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Regulation of Aurora-A kinase on the mitotic spindle.

The error-free segregation of duplicated chromosomes during cell division is essential for the maintenance of an intact genome. This process is brought about by a highly dynamic bipolar array of microtubules, the mitotic spindle. The formation and function of the mitotic spindle during M-phase of the cell cycle is regulated by protein phosphorylation, involving multiple protein kinases and phosphatases. Prominent among the enzymes implicated in spindle assembly is the serine/threonine-specific protein kinase Aurora-A. In several common human tumors, Aurora-A is overexpressed, and deregulation of this kinase was shown to result in mitotic defects and aneuploidy. Moreover, recent genetic evidence directly links the human Aurora-A gene to cancer susceptibility. Several of the physiological substrates of Aurora-A presumably await identification, but recent studies are beginning to shed light on the regulation of this critical mitotic kinase. Here, we review these findings with particular emphasis on the role of TPX2, a prominent spindle component implicated in a Ran-GTP-mediated spindle assembly pathway.

Aurora Kinases↗

Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5.

Successful cell division requires proper assembly, placement and functioning of the spindle apparatus that segregates the chromosomes. The Caenorhabditis elegans gene lin-5 encodes a novel coiled-coil component of the spindle required for spindle positioning and chromosome segregation. To gain further insights into lin-5 function, we screened for dominant suppressors of the partial loss-of-function phenotype associated with the mutation lin-5(ev571ts ), and isolated 68 suppressing mutations. Eight out of the ten suppressors sequenced contained intragenic missense mutations immediately upstream of the lesion in lin-5(ev571ts ). These probably help to stabilize protein-protein interactions mediated by the coiled-coil domain. This domain was found to be required for binding to several putative LIN-5 interacting (LFI) proteins identified in yeast two-hybrid screens. Interestingly, interaction with the coiled-coil protein LFI-1 was specifically reduced by the lin-5(ev571ts ) mutation and restored by a representative intragenic suppressor mutation. Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potential interaction in vivo. The coiled-coil domain of LIN-5 was also found to mediate homodimerization, while the C-terminal region of LIN-5 was sufficient for interaction with GPR-1, a recently identified component of a LIN-5 spindle-regulatory complex. A single amino-acid substitution in the N-terminal region of LIN-5, encoded by the e1457 allele, abolished all LIN-5 interactions. Taken together, our results indicate that the spindle functions of LIN-5 depend on interactions with multiple protein partners, and that these interactions are mediated through several different domains of LIN-5.

Amino Acid Sequence↗

Fibre type distribution in the muscle spindles of cat jaw-elevator muscles.

Small samples of the masseter and temporalis muscles of six adult male cats were dissected free, snap-frozen and cryosectioned. Serial sections from areas with muscle spindles were histochemically stained for myofibrillar ATPase after incubation in acid and alkali buffer, and for reactivity to two different myosin isoform-specific antibodies. Differential reactivity to these four stains identified bag1, bag2 and chain intrafusal fibre types. The most typical distribution of fibres within the spindle was 1 bag1, 1 bag2 and 3-4 chains. Although a few spindles were found in the anterior area of the temporalis that attaches to the coronoid process of the mandible and in the anterior area of the superficial masseter, the vast majority were found in the deepest portion of the masseter. Here spindles were characteristically with a number of simple spindles fused together into complex units.

Adenosine Triphosphatases↗

The structural and functional development of muscle spindles and their connections in fetal sheep.

In this paper we have studied the structural and functional development of hindlimb muscle receptors and the connections of their afferent fibres in fetal sheep (n = 26) from 67-143 days of gestation (term = 146 days). By recording extracellular discharges in dorsal root ganglia (L7, S1) we have shown that muscle spindle afferents first respond to a ramp-and-hold stretch at mid-gestation (approximately 75 days). Silver-stained preparations of muscle spindles revealed that afferent fibres are just beginning to form annulospiral windings at this age. It therefore appears that the annulospiral formation is not a necessary requirement for the generation of the response. By 87-92 days some receptors had developed a discharge at resting muscle length. Discharges were generally more robust and easier to elicit and static and dynamic components could be identified in the response to stretch. Although static sensitivity was generally low it was more evident than dynamic sensitivity. By 107-115 days it was possible to clearly distinguish between muscle and tendon afferents and to tentatively classify muscle responses as originating from primary or secondary afferent spindle endings. With increasing gestational age there was a progressive increase in the length and complexity of the spindle innervation in parallel with the maturation of functional activity. Biocytin injections into the dorsal root ganglia revealed afferent projections to the motoneuron pools by 67 days. Silver-staining of muscles showed that innervation of extrafusal fibres was also present by this age. We therefore conclude that the neural pathways necessary for reflex activity involving muscle spindles are present and functional from early in gestation and could contribute to early fetal movements.

Afferent Pathways↗

Time-dependent fusimotor effects on the discharge of cat primary muscle spindle afferents induced by a long-lasting succinylcholine infusion.

The responses of 46 Ia afferents from the tibial anterior muscle of the cat to repetitive ramp-and-hold stretches were investigated under a succinylcholine (SCh) infusion of 120 micrograms.kg-1.min-1 lasting 15 to 25 min. It was possible to distinguish four consecutive phases of the effect of the SCh on the responsiveness of the Ia afferents. The first three of these four phases have already been described. We analysed in more detail the changes from Phase III to the end of Phase IV. Static fusimotor effects were dominant in the discharge patterns obtained during Phase III; dynamic fusimotor effects prevail at the end of Phase IV. Our observations were quantified by comparing the mean values of initial activity, final static value, dynamic response and slow receptor adaptation read from the discharge patterns obtained during Phase III with the mean values of the same parameters obtained from discharge patterns from the end of Phase IV: the two mean values were significantly different for each of the four measurements. This change from Phase III to the end of Phase IV is highly specific for each spindle. To demonstrate this spindle specificity, discharge patterns were selected from among those produced by each Ia afferent in Phase III and at the end of Phase IV. Each of these discharge patterns was assigned to one of six categories. Category I displays purely dynamic fusimotor effects and Category VI purely static fusimotor effects. Categories II, IV and V display combinations of static and dynamic fusimotor effects with an increasing admixture of static fusimotor effects. The spindle-specific change from Phase III to the end of Phase IV is defined in terms of the specific degree of change from a higher-number to a lower-number category in the case of each of the 46 Ia afferents. In the discussion a combination of activity by the two nuclear bag fibres of a spindle is deduced from the specific discharge pattern of each category. The conclusion from these considerations is that results obtained from the administration of SCh have to be interpreted with great caution in making any statement about the existence of a dynamic bag1 fibre in a spindle.

Animals↗

Effects of skin cooling and warming on stretch responses of the muscle spindle primary and secondary afferent fibers from the cat's tibialis anterior.

Fusimotor activation induced by thermal stimuli to the skin was investigated by recording the stretch responses of primary as well as secondary spindle afferent fibers from the tibialis anterior of the barbiturate-anesthetized, spinal cats. A standard ramp-and-hold stretch was repeatedly given during warming or cooling (20 to 40 degrees C) the skin of the ipsilateral foot pads. In each response of the spindle afferent fiber to stretch, the peak (P) and static firing rates (S) were measured and the dynamic index was calculated to assess the type of fusimotor fiber influence as dynamic or static. Of 47 primary spindle afferent fibers, 22 (46%) were affected by changes in skin temperature. Cooling of the skin below a certain temperature produced a marked increase in the dynamic sensitivity of their response to stretch, and this also occurred on warming above it. Such a "critical temperature" at which a directional relationship between skin temperature and muscle spindle activity was completely altered was determined (mean +/- SD: 35.10 +/- 1.66 degrees C, N = 7). However, a marked increase in static firing accompanied by a significant decrease in the dynamic index was observed at the dynamic phase of heating the skin beyond the critical temperature. None of the seven secondary afferent fibers studied showed a detectable temperature response. We conclude that the activation of primary spindle afferent fibers by thermal stimulation of the skin is to be attributed predominantly to activation of dynamic fusimotor neurons and is significant for the initiation of cold shivering.

Animals↗

The effects of 1,6-dinitropyrene on spindle morphology in transformed human cells.

The effects of 1,6-dinitropyrene (1,6-DNP) on the fidelity of cell division were studied in the transformed human fibroblast cell line MRC5VA. Over a dose range of 0.1-10 micrograms/ml of 1.6-DNP, we observed significant increases in the levels of abnormal division stages, associated with damage to the spindle apparatus of the cell. Qualitative changes in spindle morphology and a quantitative decrease in pole-to-pole spindle length were also observed with increasing doses of 1.6-DNP. Such changes in the size and morphology of the spindle corresponded with an accumulation of cells blocked at metaphase. The presence of catalase did not modify the response, suggesting that the effects on the spindle apparatus and cell division were not caused by the generation of radicals but by the direct action of 1.6-DNP.

Catalase↗

Effects of 2,5-hexanedione on deefferented muscle spindle position sensitivity.

2,5-Hexanedione (HD) produces a neurofilamentous axonpathy in humans and experimental animals. The present study was carried out to determine the time course of the effects of HD on muscle spindle position sensitivity to permit comparisons with similar effects of other toxicants which produce a neurofilamentous axonopathy. Cats were administered HD either via their drinking water or via osmotic minipumps. Muscle spindle position sensitivity was tested after a total HD dose of 2.43 or 4.85 g/kg via the drinking water or 0.96, 1.91, or 4.78 g/kg via an osmotic minipump. The position sensitivities of both primary and secondary endings were depressed regardless of the route of administration. However, secondary muscle spindle afferents in the animals intoxicated with HD via the minipumps were affected to a greater extent than primary muscle spindle afferents at all doses studied. These data show that HD alters muscle spindle function and lends support to the suggestion that the largest diameter fibers are not the most vulnerable to HD.

Analysis of Variance↗

Kinesin-related proteins required for structural integrity of the mitotic spindle.

For S. cerevisiae cells, the assembly of a bipolar mitotic spindle requires the action of either Cin8p or Kip1p, gene products related to the mechanochemical enzyme kinesin. In this paper we demonstrate that the activity of either one of these proteins is also required following spindle assembly. When their function was eliminated, preanaphase bipolar spindles rapidly collapsed, with previously separated poles being drawn together. In contrast, anaphase spindles were apparently resistant to collapse. Deletion of kinesin-related KAR3 partially suppressed the phenotypes associated with loss of Cin8p/Kip1p function. Our findings suggest that the structure of the preanaphase bipolar spindle is maintained by counteracting forces produced by kinesin-related proteins.

Anaphase↗

Differential staining of chromosomes and spindle cannot be used as an assay to determine the effect of cancer promoters on primary cultures of human fibroblasts.

The differential staining technique allows simultaneous visualization of chromosomes and spindle fibers. The relative sensitivity of this test for chemicals with high tumor-promoting activity (TPA and iodoacetic acid) or for low tumor-promoting chemicals (4 beta-PDD and alpha-phorbol) was analyzed. Moreover, to ensure a complementary reference system, metals with possible mutagenic (As greater than Hg greater than Cd) and carcinogenic (As greater than Cd = Hg) properties were tested in parallel. As and Hg, which are known as spindle poisons, do induce, as expected, a disaggregation of the spindle fibers; with Cd, no clear-cut disappearance of the spindle is observed. As far as the cancer promoters are concerned, there is no evidence that their ability to induce mitotic aneuploidy in yeast would be confirmed by the induction of spindle disappearance and/or abnormal chromosome segregation in primary cultures of human fibroblasts.

Carcinogens↗

A comparative study of changes operated by sympathetic nervous system activation on spindle afferent discharge and on tonic vibration reflex in rabbit jaw muscles.

The effect of sympathetic activation on the spindle afferent response to vibratory stimuli eliciting the tonic vibration reflex in jaw closing muscles was studied in precollicularly decerebrate rabbits. Stimulation of the cervical sympathetic trunk, at frequencies within the physiologic range, consistently induced a decrease in spindle response to muscle vibration, which was often preceded by a transient enhancement. Spindle discharge was usually correlated with the EMG activity in the masseter muscle and the tension reflexly developed by jaw muscles. The changes in spindle response to vibration were superimposed on variations of the basal discharge which exhibited different patterns in the studied units, increases in the firing rate being more frequently observed. These effects were mimicked by close arterial injection of the selective alpha 1-adrenoceptor agonist phenylephrine. Data presented here suggest that sympathetically-induced modifications of the tonic vibration reflex are due to changes exerted on muscle spindle afferent information.

Animals↗

Estimation of generator potentials in primary spindle endings evoked by brief tendon taps in man.

The ligamentum patellae in human subjects was tapped by a random triangular waveform, while single primary spindle afferent spikes were recorded from the femoral nerve. Cross-correlation between the primary spindle afferent spikes and the taps revealed a prominent peaked distribution of the discharge probability. The cross-correlograms showed that response time from the onsets of taps to the afferent spike was 7.5 +/- 0.6 msec in 6 units and that the width between the feet of the peaked distribution was 3.3 +/- 0.2 msec in 6 units. The width of the peak implies jitter of the response time of the primary spindle spikes which appear to correspond with the time-to-peak of generator potentials elicited by the taps in primary endings of muscle spindles. Integration of the peaked distribution showed a slow slope at the beginning and near the summit and a steep slope on the way. This curve is assumed to represent the rising phase of the generator potentials in primary spindle endings.

Afferent Pathways↗

Rat muscle spindles deficient in elements of the static system.

Motor nerve supplies to 15 poles of rat lumbrical spindle were reconstructed from serial, 1-micron transverse sections of muscle embedded in resin. Neural and muscular elements associated with the modulation of static sensitivity of afferents were deficient in these spindles relative to cat tenuissimus and rat soleus spindles. Rat lumbrical spindles contained fewer static fusimotor axons, fewer static chain intrafusal fibers, fewer motor-innervated static bag2 and chain fibers and fewer secondary afferents. The sparsity of static elements in spindles of the rat lumbrical muscle may correlate with the distal location or with the delicate motor tasks performed by the muscle.

Animals↗

Spindle-membrane associations in freeze fractured HeLa cells.

Mitotic HeLa cells, obtained by means of thymidine and nocodazole synchronization, were freeze fractured, and spindle-membrane associations were studied. Except for small vesicles most membrane structures were excluded from the spindle region. A few tubular to lamellar membrane arrays were observed that projected into the spindle region from the vicinity of the spindle poles. Concentric layers of flattened, extensively fenestrated cisternae were found in the cytoplasm surrounding the spindle. The plasma membrane overlying the midbody in early G1 cells was examined.

Freeze Fracturing↗

Effects of changes in pH on the afferent impulse activity of isolated cat muscle spindles.

Muscle spindle activity has been shown to decrease in the sustained contracting muscle. The effect has been assumed to result from a declining fusimotor drive. Since accumulation of metabolites including H(+), lactate and CO(2) might also affect the receptor in the fatiguing muscle, the impulse activity of muscle spindles isolated from the cat tenuissimus muscle was characterized under varying degrees of extracellular pH, thus excluding any effect on fusimotor activity, blood supply and extrafusal muscle fibers. The isolated receptor was exposed to bathing fluids of pH 6.4, 7.4 and 8.4, and afferent discharge activity was recorded from the spindle nerve. Both primary and secondary endings responded similarly to changes in pH. Resting discharge frequency usually decreased with decreasing pH and increased with increasing pH. A sudden break-off in activity was observed with about 40% of primary endings and about 30% of secondary endings at pH 6.4. Experiments with slow stretch stimulation indicated that this effect was caused by a rising threshold of firing at the encoder site of the endings. With brief ramp-and-hold stretches, we tested the effects of changes in pH on the dynamic and static sensitivity of primary and secondary endings. When pH was reduced from 7.4 to 6.4, the initial burst activity at the beginning of the ramp phase increased in primary and secondary endings and the dynamic response increased in secondary endings, demonstrating that the dynamic properties of muscle spindle endings were usually augmented in the acidic milieu. The static properties rose as well because the static index of both types of ending increased significantly. By contrast, dynamic and static properties of both primary and secondary endings decreased significantly, when pH was increased from 7.4 to 8.4. The amplitude of tension that was measured during the passive stretch stimuli very slightly decreased in the acidic solution and very slightly increased in the alkaline solution. The decrease in the resting discharge activity at low pH supports those previous observations, which demonstrate a reduced peripheral input from muscle spindle afferents to the spinal motor nuclei during fatigue in the isometric contracting muscle. The present finding indicates that an attenuated afferent discharge is not only caused by a decreasing central activation of gamma-motorneurons, but may additionally be supported by a direct effect of protons on the muscle receptor itself. The accompanying augmentation of stretch sensitivity is suggested to correspond to the well-known increase in physiological tremor during exhaustive exercise.

Animals↗

A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly.

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.

Animals↗

The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly.

The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.

Animals↗

The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly.

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.

Animals↗