Mitosis: New Roles for Myosin-X and Actin at the Spindle

SummaryRoles for actin and myosin in positioning mitotic spindles in the cell are well established. A recent study of myosin-X function in early Xenopus embryo mitosis now reports that this unconventional myosin is required for pole integrity and normal spindle length by localizing to poles and exerting pulling forces on actin filaments within the spindle

Walking in orthostatic tremor modulates tremor features and is characterized by impaired gait stability

Primary orthostatic tremor (OT) is characterized by high-frequency lower-limb muscle contractions and a disabling sense of unsteadiness while standing. Patients consistently report a relief of symptoms when starting to ambulate. Here, we systematically examined and linked tremor and gait characteristics in patients with OT. Tremor and gait features were examined in nine OT patients and controls on a pressure-sensitive treadmill for one minute of walking framed by two one-minute periods of standing. Tremor characteristics were assessed by time-frequency analysis of surface EMG-recordings from four leg muscles. High-frequency tremor during standing (15.29 +/- 0.17 Hz) persisted while walking but was consistently reset to higher frequencies (16.34 +/- 0.25 Hz;p < 0.001). Tremor intensity was phase-dependently modulated, being predominantly observable during stance phases (p < 0.001). Tremor intensity scaled with the force applied during stepping (p < 0.001) and was linked to specific gait alterations, i.e., wide base walking (p = 0.019) and increased stride-to-stride fluctuations (p = 0.002). OT during walking persists but is reset to higher frequencies, indicating the involvement of supraspinal locomotor centers in the generation of OT rhythm. Tremor intensity is modulated during the gait cycle, pointing at specific pathways mediating the peripheral manifestation of OT. Finally, OT during walking is linked to gait alterations resembling a cerebellar and/or sensory ataxic gait disorder

Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells

Ray Rappaport spent many years studying microtubule asters, and how they induce cleavage furrows. Here, we review recent progress on aster structure and dynamics in zygotes and early blastomeres of Xenopus laevis and Zebrafish, where cells are extremely large. Mitotic and interphase asters differ markedly in size, and only interphase asters span the cell. Growth of interphase asters occurs by a mechanism that allows microtubule density at the aster periphery to remain approximately constant as radius increases. We discuss models for aster growth, and favor a branching nucleation process. Neighboring asters that grow into each other interact to block further growth at the shared boundary. We compare the morphology of interaction zones formed between pairs of asters that grow out from the poles of the same mitotic spindle (sister asters) and between pairs not related by mitosis (non-sister asters) that meet following polyspermic fertilization. We argue growing asters recognize each other by interaction between antiparallel microtubules at the mutual boundary, and discuss models for molecular organization of interaction zones. Finally, we discuss models for how asters, and the centrosomes within them, are positioned by dynein-mediated pulling forces so as to generate stereotyped cleavage patterns. Studying these problems in extremely large cells is starting to reveal how general principles of cell organization scale with cell size. V C 2012 Wiley Periodicals, Inc Key Words: aster, embryo, microtubule, cleavage, centrosome Introduction M icrotubule asters-radial arrays of microtubules radiating from centrosomes-play a central organizing role in early embryos. Ray Rappaport was fascinated by the question of how asters, in particular pairs of asters, induce cleavage furrows. One of his most celebrated discoveries The amphibian Xenopus laevis and the fish Danio rerio (Zebrafish) are easy to rear in the laboratory, and offer complementary technical advantages. Xenopus eggs cleave completely and are easy to fertilize with one or multiple sperm and to microinject. They are opaque, which precludes live imaging of internal events, but fixed embryos can be cleared for immunofluorescence imaging by immersion in a high refractive index medium REVIEW ARTICLE n 738 live imaging Xenopus and Zebrafish zygotes and early blastomeres are extremely large cells, with zygotes $1200 lm and$600 lm in diameter, respectively. They are also unusually fast compared to somatic cells, in the sense that the cell cycle takes 20-30 min to complete at room temperature (the first cell cycles are longer). These sizes and speeds represent physical extremes compared to typical somatic cells, which may require special adaptations of conserved cell organizing mechanisms, and/or reveal underappreciated intrinsic capabilities of those mechanisms. One well-studied example is adaptation of replication origins for very fast genome duplication Aster Growth in Large Cells The question of how microtubule asters grow in extremely large embryo cells has received little attention, but we believe that answering it will reveal principles of size scaling and unexpected molecular mechanisms. Figures 1 and 2 illustrate aster morphology and growth during the first and second cell cycle in frog and fish embryos. Inspection of these and similar images n 740 Mitchison et al. CYTOSKELETON Possibly consistent with this model, prometaphase asters in tissue culture cells capture and orient non-centrosomal microtubules using dynein An important question is whether the unusual aster growth mechanisms illustrated in Aster size in frog and fish embryos is temporally controlled by the cell cycle, with important implications for growth mechanisms and embryo organization. Aster radius at the poles of the first metaphase spindle is $30-40 lm in Xenopus [Figs. 1A and 1B, Wühr et al., 2008] and similar in Zebrafish [Fig. 2, 4 min, Wühr et al., 2010]. In both cases, this is much smaller than the zygote radius. Asters grow dramatically at anaphase onset, presumably due to decreased activity of Cdk1 (Cdc2.Cyclin B) kinase. In mitosis, Cdk1 acts on a complex network of microtubule interacting proteins to promote catastrophes (growing to shrinking transitions) and limit length CYTOSKELETON Growth, Interaction, and Positioning of Microtubule Asters 741 n presumably limited by the length distribution of microtubules in this bounded regime. Cdk1 levels drop shortly after fertilization, and at anaphase onset [reviewed in Cell cycle regulation of aster size has important implications for spatial organization of the early embryo. In early frog or fish blastomeres metaphase spindles are centrally located, and their short astral microtubules do not reach the cortex Aster-Aster Interactions What happens when two neighboring asters grow to touch each other? This question was of great interest to Rappaport, since cleavage furrows are typically induced where and when microtubules growing from aster pairs meet at the cortex. Asters grow into each other in early embryos under different circumstances. Two asters grow out from the poles of each mitotic spindle at anaphase, and meet each other at the midplane of the cell The most characteristic consequence of aster-aster interaction in interphase frog and fish embryos, seen for both n 742 Mitchison et al. The microtubule distribution in metaphase (A) appears radial, and microtubule density decreases rapidly with radius. In late anaphase (B), it appears more bundled and bushy, and microtubule density decreases less with radius. The dark zone in the center in B is presumably caused by a steric block to antibody penetration. A similar block is present at the center of the anaphase midzone and telophase midbody in somatic cells, and may also be present at the center of the aster-aster interaction zone in frog and fish embryos. CYTOSKELETON Growth, Interaction, and Positioning of Microtubule Asters 743 n sister ( Despite common features, there are reasons to suspect that not all aster-aster interaction zones are the same. Most notably, furrows are induced where the interaction zones between sister asters reach the cortex after 1st mitosis, and not where interaction zones between non-sisters reach the cortex, in the frogs Rana fusca and X. laevis, [Fig. 6C, Brachet, 1910; n 744 Mitchison et al. CYTOSKELETON the interaction between non-sister asters from two different spindles efficiently induced furrows if they were sufficiently close together. Non-sister asters also generated furrows at their interaction zone in tissue culture cells What molecules are likely to mediate aster-aster interactions in early frog and fish embryos? To our knowledge, no molecule has been specifically localized to aster-aster interaction zones in frog or fish embryos, but one logical set of candidates are molecules that organize cytokinesis midzone complexes in smaller cells [reviewed in Glotzer, 2005; Midzones are organized by three conserved protein modules or complexes Aster and Centrosome Positioning How asters, and the centrosomes at their centers, position themselves within embryos was also of great interest to Rappaport. Aster movement in large embryo cells is driven mainly by cytoplasmic dynein pulling on microtubules Hamaguchi and Hiramoto [1986] postulated that the sperm centrosome centers in the zygote due to length-dependent pulling forces on astral microtubules, combined with limitation of microtubule length by interaction with the cortex. Their model was based on elegant experiments CYTOSKELETON Growth, Interaction, and Positioning of Microtubule Asters 745 n where local inactivation of colcemid with UV light was used to artificially control microtubule length distribution in echinoderm embryos. Recent mathematical models support the concept that asters center by pulling forces that increase with microtubule length We hypothesized that Hiramoto&apos;s basic idea, which is cartooned in Perhaps the most intriguing unexplained aspect of aster and centrosome movement is orthogonal orientation of successive cleavage planes in embryos with an orthoradial cleavage pattern n 746 Mitchison et al. CYTOSKELETON because the yolk-free cytoplasm is laid down as a sheet in the oocyte. Successive cleavage planes are approximately orthogonal in two dimensions. Although cleavage plane geometry is stereotyped in frog eggs, it is not rigidly prespecified. Changing the shape of the egg, or the distribution of yolk within it 11 , spread between passivated coverslips and imaged by widefield fluorescence microscopy with a Â10 objective. Large asters grew with a bushy morphology at their peripheries (e.g., 32 min [Â3]). When asters grew to touch each they generated interaction zones with locally low microtubule density (e.g., arrows at 42 min, shown at higher mag. in 42 min [Â3]). These interaction zones blocked aster expansion and were stable for tens of minutes (compare 32, 52 min). When two artificial centrosomes were initially close together, they tended to initiate a single aster, and later split apart within that aster (e.g., the pair indicated by arrowheads at 2, 22, and 53 min). This splitting was reminiscent of centrosome separation within telophase asters in embryos Cell-Free Reconstitution of Interphase Aster Growth and Interaction It will be difficult to elucidate the molecular and biophysical mechanisms involved in aster dynamics using whole, living embryos as the only experimental system, especially in Xenopus where the egg is opaque. The related problem of meiosis-II spindle assembly in Xenopus eggs was tackled using cell-free extracts that accurately recapitulated the assembly process and greatly facilitated imaging and perturbation experiments Questions and Directions In closing, we will highlight key questions from each section of this review where we need to uncover new molecular and biophysical mechanisms. (i) Aster growth: what is the mechanism for keeping microtubule density constant as aster radius expands? If microtubules nucleate away from the centrosome as we suspect, what is the mechanism? (ii) Aster-aster interaction: how do growing asters recognize each other when they touch, and how does this recognition lead to inhibition of aster growth? To what extent are interaction zones between sister and non-sister asters similar at the molecular level, and why do only the former induce furrows in frog zygotes at 1st mitosis? (iii) Aster positioning: can we find further experimental validation for the Hiramoto model for aster centering? How do centrosomes split apart within growing asters, and what determines the axis on which they separate? Answering these questions will surely require interdisciplinary approaches that combine imaging, biochemistry, genetics, physical perturbation, force measurement, and computational modeling. Different biological systems have complementary advantages for these approaches, and we expect that Xenopus egg extract will prove particularly versatile. Vertebrate embryos with extremely large cells, where aster dynamics operate at a physical extreme, will help elucidate not only general principles of physical organization of cells, but also how these principles scale with cell size

The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.

The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere

Sharing tasks or sharing actions? Evidence from the joint Simon task.

In a joint Simon task, a pair of co-acting individuals divide labors of performing a choice-reaction task in such a way that each actor responds to one type of stimuli and ignores the other type that is assigned to the co-actor. It has been suggested that the actors share the mental representation of the joint task and perform the co-actor’s trials as if they were their own. However, it remains unclear exactly which aspects of co-actor’s task-set the actors share in the joint Simon task. The present study addressed this issue by manipulating the proportions of compatible and incompatible trials for one actor (inducer actor) and observing its influences on the performance of the other actor (diagnostic actor) for whom there were always an equal proportion of compatible and incompatible trials. The design of the present study disentangled the effect of trial proportion from the confounding effect of compatibility on the preceding trial. The results showed that the trial proportions for the inducer actor had strong influences on the inducer actor’s own performance, but it had little influence on the diagnostic actor’s performance. Thus, the diagnostic actor did not represent aspects of the inducer actor’s task-set beyond stimuli and responses of the inducer actor. We propose a new account of the effect of preceding compatibility on the joint Simon effect.Action Contro

Efficacy and safety of acupuncture for chronic pain caused by gonarthrosis: A study protocol of an ongoing multi-centre randomised controlled clinical trial [ISRCTN27450856]

BACKGROUND: Controlled clinical trials produced contradictory results with respect to a specific analgesic effect of acupuncture. There is a lack of large multi-centre acupuncture trials. The German Acupuncture Trial represents the largest multi-centre study of acupuncture in the treatment of chronic pain caused by gonarthrosis up to now. METHODS: 900 patients will be randomised to three treatment arms. One group receives verum acupuncture, the second sham acupuncture, and the third conservative standard therapy. The trial protocol is described with eligibility criteria, detailed information on the treatment definition, blinding, endpoints, safety evaluation, statistical methods, sample size determination, monitoring, legal aspects, and the current status of the trial. DISCUSSION: A critical discussion is given regarding the considerations about standardisation of the acupuncture treatment, the choice of the control group, and the blinding of patients and observers

Conflict in object affordance revealed by grip force

Viewing objects can result in automatic, partial activation of motor plans associated with them—“object affordance”. Here, we recorded grip force simultaneously from both hands in an object affordance task to investigate the effects of conflict between coactivated responses. Participants classified pictures of objects by squeezing force transducers with their left or right hand. Responses were faster on trials where the object afforded an action with the same hand that was required to make the response (congruent trials) compared to the opposite hand (incongruent trials). In addition, conflict between coactivated responses was reduced if it was experienced on the preceding trial, just like Gratton adaptation effects reported in “conflict” tasks (e.g., Eriksen flanker). This finding suggests that object affordance demonstrates conflict effects similar to those shown in other stimulus–response mapping tasks and thus could be integrated into the wider conceptual framework on overlearnt stimulus–response associations. Corrected erroneous responses occurred more frequently when there was conflict between the afforded response and the response required by the task, providing direct evidence that viewing an object activates motor plans appropriate for interacting with that object. Recording continuous grip force, as here, provides a sensitive way to measure coactivated responses in affordance tasks

CENP-A and topoisomerase-II antagonistically affect chromosome length

The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans , CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening