Central spindle robustness by PRC1-centralspindlin interaction

Mitotic apparatus (MA) plays central roles in cell division for both mitosis and cytokinesis. It achieves these mechanical tasks by changing its morphology under the control of cell cycle machinery. This relies on dynamic polymerization and depolymerization cycles of microtubules and their assembly into higher order structures such as bundles involving various microtubule regulators. A dramatic remodeling of the MA occurs at the metaphase to anaphase transition (Fig. 1). Before this, 2 spindle poles are connected both by interpolar microtubules and by kinetochore microtubules attaching the unsegregated chromatids. After anaphase onset, the link via kinetochore microtubules and chromosomes disappears due to loss of chromosome cohesion. As a consequence, the interpolar microtubules, which have now developed into a more prominent structure termed the central spindle, become the sole mechanical link between the 2 poles (Fig. 1, case i). Metaphase-anaphase transition also promotes the growth of astral microtubules. Dynein anchored at the cell cortex interacts with the astral microtubules and generates mechanical forces (cortical pulling forces) that pull spindle poles toward the cell cortex.1 Grill SW, et al. Nature 2001; 409(6820):630-3; PMID:11214323; http://dx.doi.org/10.1038/35054572 [CrossRef], [PubMed], [Web of Science ®] In some cell types such as the C. elegans embryos, cortical pulling force is the major driving force for chromosome separation via elongation of the pole-to-pole distance (anaphase B). In this situation, the central spindle is dispensable for chromosome segregation; it rather works as a brake against the cortical pulling force. Indeed, in C. elegans embryos, chromosome separation is accelerated when the central spindle is severed by laser manipulation or by genetic perturbation.2 Saunders AM, et al. Curr Biol 2007; 17(12):R453-4; PMID:17580072; http://dx.doi.org/10.1016/j.cub.2007.05.001 [CrossRef], [PubMed], [Web of Science ®] So, why does a cell form the central spindle? Well, this is because it has an important role in cytokinesis

Polar relaxation by dynein-mediated removal of cortical myosin II

Nearly 6 decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction (White and Borisy, 1983; Wolpert, 1960). While this mechanism has remained controversial (Rappaport, 1996), recent work has provided evidence for polar relaxation by astral microtubules (Chen et al., 2008; Dechant and Glotzer, 2003; Foe and Dassow, 2008; Murthy and Wadsworth, 2008; Werner et al., 2007), although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction whereas suppression of dynein activity delayed furrowing. We provide evidence that dynein-mediated removal of myosin II from the polar cortexes triggers cortical flow towards the cell equator, which induces the assembly of the actomyosin contractile ring. These studies for the first time provide a molecular basis for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis

Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle

During animal cell division, the central spindle, an anti-parallel microtubule bundle structure formed between segregating chromosomes during anaphase, cooperates with astral microtubules to position the cleavage furrow. Because the central spindle is the only structure linking the two halves of the mitotic spindle, it is under mechanical tension from dynein-generated cortical pulling forces, which determine spindle positioning and drive chromosome segregation through spindle elongation. The central spindle should be flexible enough for efficient chromosome segregation while maintaining its structural integrity for reliable cytokinesis. How the cell balances these potentially conflicting requirements is poorly understood. Here, we demonstrate that the central spindle in C. elegans embryos has a resilient mechanism for recovery from perturbations by excess tension derived from cortical pulling forces. This mechanism involves the direct interaction of two different types of conserved microtubule bundlers that are crucial for central spindle formation, PRC1 and centralspindlin

A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans.

BACKGROUND: Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the Δ9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. RESULTS: Liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to define the metabolome of all the possible knock-outs for the Δ9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence Δ9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. CONCLUSIONS: The propagation of metabolic changes across the network of metabolism demonstrates that modification of the Δ9 desaturases places C.elegans into a catabolic state compared with wildtype controls.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Neural correlates of a mindfulness-based intervention in anorexia nervosa

神経性やせ症患者の不安に対するマインドフルネス瞑想の効果 --脳活動の変化を明らかに--. 京都大学プレスリリース. 2023-02-02.Accepting anxiety for peace of mind. 京都大学プレスリリース. 2023-02-08.We examined the neural underpinnings of the effects of mindfulness on anxiety in anorexia nervosa using functional magnetic resonance imaging in 21 anorexia patients. We used a functional magnetic resonance imaging task designed to induce weight-related anxiety and asked participants to regulate their anxiety either using or not using an acceptance strategy. Our results showed reduced activity in the amygdala, anterior cingulate cortex, putamen, caudate, orbital gyrus, middle frontal gyrus, posterior cingulate cortex and precuneus following a mindfulness-based intervention. The present study provides new insight regarding the neural mechanisms underlying the effect of mindfulness-based intervention in ameliorating anorexia nervosa

CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules

The midbody interactome reveals unexpected roles for PP1 phosphatases in cytokinesis

Abstract: The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1β-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins

A monomeric StayGold fluorescent protein

StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from S. cerevisiae, S. pombe, and the β- and γ- isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate actin dendritic networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton