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PubMed1983

Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation.

Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.

Anaphase↗

PubMed2001

Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy.

BACKGROUND: Spindles are formed from microtubules and are exquisitely sensitive to changes in temperature. An orientation-independent polarized light microscope, the Polscope, can be used to image spindles in living oocytes allowing analysis of spindle kinetics in the living state. This study examined the effects of cooling on spindle disassembly in living human oocytes and spindle recovery after rewarming. METHODS: Oocytes were imaged continuously with the Polscope during cooling and rewarming. The quantity of microtubules in the spindles was measured by its birefringence using the Polscope. RESULTS: Spindles had completely disassembled by 5 min after cooling and recovered by 20 min after rewarming to 37 degrees C if rewarming started soon after the oocyte's temperature dropped to room temperature. However, when oocytes were cooled and kept at 33, 28 or 25 degrees C for 10 min and then warmed, it was found that warming allowed 5/5, 2/5 and 0/5 oocytes of the spindles to recover respectively. CONCLUSIONS: These results indicate that human meiotic spindles are exquisitely sensitive to alterations in temperature. The maintenance of temperature at 37 degrees C during in-vitro manipulation is important for spindle integrity and, therefore, is likely to be important for normal fertilization and subsequent embryo development.

Cold Temperature↗

PubMed2005

The effects of hyperbilirubinemia on sleep-spindle characteristics in infants.

OBJECTIVE: The purpose of this study is to investigate the effect of hyperbilirubinemia on sleep-spindle characteristics. Rhythmic activities, such as sleep spindles, may be abolished by hyperbilirubinemia. STUDY DESIGN: Electroencephalogram records were taken from 15 infants with hyperbilirubinemia and 18 healthy infants at the 12th week after birth. Sleep spindles of the 2 groups were compared according to location, density, duration, amplitude, frequency, asynchrony, and asymmetry. RESULTS: In the study and control groups, the density of the spindles was found to be 76.9 +/- 23.7 and 105.2 +/- 33.9, respectively, in a 1-hour non-rapid eye movement sleep period. The mean durations of the sleep spindles in the study and control groups were found to be 4105 +/- 802 milliseconds and 5162 +/- 1075 milliseconds, respectively. There was not any difference between the groups according to the amplitude and asymmetry. However, there was a significant difference between the 2 groups in the frequency of spindles. The mean frequency was found to be 12.5 +/- 0.6 Hz in the study group and 13.2 +/- 0.9 in the control group. The percentage of asynchronous spindles was higher in the study group than in the control group. There was a significant negative correlation between the bilirubin levels during the newborn period and density, duration, and frequency of spindles. However, there was a significantly positive correlation between the bilirubin levels and percentage of asynchronous spindles. There was a significant negative correlation between the duration of hyperbilirubinemia and spindleamplitude. CONCLUSION: We suggest that studies on the critical maturation periods of sleep-spindle patterns might provide a sensitive tool for early diagnosis of neurophysiologic brain alterations during the first trimester of life in a population of at-risk children, such as jaundiced infants.

Aged↗

PubMed1996

Pontine cholinergic mechanisms modulate the cortical electroencephalographic spindles of halothane anesthesia.

BACKGROUND: Halothane anesthesia causes spindles in the electroencephalogram (EEG), but the cellular and molecular mechanisms generating these spindles remain incompletely understood. The current study tested the hypothesis that halothane-induced EEG spindles are regulated, in part, by pontine cholinergic mechanisms. METHODS: Adult male cats were implanted with EEG electrodes and trained to sleep in the laboratory. Approximately 1 month after surgery, animals were anesthetized with halothane and a microdialysis probe was stereotaxically placed in the medial pontine reticular formation (mPRF). Simultaneous measurements were made of mPRF acetylcholine release and number of cortical EEG spindles during halothane anesthesia and subsequent wakefulness. In additional experiments, carbachol (88 mM) ws microinjected in the the mPRF before halothane anesthesia to determine whether enhanced cholinergic neurotransmission in the MPRF would block the ability of halothane to induce cortical EEG spindles. RESULTS: During wakefulness, mPRF acetylcholine release averaged 0.43 pmol/10 min of dialysis. Halothane at 1 minimum alveolar concentration decreased acetylcholine release (0.25 pmol/10 min) while significantly increasing the number of cortical EEG spindles. Cortical EEG spindles caused by 1 minimum alveolar concentration halothane were not significantly different in waveform, amplitude, or number from the EEG spindles of nonrapid eye movement sleep. Microinjection of carbachol into the mPRF before halothane administration caused a significant reduction in number of halothane-induced EEG spindles. CONCLUSIONS: Laterodorsal and pedunculopontine tegmental neurons, which provide cholinergic input to the mPRF, play a causal role in generating the EEG spindles of halothane anesthesia.

Acetylcholine↗

PubMed1967

Nature of thalamo-cortical relations during spontaneous barbiturate spindle activity.

1. The relation between thalamic and cortical spontaneous spindles was investigated in cats anaesthetized with barbiturates.2. Simultaneous recordings with multiple electrodes in the thalamus and cortex revealed a high correlation between thalamic and cortical spindle activity, both with regard to the occurrence of the spindles as well as to their individual waves, provided a critical location of the electrodes was secured. The results indicate a point-to-point relation between a group of thalamic cells and a small cortical area to which these cells project. In the spontaneous rhythm, the cortical columns are probably individually controlled by a thalamic rhythmic entity. This point-to-point relation was found in all the major sensory projection systems and in one thalamic ;association' nucleus and its corresponding ;association' cortex.3. Cortical barbiturate spindles appeared either as local spindles in a restricted cortical area or as compound spindles in several areas. Spindles recorded from electrodes separated by 2 mm or more were clearly different with regard to intraspindle wave frequency, duration, and the time of start and stop of the spindle. These differences increased with increasing distance between the electrodes, and were most pronounced when the corticograms of the two hemispheres were compared.4. Spontaneous spindle activity interfered with orthodromic transmission through n. ventralis posterolateralis (VPL) and medial geniculate nucleus (MG), judged by depression of the thalamic and cortical responses to peripheral nerve volleys or clicks. Such inhibition required the afferent volley to be delivered at a particular time of the spontaneous oscillations. Further, orthodromic volleys reset the rhythmic spindle waves in the appropriate thalamic and cortical areas.5. These findings lead to a new concept of the thalamic pace-maker function. During barbiturate anaesthesia, small assemblages of thalamic neurones seem to have the ability to generate independent rhythmic discharges, and thereby control the rhythm of the particular cortical column to which this thalamic group projects.6. During barbiturate anaesthesia, many facultative pace-makers seem to be present in the thalamus. The total number may be as large as 25,000-35,000. Usually, many of these rhythmic thalamic units beat in synchrony or near synchrony.

Animals↗

PubMed1996

Forces acting on the fission yeast anaphase spindle.

The fission yeast mitotic spindle consists of three sets of microtubules: one that extends between the chromosomes and the spindle pole bodies (SPBs); one that extends between the two SPBs forming a region of overlap; and a third, the so-called astral microtubules, that associates laterally with the cytoplasmic face of the SPBs, during anaphase B. The major bundles of the latter can exist with equal probability in two configurations which we have termed parallel and convergent. Mitosis in fission yeast is characterised by an extended anaphase B during which the spindle elongates from 2 mu m (the diameter of the interphase nucleus) to about 14 mu m, spanning the entire length of the cell. Anaphase B spindles viewed by indirect immunofluorescence microscopy frequently appeared bowed but only when the astral microtubules were in the convergent orientation. To investigate the possible significance of this observation, we have examined the situation in the abnormally long spindles that are formed in cells in which cell length has been extended either by overexpression of the weel + gene or by inactivation of the cdc25 + gene. The spindles in these strains were often considerably longer (up to 30 mu m) than in wild type cells but, unlike the latter, did not extend the entire length of the cell. Bowed spindles were again observed but only when the astral microtubules were convergent. We discuss these findings in the context of the astral microtubules either exerting a pulling force on the poles of the anaphase B spindle, or counteracting a pushing force generated by sliding of anti-parallel pole to pole microtubules of the mitotic spindle, or both of the above.

Anaphase↗

PubMed1997

Traction fibre: toward a "tensegral" model of the spindle.

Most current hypotheses of mitotic mechanisms are based on the "PAC-MAN" paradigm in which chromosome movement is generated and powered by disassembly of kinetochore microtubules (k-MTs) by the kinetochore. Recent experiments demonstrate that this model cannot explain force generation for anaphase chromosome movement [Pickett-Heaps et al., 1996: Protoplasma 192:1-10]. Another such experiment is described here: a UV-microbeam cut several kinetochore fibres (k-fibres) in newt epithelial cells at metaphase and the half-spindle immediately shortened: in several cells, the remaining intact spindle fibres bowed outwards as they came under increased compression. Thus, severing of k-MTs can lead to increased tension between chromosomes and poles. This observation cannot be explained by models in which force is produced by motor molecules at the kinetochore actively disassembling k-MTs. Rather, we argue that tensile forces act along the whole k-fibre, which, therefore, can be considered as a classic "traction fibre." We suggest that anaphase polewards force is generated by MTs interacting with the spindle matrix and when k-MTs are severed, polewards force continues to act on the remaining kMT-stub; spindle MTs act as rigid struts concurrently resisting and being controlled by these forces. We suggest that the principles of "cellular tensegrity" [Ingber, 1993: J. Cell Sci. 104:613-627] derived from the behaviour and organization of the interphase cell apply to the spindle. In an evolutionary context, this argument further suggests that the spindle might originally have evolved as the mechanism by which a single tensegral unit (cytoplast) is divided into two cytoplasts; use of the spindle for segregating chromosomes might represent a secondary, more recent development of this primary function. If valid, this concept has implications for the way the spindle functions and for the spindle's relationship to cytokinesis.

Animals↗

PubMed1998

Early stages of spindle formation and independence of chromosome and microtubule cycles in Haemanthus endosperm.

We analyzed transformation of the interphase microtubular cytoskeleton into the prophase spindle and followed the pattern of spindle axis determination. Microtubules in endosperm of the higher plant Haemanthus (Scadoxus) were stained by the immunogold and immunogold silver-enhanced methods. Basic structural units involved in spindle morphogenesis were "microtubule converging centers." We emphasized the importance of relative independence of chromosomal and microtubular cycles, and the influence of these cycles on the progress of mitosis. Cells with moderately desynchronized cycles were functional, but extreme desynchronization led to aberrant mitosis. There were three distinct phases of spindle development. The first one comprised interphase and early to mid-prophase. During this phase, the interphase microtubule meshwork radiating from the nuclear surface into the cytoplasm rearranged and formed a dense microtubule cage around the nucleus. The second phase comprised mid to late prophase, and resulted in the formation of normal (bipolar) or transitory aberrant (apolar or multipolar) prophase spindles. The third phase comprised late prophase with prometaphase. The onset of prometaphase was accompanied by a rapid association of microtubule converging centers with kinetochores. In this stage aberrant spindles transformed invariably into bipolar ones. Lateral association of a few bipolar kinetochore fibers at early prometaphase established the core of the bipolar spindle and its alignment. We concluded that (1) spindle formation is a largely independent microtubular process modified by the chromosomal/kinetochore cycle; and (2) the initial polarity of the spindle is established by microtubule converging centers, which are a functional substitute of the centrosome/MTOC. We believe that the dynamics of microtubule converging centers is an expression of microtubule self-organization driven by motor proteins as proposed by Mitchison [1992: Philos. Trans. R. Soc. Lond. B. 336:99].

Chromosomes↗

PubMed1983

Differences in muscle spindle structure between pigeon muscles used in aerial and terrestrial locomotion.

Muscle spindle density (number of spindles per gram of muscle) of all 29 muscles of the forearm and leg of the domestic pigeon was evaluated by counting receptors in van Gieson-stained serial cross sections. Extra- and intra-fusal fiber-type profiles were determined from histochemical preparations. Muscles of the leg had on the average significantly more avian slow-twitch oxidative extrafusal fibers (22.5 vs. 0.8%) and slower contraction times than muscles of the forearm, but fiber-type profiles and gross actions of muscles showed no consistent relation to the relative abundance of receptors. Differences in intrafusal fiber-type composition among spindles were sought because of their potential effect on the quality of the afferent discharge. The number of intrafusal fibers per spindle was on the average significantly less (4.57 vs 5.99) in the muscles of the leg than in those of the forearm; and of spindles with the same number of intrafusal fibers, those in the leg had smaller periaxial spaces. Distribution of intrafusal fiber types identified with the myofibrillar adenosine triphosphatase reaction differed among spindles of varying sizes. An acid- and alkali-labile type occurred most frequently (P = 0.05) in spindles with one to three intrafusal fibers, and an acid-labile and alkali-stable type was most often seen (P = 0.05) in spindles with 4 to 7 intrafusal fibers. The smaller receptors were more abundant in the leg, while the larger ones were about equally distributed between the two extremities. Muscle fibers with dimensions that sometimes approached small extrafusal fibers were present in about 3% of the axial bundles examined, most of them in the forearm. The selective morphological variation of avian muscle spindles may represent the structural basis for qualitatively different afferent discharges that relate to the characteristic types of locomotion served by the two extremities.

Animals↗

PubMed1989

Role of nerve and muscle factors in the development of rat muscle spindles.

The soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent-muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (alpha) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (gamma) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when gamma axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not gamma motor innervation. However, transient innervation of future bag2 fibers by alpha axons suggests that both sensory and alpha motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.

Animals↗

PubMed1995

The formation and functioning of yeast mitotic spindles.

The mitotic spindle contains the machinery responsible for sister chromatid segregation. It is composed of a complex and dynamic array of microtubules, which are nucleated from the spindle poles. Studies of yeast spindle functions by molecular genetic analysis and by in vitro functional analysis have identified proteins that are mitosis-specific and present at very low concentrations in the cell, and have revealed the molecular bases of several processes required for the formation and functioning of the mitotic spindle. Here I review the current knowledge of the processes that are common to most eukaryotes: microtubule nucleation at the spindle poles, bipolar spindle assembly, maintenance of the spindle structure, chromosome attachment to the spindle and chromosome separation on the spindle.

Saccharomyces cerevisiae↗

PubMed1984

Taxol stabilization of mitotic spindle microtubules: analysis using calcium-induced depolymerization.

Taxol stabilizes or promotes the assembly of microtubules. In this report we characterize the rate, extent, and reversibility of taxol stabilization of calcium-labile microtubules in isolated mitotic spindles, principally from embryos of the sand dollar Echinarachnius parma. The intense depolymerizing action of 100 microM Ca2+ was used to assess the extent of stabilization by taxol. Changes in spindle microtubule assembly were evaluated and recorded by measuring changes in spindle birefringent retardation (BR). Membrane-free mitotic spindles, isolated with a calcium-chelating, nonionic detergent buffer, were stored in an EGTA-glycerol storage buffer to prevent microtubule depolymerization. When perfused with an EGTA-buffer without glycerol, microtubules in these isolated spindles depolymerized gradually over 60-120 min; but in isolated spindles perfused with buffer that contained 100 microM Ca2+, BR decreased by 90% within 2-5 sec. In contrast, spindles that were pretreated for 3 min with 1 microM taxol, or for about 30 sec with 10 microM taxol, lost less than 10% of their initial BR when perfused with buffer containing 100 microM Ca2+. The rate and extent of microtubule stabilization by taxol depended on both the concentration and the duration of exposure to taxol. Taxol stabilization was reversible. After a 15 min preincubation with 1 microM or 10 microM taxol then washout, stability of spindle BR to 100 microM Ca2+ decreased exponentially with a time constant of 30-60 min. Thus taxol dissociates from spindle microtubules at significant rates; taxol-stabilized microtubules are not "fixed."

Alkaloids↗

PubMed1986

Incorporation and turnover of labeled exogenous tubulin in the mitotic spindles of Chaetopterus oocytes and HeLa cells.

The incorporation of tubulin into mitotic spindles in situ was studied by incubating permeabilized mitotic cells in solutions containing [3H]GTP-labeled or dichlorotriazinylamino fluorescein (DTAF)-labeled tubulin. Metaphase HeLa cells or spindle-containing "minicells" from Chaetopterus oocytes were lysed in a microtubule-assembly buffer plus 0.5% Nonidet P-40, 1 mg/ml 120,000g supernatant mammalian brain tubulin, and [3H]GTP. After different periods of incubation, mitotic spindles were isolated in 2 M-glycerol-containing assembly buffer and separated from unbound counts by centrifugation through a 4 M-glycerol cushion; 3H counts per mg protein increase linearly for 8-12 min and then reach a plateau or steady state in both Chaetopterus oocytes and HeLa cells. Addition of 4 mM CaCl2 blocks or reverses incorporation. Little or no [3H]GTP is incorporated if exogenous tubulin or lysed cells are omitted from the assembly mixture. To measure the loss rate of [3H]GTP-tubulin from mitotic spindles, cells were incubated in tubulin plus [3H]GTP for 30 min, and a 20-fold excess of cold GTP (2 mM) was added. Samples were removed after incubation for different periods, and spindles were isolated as described above and counted for 3H content. [3H]GTP is lost from spindles at a rate of about 16%/min until a new steady state is reached in about 8 min. These results are consistent with an incorporation and turnover of [3H]GTP-tubulin in spindle microtubules of these lysed-cell models. The location of this newly incorporated tubulin in the spindle was investigated by incorporating fluorescent DTAF-tubulin into mitotic spindles of these lysed cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg.

Spindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.

Adenosine Triphosphate↗

PubMed1989

Distribution of phosphorylated spindle-associated proteins in the diatom Stephanopyxis turris.

Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATP gamma S at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATP gamma S. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.

Antibodies↗

PubMed1995

Cytochalasin J affects chromosome congression and spindle microtubule organization in PtK1 cells.

PtK1 cells were treated with 10 micrograms/ml cytochalasin J (CJ) for 15 min at various stages of mitosis. When applied at nuclear envelope breakdown (NEB) chromosome congression was blocked or substantially slowed, and chromosomes failed to show organization patterns typical of prometaphase. Spindle microtubule (MT) numbers appeared unaffected as judged by the pattern of birefringent retardation. However, ultrastructural analysis showed MTs to be reorganized within the spindle domain with some exhibiting fragmentation and others failing to interact with poorly defined kinetochore laminae. The spindle domain took on a curved, almost banana-like shape, as related to the position of the centrosomes and lack of orientation of chromosomes. Serial section analysis of kinetochore regions showed reduced contour length and maturation of the kinetochore plate with few MTs associated with this structure. Cells similarly treated with 10 micrograms/ml CJ at NEB for 15 min and then released into conditioned medium for 15 min showed the most chromosomes resumed congression to the metaphase plate. Ultrastructural analysis revealed a more normal organization of spindle MTs, but kinetochore structure remained affected. CJ treatment of cells in prometaphase slightly affected chromosomes congression with most chromosomes aligning at the metaphase plate after 10-15 min of treatment. Ultrastructural analysis showed that astral MTs were disrupted and spindle MTs were fragmented; few MTs coursed from kinetochore to pole. Kinetochore structure was also affected with only small numbers of short MTs seen associated with kinetochores. Application of CJ at anaphase onset had little effect on anaphase A and B, but cytokinesis failed to occur. Anti-tubulin staining of a monolayer of cells treated with 10 micrograms/ml CJ for 15 min showed that over 60% of mitotic figures exhibited changes in MT organization. Cells showing the greatest effect of treatment had several foci of bundles of MTs, as if the spindle were multipolar. Chromosomes were arranged near the periphery of the spindle which could be a result of abnormalities of kinetochore structure. Improper association of spindle MTs with kinetochores and, thus, changes in kinetochore position could account for these changes in spindle architecture.

Actins↗

PubMed2004

Functional role of centrosomes in spindle assembly and organization.

The centrosome is the main MT organizing center in animal cells, and has traditionally been regarded as essential for organization of the bipolar spindle that facilitates chromosome segregation during mitosis. Centrosomes are associated with the poles of the mitotic spindle, and several cell types require these organelles for spindle formation. However, most plant cells and some female meiotic systems get along without this organelle, and centrosome-independent spindle assembly has now been identified within some centrosome containing cells. How can such observations, which point to mutually incompatible conclusions regarding the requirement of centrosomes in spindle formation, be interpreted? With emphasis on the functional role of centrosomes, this article summarizes the current models of spindle formation, and outlines how observations obtained from spindle assembly assays in vitro may reconcile conflicting opinions about the mechanism of spindle assembly. It is further described how Drosophila mutants are used to address the functional interrelationships between individual centrosomal proteins and spindle formation in vivo.

Animals↗

PubMed2002

Effects of spindle removal on MPF and MAP kinase activities in porcine matured oocytes.

Intracellular localization of maturation/M-phase promoting factor (MPF) and mitogen activated protein (MAP) kinase in mature oocytes has been examined by immunocytochemical methods and the authors of these studies have reported that they are localized on spindles during M-phase. Although these reports showed the relative localization of MPF and MAPK on spindles, it has never been shown whether these kinases are present in the cytoplasm and, if they are present, how many parts of the kinases are localized on the metaphase spindle. In the present study, we made quantitative analyses of MPF and MAP kinase localized on oocyte spindles by kinase assays and immunoblotting after removal of the spindles from porcine mature oocytes. First, we certified their intracellular distribution by immunocytochemical methods and observed sharp signals of cyclin B1 on spindle poles and MAP kinase signals on the microtubule of metaphase spindles. In contrast to these results by immunostaining, the amounts of MPF and MAP kinase localized on spindles examined by immunoblotting and kinase assays were undetectable and less than 20%, respectively. These results indicate that the immunocytochemical technique is a powerful method for showing relative localization, but it is not suitable for quantitative analysis, and that the removal of metaphase spindles from mature oocytes does not have a severe negative impact on the subsequent MPF and MAP kinase activity and on the cell cycle progression in early embryo development.

Animals↗