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

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

SWI/SNF Deficiency Results in Aberrant Chromatin Organization, Mitotic Failure, and Diminished Proliferative Capacity

Switch (SWI)/sucrose nonfermentable (SNF) is an evolutionarily conserved complex with ATPase function, capable of regulating nucleosome position to alter transcriptional programs within the cell. It is known that the SWI/SNF complex is responsible for regulation of many genes involved in cell cycle control and proliferation, and it has recently been implicated in cancer development. The ATPase action of SWI/SNF is conferred through either the brahma-related gene 1 (Brg1) or brahma (Brm) subunit of the complex, and it is of central importance to the modification of nucleosome position. In this study, the role of the Brg1 and Brm subunits were examined as they relate to chromatin structure and organization. Deletion of the Brg1 ATPase results in dissolution of pericentromeric heterochromatin domains and a redistribution of histone modifications associated with these structures. This effect was highly specific to Brg1 and is not reproduced by the loss of Brm or SNF5/BAF47/INI1. Brg1 deficiency is associated with the appearance of micronuclei and aberrant mitoses that are a by-product of dissociated chromatin structure. Thus, Brg1 plays a critical role in maintaining chromatin structural integrity