A guiding hypothesis for cell cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell cycle transitions 1, 2 . We have tested this hypothesis for mitotic exit, which is regulated by degradation of the Cdk1 activator, cyclin B 3-5 . Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with proteasome inhibitor or by expression of nondegradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. If after mitotic exit, the Cdk1 inhibitor is washed free from cells where cyclin B degradation is blocked, cells can reverse back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent protein degradation provides directionality for the M phase to G1 transition.Cdk1, the major regulator of mitotic progression is activated through binding of cyclin A or B. Cyclin A is degraded during prometaphase when chromosomes move to align at the metaphase plate 6, 7 . Cyclin B degradation begins at metaphase and continues during chromatid segregation in anaphase and exit from M phase 5 . Cytokinesis is initiated shortly after anaphase onset. Cdk1 inactivation and dephosphorylation of Cdk1 substrates during mitotic exit likely serve as timing mechanisms to ensure that cytokinesis occurs after chromatid separation 8-12 . For example prior to anaphase, high Cdk1 activity blocks the accumulation of the cytokinetic regulators Aurora B and MKLP1 at the cleavage furrow and on the microtubules of the spindle midzone 13-15 .Flavopiridol is a potent inhibitor of Cdk1 16 . We found that treatment of vertebrate cells in mitosis with Flavopiridol resulted in premature mitotic exit accompanied by cytokinesis (Fig 1a and Supplementary Video 1). Similar results were recently found for the Cdk inhibitor, BMI-1026 17 . Flavopiridol induced the microtubule network to undergo changes characteristic of anaphase and mitotic exit. The spindle poles moved apart, and microtubule bundles formed in the spindle midzone and at the equatorial cortex. Even though chromatid separation did not occur, cytokinetic furrows formed and ingressed to completion. The cleavage furrow trapped chromosomes in the midbody resulting in a "cut" phenotype. Nevertheless, the chromosomes decondensed and nuclear envelopes reformed. Eventually cytoplasmic contractile activityCorrespondence and requests for materials should be addressed to GJG (GJG@omrf.ouhsc.edu).. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. A figure summarizing the primary finding of the pa...
The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.
Rationale: Modulation of the activity of sarcoendoplasmic reticulum calcium ATPase (SERCA) can profoundly affect Ca 21 homeostasis. Although altered calcium homeostasis is a characteristic of cystic fibrosis (CF), the role of SERCA is unknown. Objectives: This study provides a comprehensive investigation of expression and activity of SERCA in CF airway epithelium. A detailed study of the mechanisms underlying SERCA changes and its consequences was also undertaken. Methods: Lung tissue samples (bronchus and bronchiole) from subjects with and without CF were evaluated by immunohistochemistry. Protein and mRNA expression in primary non-CF and CF cells was determined by Western and Northern blots. Measurements and Main Results: SERCA2 expression was decreased in bronchial and bronchiolar epithelia of subjects with CF. SERCA2 expression in lysates of polarized tracheobronchial epithelial cells from subjects with CF was decreased by 67% as compared with those from subjects without CF. Several non-CF and CF airway epithelial cell lines were also probed. SERCA2 expression and activity were consistently decreased in CF cell lines. Adenoviral expression of mutant F508 cystic fibrosis transmembrane regulator gene (CFTR), inhibition of CFTR function pharmacologically (CFTR inh 172), or stable expression of antisense oligonucleotides to inhibit CFTR expression caused decreased SERCA2 expression. In CF cells, SERCA2 interacted with Bcl-2, leading to its displacement from caveolae-related domains of endoplasmic reticulum membranes, as demonstrated in sucrose density gradient centrifugation and immunoprecipitation studies. Knockdown of SERCA2 using siRNA enhanced epithelial cell death due to ozone, hydrogen peroxide, and TNF-a. Conclusions: Reduced SERCA2 expression may alter calcium signaling and apoptosis in CF. These findings decrease the likelihood of therapeutic benefit of SERCA inhibition in CF.
The multiprotein Mediator coactivator complex is universally required for transcription of metazoan genes. It has been proposed to function by interfacing between transcriptional activators and the RNA polymerase II machinery. However, in vitro transcription systems reconstituted from homogeneous preparations of RNA polymerase II, the general transcription initiation factors, and the cofactor PC4 display relatively robust activator (HNF-4)-dependent activity, which, nonetheless, can be further stimulated by Mediator. By contrast, an unfractionated nuclear extract-based system in which Mediator has been immunodepleted displays a nearabsolute dependence on ectopic Mediator. Here, we identified and purified an activity, MSA-2, that confers extract-like Mediator responsiveness to our reconstituted system. Mass spectrometric analyses identified its two constituent polypeptides as hSpt5 and hSpt4, which also comprise the elongation factor DSIF. Mechanistically, MSA-2/DSIF acts by restricting overall transcription in the pure system, thereby imposing a strong Mediator dependence. Our data thus point to potential mechanisms for Mediator function beyond its presently believed role in promoting the initial formation of the RNA polymerase II-containing preinitiation complex.DRB sensitivity-inducing factor ͉ hepatocyte nuclear factor-4 ͉ positive cofactor 2 ͉ RNA polymerase II
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