Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements

Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved

Dynamic and Polarized Muscle Cell Behaviors Accompany Tail Morphogenesis in the Ascidian Ciona intestinalis

BACKGROUND: Axial elongation is a key morphogenetic process that serves to shape developing organisms. Tail extension in the ascidian larva represents a striking example of this process, wherein paraxially positioned muscle cells undergo elongation and differentiation independent of the segmentation process that characterizes the formation of paraxial mesoderm in vertebrates. Investigating the cell behaviors underlying the morphogenesis of muscle in ascidians may therefore reveal the evolutionarily conserved mechanisms operating during this process. METHODOLOGY/PRINCIPLE FINDINGS: A live cell imaging approach utilizing subcellularly-localized fluorescent proteins was employed to investigate muscle cell behaviors during tail extension in the ascidian Ciona intestinalis. Changes in the position and morphology of individual muscle cells were analyzed in vivo in wild type embryos undergoing tail extension and in embryos in which muscle development was perturbed. Muscle cells were observed to undergo elongation in the absence of positional reorganization. Furthermore, high-speed high-resolution live imaging revealed that the onset and progression of tail extension were characterized by the presence of dynamic and polarized actin-based protrusive activity at the plasma membrane of individual muscle cells. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that in the Ciona muscle, tissue elongation resulted from gradual and coordinated changes in cell geometry and not from changes in cell topology. Proper formation of muscle cells was found to be necessary not only for muscle tissue elongation, but also more generally for completion of tail extension. Based upon the characterized dynamic changes in cell morphology and plasma membrane protrusive activity, a three-phase model is proposed to describe the cell behavior operating during muscle morphogenesis in the ascidian embryo

Large Isoforms of UNC-89 (Obscurin) Are Required for Muscle Cell Architecture and Optimal Calcium Release in Caenorhabditis elegans

Calcium, a ubiquitous intracellular signaling molecule, controls a diverse array of cellular processes. Consequently, cells have developed strategies to modulate the shape of calcium signals in space and time. The force generating machinery in muscle is regulated by the influx and efflux of calcium ions into the muscle cytoplasm. In order for efficient and effective muscle contraction to occur, calcium needs to be rapidly, accurately and reliably regulated. The mechanisms underlying this highly regulated process are not fully understood. Here, we show that the Caenorhabditis elegans homolog of the giant muscle protein obscurin, UNC-89, is required for normal muscle cell architecture. The large immunoglobulin domain-rich isoforms of UNC-89 are critical for sarcomere and sarcoplasmic reticulum organization. Furthermore, we have found evidence that this structural organization is crucial for excitation-contraction coupling in the body wall muscle, through the coordination of calcium signaling. Thus, our data implicates UNC-89 in maintaining muscle cell architecture and that this precise organization is essential for optimal calcium mobilization and efficient and effective muscle contraction

The Enhancer of Trithorax and Polycomb Corto Interacts with Cyclin G in Drosophila

BACKGROUND: Polycomb (PcG) and trithorax (trxG) genes encode proteins involved in the maintenance of gene expression patterns, notably Hox genes, throughout development. PcG proteins are required for long-term gene repression whereas TrxG proteins are positive regulators that counteract PcG action. PcG and TrxG proteins form large complexes that bind chromatin at overlapping sites called Polycomb and Trithorax Response Elements (PRE/TRE). A third class of proteins, so-called "Enhancers of Trithorax and Polycomb" (ETP), interacts with either complexes, behaving sometimes as repressors and sometimes as activators. The role of ETP proteins is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In a two-hybrid screen, we identified Cyclin G (CycG) as a partner of the Drosophila ETP Corto. Inactivation of CycG by RNA interference highlights its essential role during development. We show here that Corto and CycG directly interact and bind to each other in embryos and S2 cells. Moreover, CycG is targeted to polytene chromosomes where it co-localizes at multiple sites with Corto and with the PcG factor Polyhomeotic (PH). We observed that corto is involved in maintaining Abd-B repression outside its normal expression domain in embryos. This could be achieved by association between Corto and CycG since both proteins bind the regulatory element iab-7 PRE and the promoter of the Abd-B gene. CONCLUSIONS/SIGNIFICANCE: Our results suggest that CycG could regulate the activity of Corto at chromatin and thus be involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG

SS18 Together with Animal-Specific Factors Defines Human BAF-Type SWI/SNF Complexes

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Cyclin A repression in quiescent cells is associated with chromatin remodeling of its promoter and requires Brahma/SNF2alpha

International audienceCell cycle-dependent expression of cyclin A is controlled by transcriptional repression in early phase of the cell cycle. In this study, we directly examine the chromatin structure of the mouse cyclin A promoter through in vivo micrococcal nuclease footprinting. We describe here that cyclin A repression is associated with two positioned nucleosomes and that histones progressively lose DNA contact synchronously with gene activation. This particular nucleosomal organization is disrupted by mutations of the cyclin A bipartite repressor sequence. Moreover, the same sequence recruits the chromatin remodeling factor Brahma/SNF2alpha (Brm) onto the cyclin A promoter. Accordingly, cyclin A proximal promoter is not wrapped around nucleosomes and not repressed in quiescent cells lacking Brm. These results provide molecular explanations for the transcriptional repression state of cyclin A, as well as insights into the action of Brm chromatin remodeling factor as cell cycle regulator

Cyclin E drives human keratinocyte growth into differentiation

Human epidermis is continuously exposed to environmental mutagenic hazard and is the most frequent target of human cancer. How the epidermis coordinates proliferation with differentiation to maintain homeostasis, even in hyperproliferative conditions, is unclear. For instance, overactivation of the proto-oncogene MYC in keratinocytes stimulates differentiation. Here we explore the cell cycle regulation as proliferating human keratinocytes commit to terminal differentiation upon loss of anchorage or overactivation of MYC. The S-phase of the cell cycle is deregulated as mitotic regulators are inhibited in the onset of differentiation. Experimental inhibition of mitotic kinase cdk1 or kinases of the mitosis spindle checkpoint Aurora B or Polo-like Kinase, triggered keratinocyte terminal differentiation. Furthermore, hyperactivation of the cell cycle by overexpressing the DNA replication regulator Cyclin E induced mitosis failure and differentiation. Inhibition of Cyclin E by shRNAs attenuated the induction of differentiation by MYC. In addition, we present evidence that Cyclin E induces DNA damage and the p53 pathway. The results provide novel clues for the mechanisms committing proliferative keratinocytes to differentiate, with implications for tissue homeostasis maintenance, HPV amplification and tumorigenesis

Skeletal muscle effects of electrostimulation after COPD exacerbation: a pilot study: a pilot study

International audienceMuscle dysfunction is a major problem in chronic obstructive pulmonary disease (COPD), particularly after exacerbations. We thus asked whether neuromuscular electrostimulation (NMES) might be directly useful following an acute exacerbation and if such a therapy decreases muscular oxidative stress and/or alters muscle fibre distribution. A pilot randomised controlled study of NMES lasting 6 weeks was carried out in 15 in-patients (n=9 NMES; n=6 sham) following a COPD exacerbation. Stimulation was delivered to the quadriceps and hamstring muscles (35 Hz). Primary outcomes were quadriceps force and muscle oxidative stress. At the end of the study, quadriceps force improvement was statistically different between groups (p=0.02), with a significant increase only in the NMES group (median (interquartile range) 10 (4.7-11.5) kg; p=0.01). Changes in the 6-min walking distance were statistically different between groups (p=0.008), with a significant increase in the NMES group (165 (125-203) m; p=0.003). NMES did not lead to higher muscle oxidative stress, as indicated by the decrease in total protein carbonylation (p=0.02) and myosin heavy chain carbonylation (p=0.01) levels. Finally, we observed a significant increase in type I fibre proportion in the NMES group. Our study shows that following COPD exacerbation, NMES is effective in counteracting muscle dysfunction and decreases muscle oxidative stress

Ibrutinib monotherapy for relapse or refractory primary CNS lymphoma and primary vitreoretinal lymphoma Final analysis of the phase II ‘proof-of-concept’ iLOC study by the Lymphoma study association (LYSA) and the French oculo-cerebral lymphoma (LOC) network

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