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The meiotic spindle of the Drosophila oocyte: the role of centrosomin and the central aster.

We provide here the first evidence that a distinct midzone is present in the Drosophila melanogaster female meiosis I spindle. This region has the ability to bind the Pavarotti kinesin-like (PAV-KLP) and Abnormal spindle (Asp) proteins, indicating a correct organization of the central spindle microtubules. We also identified the core component centrosomal protein centrosomin (CNN) at an unexpected site within the anaphase I spindle, indicating a role for CNN during the biogenesis of the female meiotic apparatus. However, there are no apparent defects in the midzone organization of cnn oocytes, whereas defects occur later when the central aster forms. The primary mutant phenotype of cnn oocytes is the failure to form a developed central microtubule organizing center (MTOC), although twin meiosis II spindles usually do form. Thus the central MTOC may not be essential for the formation of the inner poles of twin meiosis II spindles, as generally proposed, but it might be involved in maintaining their proper spacing. We discuss the proposal that, in the presence of a central MTOC, a chromatin-driven mechanism of spindle assembly like that described during meiosis I may control the morphogenesis of the twin meiosis II spindles.

Animals↗

Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo.

The metaphase-spindle steady-state length occurs as spindle microtubules ;flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.

Anaphase↗

Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.

BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly. Two related human protein kinases, Bub1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle. However, how the activities of Bub1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known. To investigate how spindle events regulate Bub1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalise to the same domain of the kinetochore. However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of Bub1 is often asymmetrical. This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker Bub1 staining is typically closer to the nearest spindle pole. In addition, a 30 minute nocodazole treatment dramatically increases the amount of Bub1 localising to kinetochores but has little effect on BubR1. Furthermore, Bub1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment. Thus, these observations suggest that Bub1 localisation is sensitive to changes in both tension and microtubule attachment. Consistent with this, we also show that Bub1 is rapidly phosphorylated following brief treatments with nocodazole or taxol. In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect. Although these observations indicate that Bub1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis. We suggest therefore that Bub1 and BubR1 may integrate different 'spindle assembly signals' into a single signal which can then be interpreted by downstream cell cycle regulators.

Animals↗

Localization of a mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells.

mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu(434) and Leu(455) in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.

Adaptor Proteins, Signal Transducing↗

Distribution of pericentriolar material in multipolar spindles induced by colcemid treatment in Chinese hamster ovary cells.

Mitotic Chinese hamster ovary cells were obtained by treatment with microtubule drugs under various conditions, and the shape of spindles was analysed by phase-contrast microscopy of isolated spindles, and by indirect immunofluorescence staining of whole mitotic cells with anti-tubulin antibody. Bipolarity of spindles was maintained after treatment with 0.05 microM of colcemid for 3.5 h, but increased exposure to higher concentrations (0.32 microM) and for longer durations (5.5 h) led to a marked rise in multipolar spindles. Nocodazole treatment, on the other hand, failed to show a multiplicity of spindle poles even at 3.3 microM. Each pole of a multipolar spindle was associated with pericentriolar material, as shown by staining with an autoimmune serum specific for pericentriolar material. The number of locations with free pericentriolar material capable of polymerizing microtubules in vitro also increased with increasing numbers of spindle poles, suggesting that dispersion of the pericentriolar material resulted in the production of many microtubule-nucleating sites in multipolar spindles. The different efficiencies of recovery from different drugs, which have been known to be quite variable, may be partly due to the different extent of dispersion of the pericentriolar material.

Animals↗

The transition of cells of the fission yeast beta-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy. Requirement of functional tubulin for spindle pole body duplication.

A previous fluorescence light-microscopic study showed that the fission yeast cold-sensitive beta-tubulin mutant nda3-311 was arrested with rod-like condensed chromosomes in a mitotic state at the restrictive temperature. Upon transfer to the permissive temperature, a spindle was formed and the nucleus was divided. In the present study, we employed freeze-substitution electron microscopy to examine the ultrastructure of arrested and released nda3-311 cells. In arrested cells, a single, displaced nucleus was seen with a single spindle pole body. Therefore, spindle pole body duplication seemed to require functional beta-tubulin. The nuclear membrane was highly deformed with a leaf-like profile in cross-section, possibly due to an interaction with the rod-like, condensed chromosomes. Upon transfer to the permissive temperature, the spindle pole duplicated and the daughter spindle pole bodies rapidly migrated to the opposite ends of the nucleus, accompanied by the formation of the mitotic spindle. Elongation of the nuclear envelope occurred with concomitant spindle extension, as in a wild-type mitosis. The deformed nuclear membrane became smooth and described a convex curve. The numerous vacuoles that are seen in the arrested cells decreased in number and increased in size. Septation was completed, leaving the two divided nuclei in one half of the cell. Hexagonally arranged microtubules, apparently forming the mitotic spindle, were observed in a cross-section of a cell after return to the permissive conditions.

Freezing↗

Aberrant spindle assembly checkpoint in bovine somatic cell nuclear transfer oocytes.

Nuclear, microtubular dynamics and spindle assembly checkpoint (SAC) in bovine somatic cell nuclear transfer (SCNT) oocytes receiving G1/0 or M phase somatic cell nuclei were studied. SCNT oocytes assembled microtubules, however, the spindles were structurally abnormal, including bi-, tri-polar or elongated spindles with scattered premature chromosome condensation (PCC) in G1/0 phase nuclei, and some miniature spindles with unaligned chromosomes in M phase nuclei. In contrast, demecolcine-treated SCNT oocytes formed chromosome clusters with membrane protrusion and significantly induced maturation-promoting factor (MPF) activity elevation (up to 177%) for 3 hours, indicating that first SAC at second metaphase (MII) is established upon spindle disruption in SCNT oocytes. After parthenogenetic stimuli, unlike MII oocytes which prevent exit from MII arrest with high MPF activity upon spindle disruption by second SAC, demecolcine-treated SCNT oocytes could not prevent exit from MII arrest with inactivation of MPF activities, whereas MG132-treated SCNT oocytes could persist at MII arrest, indicating that SCNT oocytes lack the ability for second SAC establishment, however, two G1/0 phase nuclei in an ooplasm restored second SAC establishment upon spindle disruption. Furthermore, the developmental potential of demecolcine-treated SCNT oocytes receiving G1/0 phase nuclei to blastocyst stage was not significantly different than untreated SCNT oocytes (29% vs 31%). These results indicate that unlike MII oocytes, SCNT oocytes have aberrant spindle morphology and SAC at MII due to insufficient SAC signals from somatic cell nuclei, thus aberrant remodeling has started immediately after somatic cell nuclear transfer and may be responsible for chromosome instability in SCNT embryos as well as the low successful efficiency of cloning.

Animals↗

Effects of Ba ions on frog's isolated muscle spindle.

Dilute solutions of Barium Chloride have been tested on frog's isolated muscle spindles with the aim of clarifying the processes leading to the afferent discharge. In the presence of Ba++ the spindle properties are modified as follows: 1. The discharge of slack spindles increases and becomes extremely regular. 2. The phasic response to stretching is enhanced, the tonic responses being on the contrary virtually suppressed. Under Ba-treatment therefore the spindle changes from a phasic-tonic receptor to a merely phasic receptor. 3. The "silent period" following every propagated spike is prolonged while the safety margin of the "abortive" into propagated spike conversion becomes maximal under Ba-treatment. Additionally, this analysis showed that, when a "true" action potential arises in the spindle, it propagates not only to the stem fibre, but also to all the sensory endings in the spindle intracapsular network. Comparison between the effects of Ba++ and those produced by TEA and ouabain indicates that the action of Ba++ on muscle spindles results from conjunction of two factors : a) reduction of K permeability, and b) enhanced efficiency of an electrogenic Na/K-pump in spindle nerve membranes.

Action Potentials↗

Thalamo-cortical systems regulating spindle bursts and recruiting responses. II. Effect of thalamic lesions.

The effects of thalamic lesions on recruiting responses and spindle bursts have been studied in 12 cats immobilized with gallamine. Recruiting responses were elicited by stimulation of the midline thalamic nuclear complex. Spindle bursts were produced by lesions in the mesencephalic tegmentum. Recruiting responses and spindle bursts were recorded from the cortex and thalamus. The effect of thalamic lesions on such spontaneous and induced potentials was evaluated by making lesions anteriorly in the rostral pole of the thalamus, medially in the midline thalamic nuclear group, and laterally in the primary and association nuclei of the thalamus. 1. Small, anterior, thalamic lesions blocked spindle bursts and recruiting responses both at the cortex and in the thalamus. 2. Massive, medial, thalamic lesions blocked spindle bursts in the suprasylvian cortex, but did not affect either frontal cortex spindle bursts or frontal and thalamic recruiting responses induced by stimulation of N. ventralis anterior. 3. Massive, lateral, thalamic lesions including specific relay and association nuclei, failed to block spontaneous spindles and recruiting responses in frontal cortex and midline thalamic nuclei. It has been postulated that the rostral pole of the thalamus constitutes an important part of the thalamic portion of a thalamo-orbitocortical system which mediates and regulates electrocortical synchronization as manifested in spindle bursts, recruiting responses and synchronized wave-like potentials.

Animals↗

Differential kinetochore requirements for establishment and maintenance of the spindle checkpoint are dependent on the mechanism of checkpoint activation in Saccharomyces cerevisiae.

The spindle checkpoint in the yeast Saccharomyces cerevisiae is an intracellular signal transduction pathway comprised of two branches that inhibit two different mitotic transitions in cells treated with benzimidazole drugs such as nocodazole. The kinetochore is an integral component of the MAD2 branch of the spindle checkpoint pathway. Current models propose that the kinetochore is required for both the establishment and maintenance of the spindle checkpoint but a role for the kinetochore in the maintenance of spindle checkpoint in yeast has never been directly tested. We used a temperature sensitive ndc10-1 mutant to inactivate kinetochores before and after arresting cells in mitosis to determine the role of kinetochores in the establishment and maintenance of the spindle checkpoint. We show that both establishment and maintenance requires kinetochore function in response to spindle damage induced by benzimidazole drugs. Excess expression of the Mps1 protein kinase causes wild type cells and ndc10-1 cells to arrest in mitosis. Unlike the spindle checkpoint arrest activated by benzimidazoles, this arrest can be maintained independently of kinetochores. The arrest induced by excess Mps1p is independent of BUB2. Therefore, mitotic arrest induced by excess Mps1p expression is due to the action of the MAD2 branch of the spindle checkpoint pathway and excess Mps1p acts downstream of the kinetochore.

Carrier Proteins↗

A histochemical study of the neuro-muscular spindles in the tongue of some rodents.

An investigation was undertaken to study the Neuro-muscular spindles in the tongue (Rattus rattus rufescens and Suncus murinus) by cholinesterase technique, under maintained pH of 5.2, incubation period 20 h., and temperature 38 degrees C (Rattus) and 39 degrees C (Suncus). Neuro-muscular spindles were observed frequently distributed from anterior to posterior of the tongue and took a little darker stain than the ordinary muscle fibres (negatively stained) while the nerve fibres and ganglia took still darker stain. Nerve fibres (myelinated, non-myelinated and tortuous) were seen penetrating and embracing the muscle spindles and most of them ran parallel to the spindles. Ganglia of various shapes (oval, irregular, elongated and rounded) and sizes (large, Rattus and medium and small, Suncus) were AChE-positive. Ganglia small and medium in size, rounded and elongated in shape were arranged in chain-like fashion on the muscle spindles in Suncus (Fig. 9, 10). The blood vessels (artery, vein, and capillary) were recorded either in close association with the muscle spindles or lying parallel to it. In all cases they were closely related and deeply associate either with the nerve fibres of the plexus of the muscle spindles or with the nerve fibres of the neural network of vessel. Perivascular and perimuscular ganglia with pre- and postganglionic nerve fibres were closely related to each other. Nerve endings, dot-like (Rattus) and bulb-like (Suncus) were occasionally recorded. AChE activity was found randomly in the muscle spindles (Fig. 2, 5.) and in the form of dark brown patches in the ordinary muscle fibres.

Animals↗

Relationships between spindle density, muscle architecture and fibre type composition in different parts of the rabbit masseter.

The occurrence of muscle spindles was studied in the masseter muscle of rabbits by light-microscopy of whole muscle sections. The distribution of the spindles appeared to be heterogeneous. Most spindles lay in the anterior/deep and mid-parts of the masseter. The lateral/superficial part contained only a few muscle spindles. The distribution of the spindles is correlated to the distribution of slow twitch (type I) extrafusal fibres. This means that spindles, like type I fibres might be involved in the control of fine movements and posture. Spindle density and type I fibre density increase with distance from the temporomandibular joint. This could mean that spindles are involved in controlling bite force.

Animals↗

[Ultrastructure of the mitotic nuclei in Leishmania mexicana ssp. Tridimensional reconstruction of the mitotic spindle and dense plaques].

The ultrastructure of mitotic nuclei of the promastigote Leishmania mexicana ssp. was studied by serial thin sections and three-dimensional reconstructions of each divisional stage. At the beginning of nuclear division (equatorial stage), a set of six dense plaques located about the equatorial region of the nucleus and a microtubular spindle develops in the two opposing poles of the nucleus (two sets of polar microtubules). The microtubular mitotic spindle is entirely intranuclear with the nuclear membrane persisting through mitosis. The polar spindle consists of a discrete bundle of about 50 microtubules and the equatorial spindle is formed by about 100 microtubules. The spindle may contain several continuous microtubules, but no microtubular organizing centres were observed in association with the spindle. The plaques and hemiplaques are associated with microtubular bundles; some of the spindle microtubules converge on kinetochore-like plaques. It is suggested that the spindle has a special significance in the physiology of mitosis. The two sets of hemiplaques may guide the separation of the daughter genomes. At the beginning of the elongational stage the mitotic plaques split into halves and each set of half-plaques migrates to one pole. It is concluded that the dense plaques play a kinetochore-like role and thus Leishmania mexicana ssp. may have six chromosomal units. Mitotic events of this species are essentially similar to those of Trypanosoma cruzi.

Animals↗

[Effects of muscle relaxant E.M.P.P. on afferent discharges of muscle spindle in man--an microneurographic analysis (author's transl)].

Effects of a new muscle relaxant E.M.P.P.(4'-ethyl-2-methyl-3-piperidino-propiophenone . hydrochloride) on afferent discharges of muscle spindle were studied in 10 healthy volunteer subjects. Single unit afferent discharges of muscle spindle were recorded microneurographically from the median or tibial nerve using a tungsten microelectrode with a tip diameter of about 1 micrometer (impedence: about 10 M omega). Effects of an oral administration of 150--300 mg of E.M.P.P. on spontaneous afferent discharges as well as dynamic and static responses of muscle spindle were analysed in the present study. 1. E.M.P.P. suppressed remarkably the frequency of spontaneous afferent discharges of muscle spindle. Meanwhile, mental activations such as speech of mental arithmetic recovered temporarily the frequency of afferent discharges to that before E.M.P.P. administration. 2. E.M.P.P. suppressed the dynamic and static responses of muscle spindle to stretch. It also suppressed the dynamic responses of muscle spindle in the decontraction phase of electrically-induced twitch contractions of the receptor-bearing muscle. Based on these results, it is concluded that E.M.P.P. suppresses the static and dynamic activities of muscle spindle in man. These effects seem to be due to modifications of descending influences from central structures on the static and dynamic gamma motoneurons which innervate the muscle spindle.

Adult↗

Spindle cell carcinoma of the lung. A clinicopathologic study of three cases.

Two cases of monophasic spindle cell carcinomas and one case of adenosquamous carcinoma with the spindle cell component located in the lower respiratory tract are presented. In the biphasic tumor, areas of transition from carcinoma to sarcomatous spindle cells were clearly found. The two monophasic tumors and the spindle cell component of the biphasic tumor were histologically characterized by sheets of spindle cells. However, by electron microscopic and immunohistochemical study, several features of squamous epithelial differentiation were found in the spindle cell areas of all cases. Keratin and vimentin were, in various degrees, coexpressed in all the cases. Therefore, it is supposed that the spindle cell component displays a spectrum of phenotypes originating from squamous cell carcinoma, and monophasic spindle cell carcinoma is considered as a kind of the extreme phenotype of squamous cell carcinoma pretending mesenchymal differentiation.

Adenocarcinoma↗

High incidence of multiple-bag fiber muscle spindles in the articularis humeri muscle of the horse.

The articularis humeri (AH) muscle of the horse is a small muscle composed of histochemically identified type I and IIA extrafusal fibers and a large number of muscle spindles. A total of 150 complete spindles with both spindle poles available were examined in serial transverse sections. On the basis of myosin ATPase-staining reactions after alkaline and acid preincubations, four types of intrafusal fibers, namely, bag1, bag2, "mixed" bag, and chain fibers, were identified. A high proportion of the spindle population (62.6%) consisted of multiple-bag spindles containing three or more (up to six) bag fibers. Also one-bag-fiber spindles were observed. The one-bag-fiber spindles containing a bag2 fiber could be traced into tandem linkages. "Mixed" bag intrafusal fibers, differing in their ATPase staining profile at the two poles, were found in spindles containing also at least one bag1 and one bag2 fiber. An unusually long extracapsular tract (up to 5,500 microns) of the bag intrafusal fibers was observed.

Animals↗

Morphometry and histoenzymology of the hamster tenuissimus and its muscle spindles.

Muscle spindles and extrafusal fibers in the tenuissimus muscle of mature golden Syrian hamsters were studied morphologically and quantitatively using several light microscopic techniques. Muscle spindles were identified in serial-transverse frozen-sections of whole muscles stained with hematoxylin and eosin. Five tenuissimus muscles were examined from origin to insertion, and the locations of individual receptors were plotted in camera-lucida reconstructions. Spindles were found in proximity to the main neurovascular bundle in the central core of each muscle. A range of 16-20 receptors was noted per muscle. The mean muscle spindle index (the total number of spindles per gram of muscle weight) was 503 and the average spindle length was 7.5 mm. Oxidative enzyme and myosin adenosine-triphosphatase (ATPase) staining profiles were also evaluated in the intrafusal and extrafusal fibers in each muscle. Even numbers of type I and type IIA extrafusal fibers were distributed homogeneously throughout all muscle cross-sections. Histochemical staining patterns varied along the lengths of the three intrafusal fiber types. Nuclear chain fibers possessed staining properties similar to the type IIA extrafusal fibers and exhibited no regional variations. Bag1 fibers displayed staining variability, particularly when treated for myosin ATPase under acid preincubation conditions. Some spindles were isolated under darkfield illumination and then either treated with 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to detect filamentous actin by fluorescence microscopy, or prepared for conventional scanning electron microscopy (SEM). By fluorescence microscopy, a registered actin banding-pattern was observed in the sarcomeres of the intrafusal fibers, and variations in the intensity of banding were noted amongst different fibers. SEM revealed punctate sensory nerve endings that adhered intimately to the surfaces of underlying intrafusal fibers in the equatorial and juxtaequatorial regions. By transmission electron microscopy (TEM) these endings appeared crescent-shaped and were enveloped by external laminae. Each profile contained numerous mitochondria and cytoskeletal organelles. The high spindle density observed in this muscle suggests that the hamster tenuissimus may function in hindlimb proprioception.

Animals↗

Function and structure of atypical muscle spindles after neonatal nerve crush in rats.

During the early postnatal period, the differentiation and maturation of muscle spindles in the rat is still dependent on their sensory innervation. When a nerve is crushed during this period, most spindles in the denervated muscles degenerate and after reinnervation only occasional spindles of atypical structure are to be found in these muscles. We determined the basic functional properties of these atypical spindles in adult rats and attempted to correlate them with their structural characteristics. The discharge rates of 13 afferent units from the soleus or lateral gastrocnemius muscles were evaluated in response to stretch. These units were capable of a slowly adapting response to 2-4 mm stretches. Their mean discharge frequencies at any point of the ramp-and-hold stretch were, however, on an average 50% lower than normal values. The conduction velocities of afferents from the atypical spindles were in the range of 10-40 m/s. Histological examinations revealed that 90% of the atypical muscle spindles found in the soleus or lateral gastrocnemius muscles had only 1 or 2 intrafusal fibres without any nuclear accumulations as compared to four intrafusal fibres in normal muscle spindles in the rat. The proportional decrease of the discharge rate in both the dynamic and static part of the response of these atypical spindles could be due to the decreased synaptic area between the sensory terminals and the intrafusal fibres and/or to altered structural properties of the intrafusal fibres.

Action Potentials↗