(B) Average roGFP2-NLS signal integrated across the nuclear area in non-Target control or IPO9 hairpin lines in untreated and 0

(B) Average roGFP2-NLS signal integrated across the nuclear area in non-Target control or IPO9 hairpin lines in untreated and 0.01% MMS-treated cells. molecular mechanisms ALLO-1 creating these nuclear filaments. DOI: http://dx.doi.org/10.7554/eLife.07735.001 oocytes, this germinal vesicle actin forms a filamentous mesh that protects nucleoli from gravity-induced aggregation (Feric and Brangwynne, 2013). Actin filaments associated with germinal vesicles of starfish oocytes facilitate nuclear envelope breakdown and form a contractile net that facilitates chromosome capture during mitosis (Lnrt et al., 2005; Mori et al., 2014). Several studies have also implicated nuclear actin filaments in oocyte transcription (reviewed in Belin and Mullins, 2013). In contrast, most somatic cells express some amount of XPO6, and therefore, have a much lower concentration of actin in the nucleus than in the cytoplasm. Also, unlike germinal vesicles, mammalian somatic nuclei contain relatively small amounts of filamentous actin (Belin et al., 2013), suggesting that monomeric actin may play an important role. Monomers of actin and several actin-related proteins (Arps), for example, are conserved components of chromatin-remodeling complexes (Farrants, 2008), and nuclear actin monomers inhibit the activity of the serum-responsive transcriptional co-activator MRTF (myotonin-related transcription factor) (Vartiainen et al., 2007; Mouilleron et al., 2008). Many reports have also linked actin to the regulation of RNA polymerases, although there are conflicting data on whether this activity depends on monomers or filaments (Belin and Mullins, 2013). Functions for filamentous actin in somatic cell nuclei are slowly beginning to emerge. Serum stimulation of quiescent fibroblasts (Baarlink et al., 2013) and integrin engagement ALLO-1 in spreading cells (Plessner et al., 2015) induce transient (<60 s) bursts of nuclear actin polymerization, driven by the nucleation activity of formin-family proteins mDia1 and mDia2. These short-lived filaments appear to promote activity of the transcriptional co-activator MRTF by depleting monomeric actin from the nucleus. Serum stimulation also activates the actin-severing protein MICAL-2, which reversibly oxidizes actin monomers, rendering them incapable of inhibiting MRTF-dependent transcription (Lundquist et al., 2014). Environmental stresses also promote actin assembly in somatic cell nuclei. Heat shock, dimethyl sulfoxide (DMSO), depletion of ATP, and oxidative stress all induce formation of nuclear filament bundles that contain large amounts of cofilin (Fukui, 1978, Fukui and Katsumaru, 1980; Iida et al., 1992; Pendleton et al., 2003; Kim et al., 2009). In addition to its function as a co-factor for nuclear import, cofilin appears to play a structural role in these cofilinCactin rods, which are highly oxidized and appear to be held together by intermolecular disulfide bonds between cofilin molecules (Pfannstiel et al., 2001; Bernstein et al., 2012; Zhang et al., 2013). Little is known about the physiological role of these cofilinCactin rods but they sense and perhaps regulate the reducing potential of the nucleus (Bernstein et al., 2012; Munsie et al., 2012). Many functions proposed for nuclear actin have been controversial, due in part to a lack of molecular tools for visualizing and perturbing actin inside the nucleus without affecting cytoplasmic actin (Belin et al., 2013). The discovery of actin's nuclear import and export factors, along with the recent identification of some of the molecular mechanisms that create nuclear actin filaments, now enable us to make more specific perturbations of actin inside the nucleus. In addition, we and others have developed fluorescent probes that enable us to visualize actin monomers and filaments in the nuclei of live cells (Baarlink et al., 2013; Belin et al., 2013; Plessner et al., 2015). Using these recently developed tools, we discovered that DNA damage induced by various genotoxic agents triggers formation of actin filaments inside the nucleus of mammalian cells. These filaments promote efficient repair of DNA double-strand breaks (DSBs) and are required for a DNA damage-associated burst of oxidation in the nucleus. DNA damage-induced nuclear actin structures differ in both composition and mechanism of assembly from those triggered by serum stimulation or by non-specific cell stresses. Specifically, we find that the actin regulators Formin-2 (FMN2) and Spire-1/2 nucleate nuclear actin assembly in response to ALLO-1 DNA damage. Homologs of Formin-2 and Spire-1/2 Rabbit Polyclonal to PNPLA6 interact directly (Quinlan et al., 2007; Vizcarra et al., 2011; Montaville et al., 2014) and collaborate to form functional actin networks in mouse and oocytes. Murine oocyte FMN2 and Spire are required for migration of meiotic spindles to the cortex, extrusion of polar bodies, and radial transport of vesicles (Schuh and Ellenberg, 2008; Schuh, 2011). In The roGFP2 excitation spectrum contains 2 peaks, at 488 nm and at 405 nm..