypertonic solutions was increased compared to the activity in hypotonic solutions in LSC and decreased in cells with a high sodium content (high-sodium cells; HSC). The concentration of ouabain which led to maximal inhibition of active 22Na efflux from the neurons was 10(-4) M. Lower concentrations of ouabain (10(-8) M and lower) did not inhibit the sodium pump but stimulated it. The swelling of neurons in hypotonic solutions was accompanied by an increase in the number of binding sites for ouabain, while shrinking in hypertonic solutions led to the opposite effect--a decrease in binding sites. An increase in the number of binding sites also took place in normal isotonic potassium-free solutions compared with normal Ringer's solution. Two saturable components of ouabain binding were detectable in all solutions examined. gamma-Aminobutyric acid (GABA) and acetylcholine (ACh) increased the number of ouabain binding sites on the membrane. The results suggest that there are two opposite mechanisms by which cell volume changes can modulate the pump activity. One of them depends on the intracellular sodium ion concentration and causes pump activation in hypertonic solutions in LSC and saturation in HSC, while a second mechanism mediates the activating effect of cell swelling on the sodium pump in HSC. In addition, there may be a negative feedback between the pump activity and the number of functioning pump units in the membrane.     

Separate to operate: control of centrosome positioning and separation

The centrosome is the main microtubule (MT)-organizing centre of animal cells. It consists of two centrioles and a multi-layered proteinaceous structure that surrounds the centrioles, the so-called pericentriolar material. Centrosomes promote de novo assembly of MTs and thus play important roles in Golgi organization, cell polarity, cell motility and the organization of the mitotic spindle. To execute these functions, centrosomes have to adopt particular cellular positions. Actin and MT networks and the association of the centrosomes to the nuclear envelope define the correct positioning of the centrosomes. Another important feature of centrosomes is the centrosomal linker that connects the two centrosomes. The centrosome linker assembles in late mitosis/G1 simultaneously with centriole disengagement and is dissolved before or at the beginning of mitosis. Linker dissolution is important for mitotic spindle formation, and its cell cycle timing has profound influences on the execution of mitosis and proficiency of chromosome segregation. In this review, we will focus on the mechanisms of centrosome positioning and separation, and describe their functions and mechanisms in the light of recent findings.     

Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.     

Localization of Golgi 58K protein (formiminotransferase cyclodeaminase) to the centrosome

In vertebrate cells, the centrosome consists of a pair of centrioles and surrounding pericentriolar material. Using anti-Golgi 58K protein antibodies that recognize formiminotransferase cyclodeaminase (FTCD), we investigated its localization to the centrosome in various cultured cells and human oviductal secretory cells by immunohistochemistry. In addition to the Golgi apparatus, FTCD was localized to the centrosome, more abundantly around the mother centriole. The centrosome localization of FTCD continued throughout the cell cycle and was not disrupted after Golgi fragmentation, which was induced by colcemid and brefeldin A. Centriole microtubules are polyglutamylated and stable against tubulin depolymerizing drugs. FTCD in the centrosome may be associated with polyglutamylated residues of centriole microtubules and may play a role in providing centrioles with glutamate produced by cyclodeaminase domains of FTCD.     

Influence of centriole behavior on the first spindle formation in zygotes of the brown alga Fucus distichus (Fucales, Phaeophyceae)

The influence of centrioles, derived from the sperm flagellar basal bodies, and the centrosomal material (MTOCs) on spindle formation in the brown alga Fucus distichus (oogamous) was studied by immunofluorescence microscopy using anti-centrin and anti-beta-tubulin antibodies. In contrast to a bipolar spindle, which is formed after normal fertilization, a multipolar spindle was formed in polyspermic zygote. The number of mitotic poles in polyspermic zygotes was double the number of sperm involved in fertilization. As an anti-centrin staining spot (centrioles) was located at these poles, the multipolar spindles in polyspermic zygotes were produced by the supplementary centrioles. When anucleate egg fragments were fertilized, chromosome condensation and mitosis did not occur in the sperm nucleus. Two anti-centrin staining spots could be detected, microtubules (MTs) radiated from nearby, but the mitotic spindle was never produced. When a single sperm fertilized multinucleate eggs (polygyny), abnormal spindles were also observed. In addition to two mitotic poles containing anti-centrin staining spots, extra mitotic poles without anti-centrin staining spots were also formed, and as a result multipolar spindles were formed. When karyogamy was blocked with colchicine, it became clear that the egg nucleus proceeded independently into mitosis accompanying chromosome condensation. A monoastral spindle could be frequently observed, and in rare cases a barrel-shaped spindle was formed. However, when a sperm nucleus was located near an egg nucleus, the two anti-centrin staining spots shifted to the egg nucleus from the sperm nucleus. In this case, a normal spindle was formed, the egg chromosomes arranged at the equator, and the associated MTs elongated from one pole of the egg spindle toward the sperm chromosomes which were scattered. From these results, it became clear that paternal centrioles derived from the sperm have a crucial role in spindle formation in the brown algae, such as they do during animal fertilization. However, paternal centrioles were not adequate for the functional centrosome during spindle formation. We speculated that centrosomal materials from the egg cytoplasm aggregate around the sperm centrioles and are needed for centrosomal activation.     

Breaking the ties that bind: new advances in centrosome biology

The centrosome, which consists of two centrioles and the surrounding pericentriolar material, is the primary microtubule-organizing center (MTOC) in animal cells. Like chromosomes, centrosomes duplicate once per cell cycle and defects that lead to abnormalities in the number of centrosomes result in genomic instability, a hallmark of most cancer cells. Increasing evidence suggests that the separation of the two centrioles (disengagement) is required for centrosome duplication. After centriole disengagement, a proteinaceous linker is established that still connects the two centrioles. In G2, this linker is resolved (centrosome separation), thereby allowing the centrosomes to separate and form the poles of the bipolar spindle. Recent work has identified new players that regulate these two processes and revealed unexpected mechanisms controlling the centrosome cycle.