p70 S6 kinase and actin dynamics: A perspective

p70 S6 kinase (p70S6K), a member of the AGC serine/threonine kinase family, was initially identified as a key player, together with its downstream effector S6, in the regulation of cellular growth and survival. The p70S6K protein has emerged in recent years as a multifunctional protein which also regulates the actin cytoskeleton and thus plays a role in cell migration. This new function is through two important activities of p70S6K, namely actin cross-linking and Rac1 and Cdc42 activation. The testis is critically dependent on an intricate balance of fundamental cellular processes such as adhesion, migration, and differentiation. It is increasingly evident that Rho GTPases and actin binding proteins play fundamental roles in regulating spermatogenesis within the testis. In this review, we will discuss current findings of p70S6K in the control of actin cytoskeleton dynamics. In addition, the potential role of p70S6K in spermatogenesis and testicular function will be highlighted

Transcriptional co-activators YAP1-TAZ of Hippo signalling in doxorubicin-induced cardiomyopathy

Aims Hippo signalling is an evolutionarily conserved pathway that controls organ size by regulating apoptosis, cell proliferation, and stem cell self-renewal. Recently, the pathway has been shown to exert powerful growth regulatory activity in cardiomyocytes. However, the functional role of this stress-related and cell death-related pathway in the human heart and cardiomyocytes is not known. In this study, we investigated the role of the transcriptional co-activators of Hippo signalling, YAP and TAZ, in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in response to cardiotoxic agents and investigated the effects of modulating the pathway on cardiomyocyte function and survival. Methods and results RNA-sequencing analysis of human heart samples with doxorubicin-induced end-stage heart failure and healthy controls showed that YAP and ERBB2 (HER2) as upstream regulators of differentially expressed genes correlated with doxorubicin treatment. Thus, we tested the effects of doxorubicin on hiPSC-CMs in vitro. Using an automated high-content screen of 96 clinically relevant antineoplastic and cardiotherapeutic drugs, we showed that doxorubicin induced the highest activation of YAP/TAZ nuclear translocation in both hiPSC-CMs and control MCF7 breast cancer cells. The overexpression of YAP rescued doxorubicin-induced cell loss in hiPSC-CMs by inhibiting apoptosis and inducing proliferation. In contrast, silencing of YAP and TAZ by siRNAs resulted in elevated mitochondrial membrane potential loss in response to doxorubicin. hiPSC-CM calcium transients did not change in response to YAP/TAZ silencing. Conclusions Our results suggest that Hippo signalling is involved in clinical anthracycline-induced cardiomyopathy. Modelling with hiPSC-CMs in vitro showed similar responses to doxorubicin as adult cardiomyocytes and revealed a potential cardioprotective effect of YAP in doxorubicin-induced cardiotoxicity

Right Ventricle Has Normal Myofilament Function But Shows Perturbations in the Expression of Extracellular Matrix Genes in Patients With Tetralogy of Fallot Undergoing Pulmonary Valve Replacement

BACKGROUND: Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. METHODS AND RESULTS: We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF‐PVR and RV donor samples, as was collagen content and cross‐linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine‐rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. CONCLUSIONS: RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria

Prelamin A mediates inflammation in dilated and HIV associated cardiomyopathies

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues yet its impact upon the heart is unknown. Here we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy and show that a novel mouse model of cardiac specific prelamin A accumulation exhibited a phenotype consistent with ‘inflammatory cardiomyopathy’ which we observed to be similar to HIV associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings: (1) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease suggesting protease inhibitors should be replaced with alternative therapies i.e. non-nucleoside reverse transcriptase inhibitors; and (3) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy

Author Correction: Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-017-13675-8, published online 01 November 201

Characterization of Engineered Actin Binding Proteins That Control Filament Assembly and Structure

Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate.We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs), are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel "reduced genetic code" phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively.From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly

Scapinin, the Protein Phosphatase 1 Binding Protein, Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton

Copyright (c) 2009 Sagara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes.ArticlePLOS ONE. 4(1):e4247 (2009)journal articl

Sex-specific control of human heart maturation by the progesterone receptor

Background: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. Methods: Single nucleus RNA-sequencing (snRNA-seq) of 54,140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA-sequencing and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. Results: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. Conclusions: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.Choon Boon Sim, Belinda Phipson, Mark Ziemann, Haloom Rafehi, Richard J. Mills, Kevin I. Watt ... et al

Simulation vs. Reality: A Comparison of In Silico Distance Predictions with DEER and FRET Measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions