Cellular Contractility Requires Ubiquitin Mediated Proteolysis

Background

Cellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC) Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility.

Principal Findings

We observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation.

Conclusions

Our results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions.     

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington’s disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs.     

Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint

The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.     

Frontal GABA levels change during working memory

Functional neuroimaging metrics are thought to reflect changes in neurotransmitter flux, but changes in neurotransmitter levels have not been demonstrated in humans during a cognitive task, and the relationship between neurotransmitter dynamics and hemodynamic activity during cognition has not yet been established. We evaluate the concentration of the major inhibitory (GABA) and excitatory (glutamate + glutamine: Glx) neurotransmitters and the cerebral perfusion at rest and during a prolonged delayed match-to-sample working memory task. Resting GABA levels in the dorsolateral prefrontal cortex correlated positively with the resting perfusion and inversely with the change in perfusion during the task. Further, only GABA increased significantly during the first working memory run and then decreased continuously across subsequent task runs. The decrease of GABA over time was paralleled by a trend towards decreased reaction times and higher task accuracy. These results demonstrate a link between neurotransmitter dynamics and hemodynamic activity during working memory, indicating that functional neuroimaging metrics depend on the balance of excitation and inhibition required for cognitive processing.     

Monoamine oxidase A polymorphism moderates stability of attention problems and susceptibility to life stress during adolescence

Attention problems affect a substantial number of children and adolescents and are predictive of academic underachievement and lower global adaptive functioning. Considerable variability has been observed with regard to the individual development of attention problems over time. In particular, the period of adolescence is characterized by substantial maturation of executive functioning including attentional processing, with the influence of genetic and environmental factors on individual trajectories not yet well understood. In the present investigation, we evaluated whether the monoamine oxidase A functional promoter polymorphism, MAOA‐LPR, plays a role in determining continuity of parent‐rated attention problems during adolescence. At the same time, a potential effect of severe life events (SLEs) was taken into account. A multi‐group path analysis was used in a sample of 234 adolescents (149 males, 85 females) who took part in an epidemiological cohort study at the ages of 11 and 15 years. Attention problems during early adolescence were found to be a strong predictor of attention problems in middle adolescence. However, in carriers of the MAOA‐LPR low‐activity variant (MAOA‐L), stability was found to be significantly higher than in carriers of the high‐activity variant (MAOA‐H). Additionally, only in MAOA‐L carriers did SLEs during adolescence significantly impact on attention problems at the age of 15 years, implying a possible gene × environment interaction. To conclude, we found evidence that attention problems during adolescence in carriers of the MAOA‐L allele are particularly stable and malleable to life stressors. The present results underline the usefulness of applying a more dynamic GxE perspective.