DNA damage induces nuclear actin filament assembly by Formin-2 and Spire-1/2 that promotes efficient DNA repair

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in eLife 4 (2015): e07735, doi:10.7554/eLife.07735.Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments—detectable by phalloidin and live-cell actin probes—with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: Formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either Formin-2 or actin’s nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments.Howard Hughes Medical Institute; National Institutes of Health, GM061010, GM079556, 5F31AG39147-2; National Science Foundatio

Visualization of actin filaments and monomers in somatic cell nuclei

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 24 (2013): 982-994, doi:10.1091/mbc.E12-09-0685.In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or noncanonical oligomer) of nuclear actin are hotly debated, and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and evaluate its potential functions, we constructed and validated fluorescent nuclear actin probes. Monomeric actin probes concentrate in nuclear speckles, suggesting an interaction of monomers with RNA-processing factors. Filamentous actin probes recognize discrete structures with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: 1) diffusive, with an average diffusion coefficient of 0.06–0.08 μm2/s, or (2) subdiffusive, with a mobility coefficient of 0.015 μm2/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh. The small size of nuclear actin filaments is inconsistent with a role in micron-scale intranuclear transport, and their localization suggests that they do not participate directly in chromatin-based processes. Our results instead suggest that actin filaments form part of a large, viscoelastic structure in the nucleoplasm and may act as scaffolds that help organize nuclear contents.This bulk of this work was supported by a grant from the National Institutes of Health to R.D.M. (5R01GM061010-12). Additional support was provided by National Institutes of Health Grant R01 CA096840 (E.H.B.), a National Science Foundation Predoctoral Fellowship (B.B.), a National Institutes of Health Ruth L. Kirschstein Predoctoral Fellowship (B.B.), and a Genentech Fellowship (B.C.)

Interdisciplinary Graduate Training in Teaching Labs

Modern research and training in the life sciences require cross-disciplinary programs, integrating concepts and methods from biology, physics, chemistry, and mathematics. We describe the structure and outcomes from an example of one such approach, the Physiology Course at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts, and discuss how similar intensive, team-building research courses are also being applied to improve graduate education in universities. These courses are based on teaching laboratories that have students address contemporary research questions by combining ideas and approaches from biology, computation, and physics

Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations

Urological referral of asymptomatic men in general practice in England

The Prostate Cancer Risk Management Programme (PCRMP) launched in November 2002 provides guidelines for general practitioners (GPs) on age-specific prostate-specific antigen (PSA) cutoff levels in asymptomatic men. The impact of the PCRMP on GP referrals is unknown. This study investigates whether there was a change in the proportion of asymptomatic men with raised PSA levels (⩾3 ng ml−1) who were referred to urologists since the launch of the guidelines. Sixty-nine general practices in four areas of England and the main pathology laboratory in each area, which had participated in our previous research, were asked to provide data. Forty-eight practices (70%) provided retrospective data on urological referrals in men who had a PSA test taken in the periods 1 December 2001 to 31 May 2002 (pre-launch) and 1 December 2003 to 31 May 2004 (post-launch). Data on referrals were completed for 709 (79%) out of 898 and 1040 (90%) out of 1157 raised records pre- and post-launch, respectively. The percentage of men with raised PSA levels who were asymptomatic was similar in both time periods (19–20%) and the proportion referred to urologists according to the PCRMP guidelines did not increase significantly over time (24% pre-launch and 29% post-launch, P=0.42). The referral rate was lower than expected if the guidelines had been followed. The influence of the guidelines seems to have been low. At the time of data collection, 56% (112 out of 200) of GP partners reported that they were aware of receiving the PCRMP pack. To ensure future, effective implementation of guidelines requires evaluation

Weighted vests in CrossFit increase physiological stress during walking and running without changes in spatiotemporal gait parameters

This study quantified the physiological and biomechanical effects of the 20 lb (9.07 kg, males) and 14 lb (6.35 kg, females) weighted vest used in CrossFit, and whether they were predisposed to injury. Twenty subjects (10 males, 10 females) undertook walking (0%, 5% and 10% gradient) and running trials in two randomised study visits (weighted vest/no weighted vest). Physiological demand during walking was increased with the vest at 10% but not 5% or 0% with no change in gait variables. In the running trial, the weighted vest increased oxygen uptake (males; females) (+0.22L/min, p < 0.01; +0.07 L/min, p < 0.05), heart rate (+11bpm, p < 0.01; +11bpm, p < 0.05), carbohydrate oxidation (+0.6 g/min, p < 0.001; +0.2 g/min, p < 0.01), and energy expenditure (+3.8 kJ/min, p < 0.001; +1.5 kJ/min, p < 0.05) whilst blood lactate was increased only in males (+0.6 mmol/L, p < 0.05). There was no change in stride length or frequency. Weighted vest training increases physiological stress and carbohydrate oxidation without affecting measured gait parameters. Practitioner summary: We examined the effect of weighted vest training prescribed in CrossFit (20 lb/9.07 kg, males and 14 lb/6.35 kg, females) in a randomised controlled trial. We found that physiological stress is increased in both sexes, although three-fold greater in males, but with no change in biomechanical gait that predisposes to lower-limb injury

Activation of Arp2/3 Complex: Addition of the First Subunit of the New Filament by a WASP Protein Triggers Rapid ATP Hydrolysis on Arp2

In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament

In Silico Reconstitution of Actin-Based Symmetry Breaking and Motility

Computational modeling and experimentation in a model system for actin-based force generation explain how actin networks initiate and maintain directional movement