Background: FUS has been implicated in the DNA damage response; however, the mechanisms are unknown. Results: FUS recruitment to DNA lesions is PARP-dependent. Depletion of FUS disrupts DNA repair. Conclusion: FUS functions downstream of PARP and promotes double-strand break repair. Significance: This work identifies FUS as a novel factor at DNA lesions and furthers our understanding of RNA-binding proteins in maintaining genomic stability.

Natural killer T (NKT) cells are innate-likeT cells that recognize specific microbial antigens and also display autoreactivity to self-antigens. The nature of NKT-cell autoreactive activation remains poorly understood. We show here that the mitogenactivated protein kinase (MAPK) pathway is operative during human NKT-cell autoreactive activation, but calcium signaling is severely impaired. This results in a response that is biased toward granulocyte macrophage colony-stimulating factor (GM-CSF) secretion because this cytokine requires extracellular signalregulated kinase (ERK) signaling but is not highly calcium dependent, whereas interferon-␥ (IFN-␥), interleukin (IL)-4, and IL-2 production are minimal. Autoreactive activation was associated with reduced migration velocity but did not induce arrest; thus, NKT cells retained the ability to survey antigen presenting cells (APCs). IL-12 and IL-18 stimulated autoreactively activated NKT cells to secrete IFN-␥, and this was mediated by Janus kinase-signal transducers and activators of transcription (JAK-STAT)-dependent signaling without induction of calcium flux. This pathway did not require concurrent contact with CD1d ؉ APCs but was strictly dependent on preceding autoreactive stimulation that induced ERK activation. In contrast, NKT-cell responses to the glycolipid antigen ␣-galactosyl ceramide (␣-GalCer) were dampened by prior autoreactive activation. These results show that NKT-cell autoreactivity induces restricted cytokine secretion and leads to altered basal activation that potentiates innate responsiveness to costimulatory cytokines while modulating sensitivity to foreign antigens. (Blood. 2008;112: 4128-4138) IntroductionNatural killer T (NKT) cells are a subset of regulatory T cells that recognizes lipid antigens presented by CD1d molecules. 1,2 Like innate lymphocytes, NKT cells are among the first responders during microbial infections, and their responses are not necessarily dependent on recognition of foreign antigens. 3 NKT cells have attracted attention because they secrete large amounts of both Th1 and Th2 cytokines rapidly on stimulation and demonstrate a potent ability to modulate immune function. 1,2,4 Remarkably, in some disease models, NKT cells promote proinflammatory immune responses, whereas in others they have a tolerogenic effect. 5 It is not clear what confers the innate-like features of NKT cells or how they mediate contrasting immunologic outcomes in different contexts.Most NKT cells can be activated to secrete cytokines by exposure to CD1d ϩ antigen-presenting cells (APCs) in the absence of microbial antigens, and this appears to depend on presentation of cellular antigens by CD1d. 6-9 NKT cells from germ-free mice and from human cord blood display a phenotype indicating prior activation, [10][11][12] suggesting that NKT cells are autoreactive to self-antigens in vivo. NKT cells have been shown to recognize certain mammalian lipids as antigens presented by CD1d 9,13 ; however, the specific self-antigens responsible for NKT cell autorea...

Weak TCR stimulation of iNKT cells, such as that resulting from self-antigen recognition, induces histone modifications at the IFNG locus that allow the iNKT cells to subsequently produce IFN-γ in response to proinflammatory cytokines alone.

, indicating these molecules likely interact under physiological conditions. Immunofluorescence microscopy confirmed a dual localization of CRHSP-28 and annexin VI, which appeared in a punctate pattern in the supranuclear and apical cytoplasm of acini. Stimulation of cells for 5 min with the secretagogue cholecystokinin enhanced the colocalization of CRHSP-28 and annexin VI within regions of acini immediately below the apical plasma membrane. Tissue fractionation revealed that CRHSP-28 is a peripheral membrane protein that is highly enriched in smooth microsomal fractions of pancreas. Further, the content of CRHSP-28 in microsomes was significantly reduced in pancreatic tissue obtained from rats that had been infused with a secretory dose of cholecystokinin for 40 min, demonstrating that secretagogue stimulation transiently alters the association of CRHSP-28 with membranes in cells. Collectively, the Ca 2؉ -dependent binding of CRHSP-28 and annexin VI, together with their colocalization in the apical cytoplasm, is consistent with a role for these molecules in acinar cell membrane trafficking events that are essential for digestive enzyme secretion.

To estimate the functional diameter of alveolar septal microvessels in zone 1, we perfused isolated rat lungs with fluorescent latex particles of specific diameters (0.24, 0.49, 1.05, or 4.0 microns) at pulmonary artery pressures (Ppulmart) that were either 5 or 10 cmH2O less than the air inflation pressure (Pinflat, 25 cmH2O). We then prepared samples for histology. Using a confocal, laser-scanning fluorescence microscope, we measured latex particle densities within the septal plane that ranged from 0.08 +/- 0.04 particles/microns2 (0.24-microns diameter particles) to 0.02 +/- 0.01 particles/microns2 (1.05-microns diameter particles). We found that 4.0-microns diameter particles were not able to enter septa at all. Latex particles were not present in all alveoli When Ppulmart was 5 cmH2O less than Pinflat, 32 +/- 6% of septa contained 0.24-microns diameter particles, but, when Ppulmart was 10 cmH2O less than Pinflat, 5 +/- 6% of septa contained these particles. Percentages were smaller for larger particles. We conclude that, when Ppulmart is both 5 and 10 cmH2O less than Pinflat, the functional diameter of accessible septal microvessels is > 1.05 but < 4.0 microns. Furthermore, the number of accessible septa decreases as the difference between Ppulmart and Pinflat widens.

In a previous study, conducted at the same value of Pinflat, but with PPA set at 15 or 20 (5 or 10 cm H2O into zone I). We estimated the capillary diameter to be 1.7 microns. Thus, the septal capillary diameter seems to increase by three- to fourfold as PPA is raised to equal Pinflat.

To estimate the functional diameter of alveolar microvessels, we perfused isolated rat lungs with fluorescent latex particles (1 diameter/lung) at inflation, pulmonary arterial, and left atrial pressures of 25, 30, and 0 cmH2O, respectively. We used confocal microscopy to count latex particles within septal microvessels and flow cytometry to count particle concentrations in venous outflow. We found 1-, 2-, and 4-micron-diameter particles within septal vessels of 45 +/- 12, 31 +/- 12, and 25 +/- 9%, respectively, of examined alveoli. Particles of 5-micron diameter were absent from septal vessels but were present within a small percentage of corner vessels. Particle concentrations in the venous outflow for 1-, 2-, 4-, and 5-micron-diameter particles were 54 +/- 28, 67 +/- 32, 2.2 +/- 0.3, and 0.4 +/- 0.3%, respectively, of the arterial inflow. Particles with diameters of 6 or 10 micron were absent from venous outflow. Our results suggest that, under these conditions, the functional diameter of the septal microvessels is approximately 4 micron and that the diameter of the adjacent corner vessels is slightly larger but <6 micron.

Mitochondrial remodeling (replication, fission/fusion) is a dynamically regulated process with diverse functions in neurons. A myelinated axon is an extension from the cell soma of a fully differentiated neuron. Mitochondria, once synthesized in the cell body, enter the axon displaying robust trafficking and accumulation at nodes of Ranvier to match metabolic needs. This long-distance deployment of mitochondria to axons raises the issue of whether myelinated axons can function independently of the cell body to execute mitochondrial remodeling to match local demands. Mitochondrial fusion has been suggested to occur in axons in simple neuronal cultures in vitro. However, whether such events occur in vivo in an intact nervous system remains unanswered. Here we describe a novel technique which allows monitoring of mitochondrial fusion in intact sciatic nerve of frog (Xenopus laevis). Mitochondrial population was labeled by injecting two different mitotracker dyes (red and green), spatially apart along sciatic nerves surgically and then allow to “meet” in vivo. At 24 hrs post-surgery, the sciatic nerves were taken out for mitochondrial imaging at the half-way point. During the post-injection periods, the anterograde-directed Green mitochondria meet with the retrograde-directed Red mitochondria. If fusion occurs, the merged of Green and Red fluorophores in the same mitochondrion will produce a Yellow color in merged images. The labeled mitochondria were observed with a Nikon A1 confocal microscope. Our new mitochondrial imaging method opens an avenue to separately assess the role of local axonal mitochondrial fusion, independent of the cell body of nerve fibers.