SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin organization.

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.

Stress relaxation in epithelial monolayers is controlled by the actomyosin cortex.

Epithelial monolayers are one-cell thick tissue sheets that line most of the body surfaces, separating internal and external environments. As part of their function, they must withstand extrinsic mechanical stresses applied at high strain rates. However, little is known about how monolayers respond to mechanical deformations. Here, by subjecting suspended epithelial monolayers to stretch, we find that they dissipate stresses on a minute timescale and that relaxation can be described by a power law with an exponential cut-off at timescales larger than ~10 s. This process involves an increase in monolayer length, pointing to active remodelling of cellular biopolymers at the molecular scale during relaxation. Strikingly, monolayers consisting of tens of thousands of cells relax stress with similar dynamics to single rounded cells and both respond similarly to perturbations of the actomyosin cytoskeleton. By contrast, cell-cell junctional complexes and intermediate filaments do not relax tissue stress, but form stable connections between cells, allowing monolayers to behave rheologically as single cells. Taken together our data show that actomyosin dynamics governs the rheological properties of epithelial monolayers, dissipating applied stresses, and enabling changes in monolayer length.

Correction: Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

[This corrects the article DOI: 10.1371/journal.pone.0145937.].

A Novel In Vitro Model of Blast Traumatic Brain Injury.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

Xenon Protects against Blast-Induced Traumatic Brain Injury in an In Vitro Model.

The aim of this study was to evaluate the neuroprotective efficacy of the inert gas xenon as a treatment for patients with blast-induced traumatic brain injury in an in vitro laboratory model. We developed a novel blast traumatic brain injury model using C57BL/6N mouse organotypic hippocampal brain-slice cultures exposed to a single shockwave, with the resulting injury quantified using propidium iodide fluorescence. A shock tube blast generator was used to simulate open field explosive blast shockwaves, modeled by the Friedlander waveform. Exposure to blast shockwave resulted in significant (p < 0.01) injury that increased with peak-overpressure and impulse of the shockwave, and which exhibited a secondary injury development up to 72 h after trauma. Blast-induced propidium iodide fluorescence overlapped with cleaved caspase-3 immunofluorescence, indicating that shock-wave-induced cell death involves apoptosis. Xenon (50% atm) applied 1 h after blast exposure reduced injury 24 h (p < 0.01), 48 h (p < 0.05), and 72 h (p < 0.001) later, compared with untreated control injury. Xenon-treated injured slices were not significantly different from uninjured sham slices at 24 h and 72 h. We demonstrate for the first time that xenon treatment after blast traumatic brain injury reduces initial injury and prevents subsequent injury development in vitro. Our findings support the idea that xenon may be a potential first-line treatment for those with blast-induced traumatic brain injury.

Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.

INTRODUCTION: Left ventricular dysfunction is a frequent and potentially severe side effect of many tyrosine kinase inhibitors (TKI). The mode of toxicity is not identified, but may include impairment of mitochondrial or sarcomeric function, autophagy or angiogenesis, either as an on-target or off-target mechanism. METHODS AND RESULTS: We studied concentration-response curves and time courses for nine TKIs in three-dimensional, force generating engineered heart tissue (EHT) from neonatal rat heart cells. We detected a concentration- and time-dependent decline in contractile force for gefitinib, lapatinib, sunitinib, imatinib, sorafenib, vandetanib and lestaurtinib and no decline in contractile force for erlotinib and dasatinib after 96 hours of incubation. The decline in contractile force was associated with an impairment of autophagy (LC3 Western blot) and appearance of autophagolysosomes (transmission electron microscopy). CONCLUSION: This study demonstrates the feasibility to study TKI-mediated force effects in EHTs and identifies an association between a decline in contractility and inhibition of autophagic flux.

Cellular control of cortical actin nucleation.

The contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division [1-3], migration [4], and tissue morphogenesis [5]. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells [6] and during division, where its misregulation can lead to aneuploidy [7]. Despite its importance, our knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence [8-15]. Here, we used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 and the Arp2/3 complex. Each nucleator contributed a similar amount of F-actin to the cortex but had very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. Our findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator.