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Formation of supernumerary muscle spindles at the expense of Golgi tendon organs in ER81-deficient mice.

ER81, a member of the ETS family of transcription factors, is essential for the formation of connections between sensory and motor neurons in the spinal cord. Mice lacking Er81 genes exhibit reduced monosynaptic sensory-motoneuron connectivity in response to muscle nerve stimulation. Proximal muscle nerve stimulation elicits fewer monosynaptic potentials than stimulation of distal nerves in hindlimbs, a deficit that is paralleled by a paucity of muscle spindles in proximal muscles (Arber et al., 2000). We examined whether a presence of spindles innervated by afferents in distal muscles correlated with the increased preservation of monosynaptic sensory-motor potentials in distal muscle nerves. Not only were spindles and Ia afferents present, but also they were supernumerary in distal muscles such as the soleus, medial gastrocnemius, and extensor hallucis longus. Concomitantly, a deficiency of Golgi tendon organs (GTOs) and Ib afferents was observed in distal muscles, as if supernumerary spindles formed at the expense of tendon organs in the absence of Er81. Thus, ER81 may be involved in mechanisms that regulate acquisition of the Ia and Ib phenotypes by subsets of proprioceptive muscle afferents. Segmental differences in muscle spindle and GTO dependence on ER81 suggest that more than one ETS transcription factor may participate in the regulation of limb proprioceptive system assembly in the mouse.

Alleles↗

Chromator, a novel and essential chromodomain protein interacts directly with the putative spindle matrix protein skeletor.

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

Amino Acid Sequence↗

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.

Animals↗

Density of muscle spindle profiles in the intrinsic forelimb muscles of the dog.

The concept of parallel muscle combinations, in which spindle density is significantly higher in small muscles compared to their larger counterparts in large-small muscle combinations acting across a joint, is supported by the results of this study regardless of the joint. Analysis of the canine data as well as previously published guinea pig forelimb and human pelvic limb data revealed no significant difference in spindle density between antigravity and non-antigravity muscles. Furthermore, a gradual increase in spindle density from proximal to distal on the limb was not found, although spindle density was significantly higher in the intrinsic manus or pes muscles compared to more proximal limb muscles in all three species. The significant differences in spindle densities in parallel muscle combinations and in manus/pes versus proximal muscles are discussed relative to their possible role in the control of locomotion.

Animals↗

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.

Animals↗

Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase II-arrested mouse oocytes.

The cell cycle is regulated by phosphorylation events via a cascade of protein kinases and phosphatases, but many of their substrates remain unknown. To study whether proteins of the metaphase II-arrested mouse oocyte meiotic spindle are substrates for phosphorylation events, we used okadaic acid (OA), a potent phosphatase inhibitor, upon fully mature spindles. Incubation of oocytes for 3 hr with 1 microM OA led to a dramatic lengthening of the spindle and a disorganization of the metaphase plate. Electron microscope studies revealed that this was due to a disruption of the interactions between the microtubules and the kinetochores. Biochemical analysis including MPM-2 immunoblotting and [32P]phosphate labeling of whole oocytes revealed several changes in the phosphorylation pattern following the OA treatment. Moreover, meiotic spindle purification or microtubule-associated proteins (MAPs) isolation showed that some of these phosphorylations occur on proteins associated with the microtubules or with structures closely related to the spindle. These results suggest that the changes occurring in the microtubule network during the cell cycle are partly due to the phosphorylation of some proteins associated with the microtubules.

Animals↗

Protein kinases involved in mitotic spindle checkpoint regulation.

A number of checkpoint controls function to preserve the genome by restraining cell cycle progression until prerequisite events have been properly completed. Chromosome attachment to the mitotic spindle is monitored by the spindle assembly checkpoint. Sister chromatid separation in anaphase is initiated only once all chromosomes have been attached to both poles of the spindle. Premature separation of sister chromatids leads to the loss or gain of chromosomes in daughter cells (aneuploidy), a prevalent form of genetic instability of human cancer. The spindle assembly checkpoint ensures that cells with misaligned chromosomes do not exit mitosis and divide to form aneuploid cells. A number of protein kinases and checkpoint phosphoproteins are required for the function of the spindle assembly checkpoint. This review discusses the recent progress in understanding the role of protein kinases of the mitotic checkpoint complex in the surveillance pathway of the checkpoint.

Animals↗

Properties of cat neck muscle spindles and their excitation by succinylcholine.

The sensitivity to sinusoidal stretching and small-amplitude vibration, and the variability of the resting discharge rate of de-efferented muscle spindles in the neck extensor muscle biventer cervicis (b.c.) of the cat have been studied. The effects of intra-arterial infusion of succinylcholine (SCh) on the response of the receptors to ramp stretches of the muscle were also determined. When activated by SCh, one group of afferents showed only a slow facilitation of their discharge rate, similar to that of spindle secondary sensory endings in hind-limb muscles. A second group of afferents developed a large dynamic response and a marked increase in their static discharge rate and were presumed to originate in "normal" b1b2 c spindles in the b.c. muscle. A third group of afferents developed only a marked increase in static discharge, without potentiation of the dynamic response, suggesting an origin in the b2c units of tandem spindles which are relatively common in the neck muscles. On the basis of their passive characteristics alone, afferents from b1b2c units could not be readily distinguished from those from b2c units. The characteristics of these receptors, and their differences from the well-studied hind-limb spindle afferents, are discussed.

Afferent Pathways↗

Increased spindle resistance to antimicrotubule agents in cells prone to chromosomal nondisjunction.

The cytological behavior of the spindle apparatus was studied in cells prone to nondisjunction (ND), i.e., PHA-stimulated lymphocytes derived from children suffering from different types of neoplasia. These cells, which exhibited a high frequency of nonspecific aneuploidy, revealed an increased resistance of the spindle fibers to colchicine, podophyllotoxin, and cold, which was several times that of lymphocytes derived from healthy children. The results are in accord with previous findings showing a high resistance of spindle microtubules to the antimicrotubular agents colchicine, podophyllotoxin, vinblastine, and cold in PHA-stimulated lymphocytes derived from individuals prone to meiotic ND. It is therefore assumed that high resistance of the spindle apparatus to antimicrotubule agents characterizes cells at high risk for aneuploidy, and possibly, the overstabilized spindle fibers are responsible for failure of chromosomal disjunction.

Aneuploidy↗

Chromosome segregation and spindle structure in crane fly spermatocytes following Colcemid treatment.

Chromosome segregation in primary spermatocytes of the crane fly Nephrotoma suturalis was studied after exposure to Colcemid at doses that did not completely inhibit spindle formation. Colcemid was added either to the medium in which larvae were cultured or to Tricine buffer in which isolated testes were incubated. Patterns of chromosome segregation were analyzed in fixed, Feulgen-stained smears of testes from Colcemid-treated larvae and in living cell preparations. Anomalies observed during the first meiotic division at higher than normal frequencies in Colcemid-treated spermatocytes included anaphase lagging of autosomes, chromosomal strands, tripolar and tetrapolar divisions, and unequal distribution of chromosomes to secondary cells. Following those doses of Colcemid that induced the above anomalies, the length of the birefringent spindle in primary spermatocytes was shorter than normal. This effect on spindle length also was apparent in Giemsa-stained preparations of fixed cells, in which the two centrosomes at the spindle poles were differentiated from the rest of the cytoplasm. The results indicate a correlation between the inhibition of spindle formation and the induction of anomalous patterns of chromosome segregation.

Animals↗

Myelinated nerve fiber supply and muscle spindles in the respiratory muscles of cat: quantitative study.

The present study was undertaken to provide quantitative data on the myelinated fibers of the phrenic and intercostal nerves and the number of spindles in the main respiratory muscles of the cat. The myelinated component of the phrenic and intercostal nerves was studied in the cat. Histograms of sequency distributions as a function of nerve fiber diameter were established for normal nerves. Certain nerves were then examined 35 to 40 days after excision of the dorsal spinal ganglia. The muscle spindles of the corresponding muscles were counted and localized, and, on the basis of several morphological criteria, were classified with those usually described in the interosseous muscles. The study of the nerves, as that of the spindles, demonstrates clear differences of proprioceptive innervation among the respirator muscles. The lateral part of the diaphragm and the Triangularis sterni have practically no spindles. The external muscles of the first thoracic spaces are very rich in spindles. Respiratory muscles can be ranged in an almost continuous manner between these two extremes.

Animals↗

Analysis of the dynamic responses of deefferented primary muscle spindle endings to ramp stretch.

A detailed analysis of the complex dynamic response of 8 deefferented primary muscle spindle endings to ramp stretches of the extensor digitorum longus muscle (EDL) was made in anaesthetized cats. The analysis was based on Lennerstand's linear muscle spindle model, in which the dynamic peak, i.e. the peak frequency at the end of the dynamic phase of a ramp stretch, is assumed to consist of three components: a position response, a slow velocity response, and a quick velocity response. The components of the dynamic peaks analysed were fairly well correlated with the static behaviour of the primary muscle spindle endings, the latter, in turn, being regarded as an indicator of the spindles' location in the non-homogeneous extrafusal muscle. The results thus provide a satisfactory explanation of the high correlations between the static and gross dynamic behaviour of deefferented primary muscle spindle endings of the EDL previously reported from our laboratory.

Animals↗

Ischemic degeneration of the avian muscle spindle.

The neurovascular supply to the pigeon's extensor digitorum communis muscle was disrupted. The muscle spindles were studied by light and electron microscopy to determine whether their degeneration was compatible with regeneration by activation of satellite cells within an intact spindle capsule. The denervation and ischemia induced intrafusal muscle fiber necrosis and degeneration of the sarcolemma and basal lamina. The muscle fibers in the equatorial region were often absent and their sites were indicated by collagenous caps which usually covered the sensory nerve terminal regions. These collagenous caps enclosed amorphous material derived from the intrafusal fibers and degenerating mitochondria from the sensory terminals. In this equatorial region, the basal lamina was present only under the collagenous cap and was disrupted elsewere . The cells of the muscle spindle capsule were more sparse or absent, but the collagen content had increased. The sheath lacked continuity, containing numerous gaps. These observations indicate that the basal lamina does not remain intact, and regeneration may not occur by activation of satellite cells within the former basal lamina, as reported for regenerating rat muscle spindles. This suggests that the mechanisms of regeneration of muscle spindles in rat and pigeon muscle may not be similar.

Animals↗

Morphogenesis of rat muscle spindles after nerve lesion during early postnatal development.

The influence of innervation on muscle spindle morphogenesis has been investigated in rat hind-limb muscles by sectioning the sciatic nerve, with suture of the stumps, at various postnatal stages. After nerve section at 4 or 7 days of age a proportion of spindles survived during the denervation phase and developed, during the subsequent reinnervation phase, into atypical structures. The reinnervated spindles were recognized by the presence of a limiting capsule but lacked the characteristic distinction of equatorial and polar regions. The intrafusal fibres were fewer than normal and were indistinguishable in size and fine structure from extrafusal fibres; they had a single motor endplate and lacked sensory nerve terminals. In reinnervated muscles of animals operated at 13 and 22 days of age there was a progressive tendency towards a restoration of normal spindle structure and innervation. These findings indicate that muscle spindle morphogenesis is profoundly altered by nerve lesion at early development stages, apparently as a result of inadequate sensory reinnervation. This study also shows that the differentiation of intrafusal fibres is dictated by their specific pattern of innervation and is not intrinsically predetermined.

Adenosine Triphosphatases↗

The effect of the local anaesthetic bupivacaine on the muscle spindle of rat.

The effects of a long-lasting local anaesthetic, bupivacaine, in combination with hyaluronidase, on the intrafusal muscle fibres of adult rat muscle spindles have been investigated by light and electron microscopy. Necrotic changes in the axial bundle are present within 4 h of the combined drug injection and are widespread by 2 days. The equatorial nucleation of the spindle is lost owing to the necrosis of the myonuclei. Satellite cells, however, appear to survive these changes, even where the underlying muscle fibres are grossly necrotic. Sensory nerve terminals undergo necrosis during this period and plate-type fusimotor nerve terminals withdraw from the degenerating muscle fibres. Macrophage infiltration and early regeneration of the axial bundle is apparent by the third day. Myoblasts first appear at the periphery of the affected muscle fibres, but decrease in number as regeneration proceeds. By the end of the third week, regeneration of the muscle component is complete and re-innervating fusimotor nerve terminals established. The encapsulated regions of the spindle remain abnormal, without recognizable bags or chains of nuclei, although the muscle fibres differ both in size and ultrastructure. Regenerating sensory axons make contacts with the intrafusal bundle that differ in their configuration and ultrastructure from normal terminals. The production of enucleated spindles is discussed in the light of the morphogenetic influences of the spindle nerve terminals, as well as the possible use of such a model in discovering the function of nuclear bags and nuclear chains.

Animals↗

The responses of frog muscle spindles during stimulation of slow motor axons.

Responses of muscle spindles of the iliofibularis muscle of frog Litoria aurea have been recorded during single shock and repetitive stimulation of single functional motor axons. Repetitive stimulation of axons which innervated slow muscle, and on four occasions, axons which innervated twitch muscle, produced a large increase in the dynamic response of the spindle to a ramp-and-hold stretch. While extrafusal slow muscle did not respond to a single motor volley, some spindles did, especially if at the same time the muscle was being stretched. In an explanation of the effect of muscle stretch on responses of spindles to slow motor volleys it was proposed that stretch acted to reduce the internal motion in muscle fibres produced by a non-uniform distribution of sarcomere lengths. It was proposed that this kind of effect may account for dynamic fusimotor actions in all vertebrate spindles.

Animals↗

Experimental study on meiotic spindles and chromosomes of mouse mature (M II) stage oocytes under laser scanning confocal microscopy.

Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope (LSCM) was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional (3D) image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of M II oocytes.

Animals↗

Analysis of the distribution of the kinetochore protein Ndc10p in Saccharomyces cerevisiae using 3-D modeling of mitotic spindles.

Ndc10p is one of the DNA-binding constituents of the kinetochore in Saccharomyces cerevisiae but light microscopy analysis suggests that Ndc10p is not limited to kinetochore regions. We examined the localization of Ndc10p using immunoelectron microscopy and showed that Ndc10p is associated with spindle microtubules from S-phase through anaphase. By serial section reconstruction of mitotic spindles combined with immunogold detection, we showed that Ndc10p interacts with microtubules laterally as well as terminally. About 50% of the gold label in serial section reconstructions of short mitotic spindles was associated with the walls of spindle microtubules. Interaction of kinetochore components with microtubule walls was also shown for kinetochore protein Ndc80p. Our data suggest that at least a subset of kinetochore-associated protein is dispersed throughout the mitotic spindle.

Anaphase↗