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PubMed1995

Ultrastructural basis for synaptic transmission between jaw-muscle spindle afferents and trigeminothalamic neurons in the rostral trigeminal sensory nuclei of the rat.

Trigeminothalamic neurons were retrogradely labeled by injection of horseradish peroxidase into the ventroposteromedial nucleus of the thalamus in rats. Jaw-muscle spindle afferent axons were then physiologically identified and intracellularly stained with biotinamide. The ultrastructure of labeled spindle afferent boutons was then studied in the caudolateral supratrigeminal region (Vsup) and dorsomedial trigeminal principal sensory nucleus (Vpdm). A total of 418 stained spindle afferent boutons were identified in Vsup and Vpdm; approximately 75% of these synapsed with dendrites, 10% synapsed with somata, and 15% synapsed with axons. Most jaw-muscle spindle afferent boutons were postsynaptic to unlabeled P-type boutons. Reciprocal synapses between spindle afferent boutons and unlabeled boutons were occasionally observed. A few dendrites in Vsup and Vpdm received synapses from multiple spindle afferent boutons. Conversely, some large (from 3 x 6 to 4 x 8 microns) and giant (from > 4 x 8 to 5 x 10 microns) spindle afferent boutons simultaneously contacted two to five dendrites and/or somata. Jaw-muscle spindle afferent boutons also formed synapses with retrogradely labeled trigeminothalamic neurons in Vsup and Vpdm. Numerous unlabeled S-and F-type boutons converged onto the same trigeminothalamic dendrite or soma contacted by a spindle afferent bouton. A small number of synaptic triads consisting of an unlabeled P-type bouton, a spindle afferent bouton, and either a dendrite or soma were also encountered. These data indicate that sensory feedback from the masticatory muscles is subject to presynaptic inhibition and integration prior to reaching the thalamus. This pathway is likely to be important in the relay of proprioceptive and kinesthetic information from the muscles of mastication to the thalamus.

Afferent Pathways↗

PubMed2007

Functional analysis of Survivin in spindle assembly in Xenopus egg extracts.

Survivin is a member of the inhibitor of apoptosis (IAP) protein family that serves critical roles in mitosis and cytokinesis. Many studies have suggested Survivin's involvement in spindle regulation, but direct biochemical evidence for this has been lacking. Using the cell-free system of Xenopus egg extracts, we tested whether Survivin was necessary for the assembly of metaphase spindles. Removal or inhibition of Xenopus Survivin causes the disruption in the formation of metaphase spindles. In particular, we observe the generation of microtubule (MT) asters or poorly formed shortened spindle structures. In the latter phenotype the spindle structures display a decrease pole-to-pole length and a reduction of MTs around the chromatin indicating that Survivin may promote the stabilization of MT-chromatin interactions. In addition, function analysis of Survivin's conserved phosphorylation site Thr34 (Thr43 in Xenopus) and tubulin-binding domain was also assessed in regulating spindle assembly. Treatment of Xenopus egg extracts with a recombinant Survivin mutant that contained an alanine residue substitution at Thr43 (SURT43A mutant) or that was missing the C-terminal tubulin-binding domain (SURCL mutant) produced an increased frequency of MT asters and shorten abnormal spindle structures in Xenopus egg extracts. Interestingly, a phosphomimetic mutation made at residue Thr43 of Survivin (SURT43E mutant) generated a high frequency of MT asters implying that premature 'activation' of Survivin may interfere with an early stage of spindle assembly. Taken together, we propose that Survivin is a necessary component of the mitotic spindle and its phosphorylation at residue Thr43 is important for Survivin function in spindle assembly.

Animals↗

PubMed2003

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes.

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.

Animals↗

PubMed2001

The transcription factor Egr3 modulates sensory axon-myotube interactions during muscle spindle morphogenesis.

The Egr family of zinc-finger transcription factors, consisting of Egr1, Egr2, Egr3, and Egr4, are involved in cellular growth and differentiation. Adult Egr3-deficient mice are ataxic and lack muscle spindle proprioceptors that normally develop at the sites of Ia afferent-myotube contacts during embryogenesis. To resolve whether spindles form and then degenerate, or whether they never form in the absence of Egr3, we examined the spatiotemporal expression of Egr3 relative to spindle development. In wild type mice, Egr3 was expressed in developing myotubes shortly after they were innervated by Ia afferents and its expression was controlled by innervation because it dissipated following nerve transection. In Egr3-deficient mice, myotubes received Ia afferent innervation and assembled normally into spindles during embryogenesis. However, newborn Egr3-deficient spindles had few internal myonuclei in intrafusal fibers and thin capsules. Moreover, slow-developmental myosin heavy chain was not induced in embryonic Egr3-deficient spindles suggesting that impairments in differentiation were present before they could be detected morphologically. After birth, sensory and motor innervation withdrew from the Egr3-deficient spindles, and the spindles disassembled. In spite of the spindle disassembly and retraction of afferents from muscles, the cell bodies of proprioceptive neurons within dorsal root ganglia were retained. We conclude that Egr3 has an essential role in regulating genes required for the transformation of undifferentiated myotubes into intrafusal fibers, and hence for the phenotypic differentiation of spindles.

Afferent Pathways↗

PubMed1995

Critical period in muscle spindle regeneration in grafts of developing rat muscles.

Extensor digitorum longus (EDL) muscles from rats at various intervals after birth were grafted into EDL muscles of adult recipients. Three to twelve months after the operation, host muscles containing the grafts were removed and examined for the presence of muscle spindles in the graft. The aim of the study was to establish when muscle spindles become capable of regeneration during development. Regenerated muscles grafted during the first week after birth were virtually spindleless. Grafts of muscles transplanted 10 and 15 days postnatally contained only 5-8 muscle spindles on average. In contrast, the regenerated grafts originating from muscles of 24- and 28-day-old rats were spindle-rich as in mature muscle grafts; the number of spindles in the transplanted EDL muscles (25.0 +/- 2.3; mean +/- SE) attained values comparable to free standard autografts of these muscles in adult animals. Thus, the critical period after grafting, which also involves the loss of a vascular supply, is considerably longer than the critical period for muscle-spindle survival after nerve injury. Fifteen days after birth, when muscle spindles still survive denervation, only a few regenerated spindles were present in the individual muscle regenerates. We assume that the low resistance of immature spindle capsules to ischaemia accounts for their massive degeneration and abortive spindle regeneration in grafts from 10- to 15-day-old rats.

Adenosine Triphosphatases↗

PubMed1984

Quantification and origin of spindles in orthotopic and heterotopic grafts of avian muscles.

Regeneration of muscle spindles was quantified in a series of orthotopically and heterotopically autografted muscles of pigeons. Significantly fewer spindles relative to numbers of extrafusal fibers were present in grafts than in normal muscles. These results are in marked contrast to observations of free-grafted muscles of rats. A majority of grafts of the metapatagialis, a muscle devoid of spindles, into the site of the anterior latissimus dorsi contained spindles. A few spindles were present in grafts of the extensor digitorum communis, which normally contains many spindles, into the site formerly occupied by the metapatagialis whereas muscle spindles were absent in orthotopic grafts of the metapatagialis muscle. These observations suggest that the spindle-like structures observed in the extensor digitorum communis muscles, which regenerated in the sites of the metapatagialis, were derived from spindles of the donor muscle. Thus muscle spindles in transplanted avian muscle can form by two distinct developmental processes.

Animals↗

PubMed2002

Intrafusal fiber type composition of muscle spindles in the first human lumbrical muscle.

We studied muscle spindles in the first lumbrical muscle of adult humans using myofibrillar ATPase (mATPase) activity. We found that muscle spindles exhibited a marked variability with respect to the number, position, length and detailed histochemical features of nuclear bag1, nuclear bag2 and nuclear chain fibers. Regarding mATPase activity, the nuclear bag2 fibers displayed lower alkali-stable mATPase activity along their length and many nuclear bag1 fibers tended to have lower acid-stable activity in the outer B region, whereas nuclear chain fibers exhibited medium acid-stable mATPase activity at pH 4.6. Almost 10% of spindle fibers displayed atypical features, as they were either located only at one spindle pole or exhibited mixed characteristics at either pole. The number of intrafusal fibers per spindle varied between 8 and 24. Strikingly, only 2 pairs from 22 muscle spindles had identical allotments of their intrafusal fibers. Muscle spindles in the first human lumbrical muscle contained more intrafusal fibers (12.3 +/- 4 per spindle on average) and especially relatively more nuclear bag fibers compared to other human skeletal muscles. Since each spindle apparently represents a unique morphological and physiological entity, the observed variability in the number and characteristics of intrafusal fibers in the first human lumbrical muscle likely reflects a wide range of finely tuned muscle spindle responses.

Adenosine Triphosphatases↗

PubMed1996

A numerical simulation of muscle spindle ensemble encoding during planar movement of the human arm.

We address the issue of what proprioceptive information, regarding movement of the human arm, may be provided to the central nervous system by proprioceptors located within muscles of this limb. To accomplish this we developed a numerical simulation which could provide estimates of the length regimes experienced by a set of model receptors located within some of the principal muscles of the human arm during planar movement of this limb. These receptors were assumed to have characteristics analogous to those associated with a simple model of muscle spindle signalling of movement. To this end each spindle had proprioceptive 'channels' associated with it. These corresponded to primary and secondary spindle afferent fibers which could provide independent afferent output regarding the parent muscle the spindle monitored. The angles of the shoulder and elbow joints attained by subjects performing a task requiring movement of the right arm in a horizontal plane to a static visual target were recorded. For this angular data the lengths and rates of change of lengths experienced by muscle fascicles, and hence the model spindles, during movement were calculated by means of the numerical simulation. The discharge rates of the simulated spindles during the movement were calculated to derive a measure of the depth of modulation, induced by the movement, for each spindle. These values were then summed for all spindles to provide a first-order approximation of spindle ensemble coding of the movement. Significant correlations (P < 0.0001, Spearman's rank order) were found between the resulting ensemble encodings and, in order of significance, the angular velocity of the shoulder joint (rS = 0.945), the tangential velocity of the hand (rS = 0.942), and the angular velocity of the elbow joint (rS = 0.917). Correlations between the angular positions of the shoulder (rS = -0.623) and elbow (rS = 0.628) were lower. These findings indicate that the ensemble profiles of the simulated muscle spindles, encode information regarding kinematic parameters of movements related to both intrinsic and extrinsic coordinate systems. This suggests that motor structures capable of deriving such an ensemble encoding would be in a position to perform the sensory-motor transformations between intrinsic and extrinsic frames of reference necessary for controlling movements planned in extrinsic coordinates.

Arm↗

PubMed1992

Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles.

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both M1 and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in M1 (0.7 microns min-1) was slower than that observed in M2 (1.8 microns min-1). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during M1, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar M1 spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes.

Animals↗

PubMed1993

Fusimotor-free spindles in reinnervated muscles of neonatal rats treated with nerve growth factor.

Crushing the nerve to the medial gastrocnemius muscle in newborn rats and administering nerve growth factor afterwards results in a reinnervated muscle containing supernumerary muscle spindles. The structure and innervation of 88 spindles in the reinnervated muscles were reconstructed from serial thick and thin transverse sections at 30-35 days after the nerve crush, and compared to those of five control spindles. The spindles consisted of one to four small-diameter encapsulated fibers with features of nuclear chain intrafusal fibers, or infrequently a nuclear bag intrafusal fiber. Some of the spindles were located within a capsule that also contained an extrafusal fiber. Each spindle was innervated by an afferent with features of the primary afferent. The density of secondary afferents was lower in reinnervated muscles than in controls. Endplates were observed on extrafusal fibers in the experimental muscles, attesting to restoration of skeletomotor (alpha) innervation after the nerve crush. However, 78% of the experimental spindles were entirely devoid of efferent innervation. The remainder received either one or two fusimotor (gamma) axons or a skeletofusimotor (beta) axon, compared to the six to eight motor axons that innervated control spindles. The presence of supernumerary spindles composed of fibers that resemble normal intrafusal fibers in the absence of motor innervation suggests that afferents alone can induce the formation and subsequent differentiation of intrafusal fibers in nerve-crushed muscles of neonatal rats. In addition, the paucity of gamma innervation in nerve-crushed muscles suggests that immature gamma neurons are more susceptible than spindle afferents or alpha efferents to cell death after axotomy at birth.

Animals↗

PubMed2006

Classification of muscle spindle afferents innervating the masseter muscle in rats.

Taylor et al. [Taylor, A., Durbaba, R., Rodgers, J.F., 1992a. The classification of afferents from muscle spindles of the jaw-closing muscles of the cat. J Physiol 456, 609-628] developed a method to classify muscle spindle afferents using succinylcholine (Sch) and ramp and hold stretches. They demonstrated that cat jaw muscle spindle afferents show high proportion of intermediate responses to ramp and hold jaw stretch. Together with observations on the responses to Sch their data suggests that the majority of jaw muscle spindle afferents are influenced by a combination of nuclear bag(2) and nuclear chain fibres. Relatively few are influenced solely by nuclear bag(1) fibres. The purpose of this study was to categorize jaw muscle spindle afferent in rodents in response to ramp and hold stretches. Several measures were used to classify spindle afferents including (1) conduction velocity, (2) coefficient of variation (C.V.) of the interspike interval during jaw opening, and (3) the dynamic sensitivity and the initial discharge of spindle afferents before and after succinylcholine infusion (Sch, 100mg/kg, i.v.). Consistent with observations in the cat jaw muscles, the distribution of the conduction velocity and the C.V. of Vmes masseter afferents were unimodal. Therefore, these parameters were of little value in functional classification of spindle innervation. Succinylcholine injection either markedly increased the dynamic sensitivity or produced no change in Vmes afferents. Unlike cat jaw muscle spindle afferents, the effect of Sch on the initial discharge was not clearly separable from those responding or not responding to Sch. These results suggest that rat jaw muscle spindle afferents, have physiological properties that are primarily intermediate in nature and are likely to reflect a predominance of influence from nuclear bag(2) and chain fibres. However, the distinction between bag(2) and chain fibres influences is not as clearly defined in the rat compared to the cat.

Action Potentials↗

PubMed2003

Peripheral, non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.

In centrosome-containing cells, microtubules utilized in spindle formation are thought to be nucleated at the centrosome. However, spindle formation can proceed following experimental destruction of centrosomes or in cells lacking centrosomes, suggesting that non-centrosome-associated microtubules may contribute to spindle formation, at least when centrosomes are absent. Direct observation of prometaphase cells expressing GFP-alpha-tubulin shows that peripheral, non-centrosome-associated microtubules are utilized in spindle formation, even in the presence of centrosomes. Clusters of peripheral microtubules moved into the centrosomal region, demonstrating that a centrosomal microtubule array can be composed of both centrosomally nucleated and peripheral microtubules. Peripheral bundles also moved laterally into the forming spindle between the spindle poles; 3D reconstructions of fixed cells reveal interactions between peripheral and centrosome-associated microtubules. The spindle pole component NuMA and gamma-tubulin were present at the foci of peripheral microtubule clusters, indicating that microtubules moved into the spindle with minus ends leading. Photobleach- and photoactivation-marking experiments of cells expressing GFP-tubulin or a photoactivatable variant of GFP-tubulin, respectively, demonstrate that microtubule motion into the forming spindle results from transport and sliding interactions, not treadmilling. Our results directly demonstrate that non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.

Animals↗

PubMed2004

The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5.

During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to approximately 6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.

Animals↗

PubMed2003

MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C elegans.

In most animals, successful segregation of female meiotic chromosomes involves sequential associations of the meiosis I and meiosis II spindles with the cell cortex so that extra chromosomes can be deposited in polar bodies. The resulting reduction in chromosome number is essential to prevent the generation of polyploid embryos after fertilization. Using time-lapse imaging of living Caenorhabditis elegans oocytes containing fluorescently labeled chromosomes or microtubules, we have characterized the movements of meiotic spindles relative to the cell cortex. Spindle assembly initiated several microns from the cortex. After formation of a bipolar structure, the meiosis I spindle translocated to the cortex. When microtubules were partially depleted, translocation of the bivalent chromosomes to the cortex was blocked without affecting cell cycle timing. In oocytes depleted of the microtubule-severing enzyme, MEI-1, spindles moved to the cortex, but association with the cortex was unstable. Unlike translocation of wild-type spindles, movement of MEI-1-depleted spindles was dependent on FZY-1/CDC20, a regulator of the metaphase/anaphase transition. We observed a microtubule and FZY-1/CDC20-dependent circular cytoplasmic streaming in wild-type and mei-1 mutant embryos during meiosis. We propose that, in mei-1 mutant oocytes, this cytoplasmic streaming is sufficient to drive the spindle into the cortex. Cytoplasmic streaming is not the normal spindle translocation mechanism because translocation occurred in the absence of cytoplasmic streaming in embryos depleted of either the orbit/CLASP homolog, CLS-2, or FZY-1. These results indicate a direct role of microtubule severing in translocation of the meiotic spindle to the cortex.

Adenosine Triphosphatases↗

PubMed1998

Bipolar meiotic spindle formation without chromatin.

Establishing a bipolar spindle is an early event of mitosis or meiosis. In somatic cells, the bipolarity of the spindle is predetermined by the presence of two centrosomes in prophase. Interactions between the microtubules nucleated by centrosomes and the chromosomal kinetochores enable the formation of the spindle. Non-specific chromatin is sufficient, however, to promote spindle assembly in Xenopus cell-free extracts that contain centrosomes [1,2]. The mouse oocyte represents an excellent model system in which to study the mechanism of meiotic spindle formation because of its size, transparency and slow development. These cells have no centrioles, and their multiple microtubule-organizing centers (MTOCs) are composed of foci of pericentriolar material [3,4]. The bipolarity of the meiotic spindle emerges from the reorganization of these randomly distributed MTOCs [4]. Regardless of the mechanisms involved in this reorganization, the chromosomes seem to have a major role during spindle formation in promoting microtubule polymerization and directing the appropriate rearrangement of MTOCs to form the two poles [5]. Here, we examined spindle formation in chromosome-free mouse oocyte fragments. We found that a bipolar spindle can form in vivo in the absence of any chromatin due to the establishment of interactions between microtubule asters that are progressively stabilized by an increase in the number of microtubules involved, demonstrating that spindle formation is an intrinsic property of the microtubule network.

Animals↗

PubMed2001

Control of spindle polarity and orientation in Saccharomyces cerevisiae.

Control of mitotic spindle orientation represents a major strategy for the generation of cell diversity during development of metazoans. Studies in the budding yeast Saccharomyces cerevisiae have contributed towards our present understanding of the general principles underlying the regulation of spindle positioning in an asymmetrically dividing cell. In S. cerevisiae, the mitotic spindle must orient along the cell polarity axis, defined by the site of bud emergence, to ensure correct nuclear division between the mother and daughter cells. Establishment of spindle polarity dictates this process and relies on the concerted control of spindle pole function and a precise program of cues originating from the cell cortex that directs cytoplasmic microtubule attachments during spindle morphogenesis. These cues cross talk with the machinery responsible for bud-site selection, indicating that orientation of the spindle in yeast cells is mechanistically coupled to the definition of a polarity axis and the division plane. Here, we propose a model integrating the inherently asymmetric properties of the spindle pathway with the program of positional information contributing towards orienting the spindle in budding yeast. Because the basic machinery orienting the spindle in higher-eukaryotic cells appears to be conserved, it might be expected that similar principles govern centrosome asymmetry in the course of metazoan development.

Chromosome Segregation↗

PubMedDate not supplied

Manipulation of the oocyte: possible damage to the spindle apparatus.

Oocytes are structured, polarized cells. For high developmental potential, it is essential that the distribution of organelles and molecules, and the function of meiotic spindles remain intact during handling of oocytes in assisted reproduction. Spindles are dynamic cell organelles. Spindle formation depends on activity of motor proteins, energy supply and temperature. Disturbances in spindle function may predispose oocytes to aneuploidy or maturation arrest. Thus, perturbation of the cytoskeletal integrity of oocytes may critically influence the fate of the embryo. Recently, enhanced polarizing microscopy has been developed for non-invasive analysis of spindle morphology in living mammalian oocytes. Chemically induced dynamic alterations have been characterized in the spindle in individual mouse oocytes and it has been shown that spindle aberrations are predictive of risks for non-disjunction. Spindle imaging identified adverse, irreversible effects of handling in living human oocytes (for instance, the extreme susceptibility of human oocytes to cooling). Also, oocyte immaturity may be detected. Selection of metaphase II oocytes and an injection site for intracytoplasmic sperm injection (ICSI) that avoids spindle damage may increase the yield of euploid embryos. The detection of genetic, environmentally induced, or treatment-related defects in oocyte maturation by non-invasive spindle imaging can improve quality control and assist in the selection of morphologically normal oocytes for assisted reproduction.

Aneuploidy↗

PubMed2002

The farnesyltransferase inhibitor, FTI-2153, inhibits bipolar spindle formation during mitosis independently of transformation and Ras and p53 mutation status.

Recently, we have shown that the farnesyltransferase inhibitor FTI-2153 induces accumulation of two human lung cancer cell lines in mitosis by inhibiting bipolar spindle formation during prometaphase. Here we investigate whether this mitotic arrest depends on transformation, Ras and/or p53 mutation status. Using DAPI staining (DNA) and immunocytochemistry (microtubules), we demonstrate that in normal primary foreskin fibroblasts (HFF), as well as in several cancer cell lines of different origins including human ovarian (OVCAR3), lung (A-549 and Calu-1) and fibrosarcoma (HT1080), FTI-2153 inhibits bipolar spindle formation and induces a rosette morphology with a monopolar spindle surrounded by chromosomes. In both malignant cancer cell lines and normal primary fibroblasts, the percentage of prometaphase cells with bipolar spindles decreases from 67-92% in control cells to 2-28% in FTI-2153 treated cells. This inhibition of bipolar spindle formation correlates with an accumulation of cells in prometaphase. The ability of FTI-2153 to inhibit bipolar spindle formation is not dependent on p53 mutation status since both wild-type (HFF, HT1080 and A-549) and mutant (Calu-1 and OVCAR3) p53 cells were equally affected. Similarly, both wild-type (HFF and OVCAR3) and mutant (HT1080, Calu-1 and A-549) Ras cells accumulate monopolar spindles following treatment with FTI-2153. However, two cell lines, NIH3T3 (WT Ras and WT p53) and the human bladder cancer cell line, T-24 (mutant H-Ras and mutant p53) are highly resistant to FTI-2153 inhibition of bipolar spindle formation. Finally, the ability of FTI-2153 to inhibit tumor cell proliferation does not correlate with inhibition of bipolar spindle formation. Taken together these results demonstrate that the ability of FTI-2153 to inhibit bipolar spindle formation and accumulate cells in mitosis is not dependent on transformation, Ras or p53 mutation status. Furthermore, in some cell lines, FTIs inhibit growth by mechanisms other than interfering with the prophase/metaphase traverse.

3T3 Cells↗