Background: Lipopolysaccharides (LPSs) up-regulate proinflammatory cytokines in macrophages, partly through a NF-Bdependent process. Results: Blocking neddylation, which helps regulate NF-B, represses LPS-induced up-regulation of proinflammatory cytokines. Conclusion: Neddylation plays a role in the up-regulation of NF-B-regulated proinflammatory cytokines produced by macrophages in response to LPS. Significance: Inhibition of neddylation represents a novel and effective method for the prevention of LPS-induced proinflammatory cytokines.

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Tight junctions (TJ) of polarized epithelial cells regulate barrier function at mucosal surfaces. Structural proteins of TJs include hyperphosphorylated occludin (HO) and the peripheral membrane protein, ZO-1. Since TJs are dynamically regulated, and lipid-modified signal transduction proteins localize to TJs, we considered the possibility that the TJ itself is composed of microdomains with unique structure. Differential detergent extraction and isopycnic sucrose density gradients were utilized to isolate TJ-enriched membranes from a polarized intestinal epithelial cell line, T84. Here we report that major pools of hyperphosphorylated occludin (HO) and ZO-1 are found in raft-like membrane microdomains with characteristics of the previously described detergent-insoluble glycolipid rafts (DIGs). Properties of such gradient fractions included Triton X-100 (TX-100) insolubility, light scattering at 600 nm, buoyant density of approximately 1.08 g/cm(3) and increased cholesterol content compared to high density fractions. Similar results were obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, other basolateral transmembrane proteins including E-cadherin, c-met and β 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-1, a scaffolding protein abundant in DIGs. Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveolin-1 localization in the apical membrane and focal colocalization with occludin in TJs. TJ disassembly by calcium chelation resulted in displacement of TJ proteins from the ‘raft-like’ compartment. Our findings suggest that raft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.

As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and fission pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Δψ m) disrupts mitochondrial fission/fusion balance, causing fragmentation of the network. However, the level of Δψ m required for mitochondrial dynamic balance, as well as the relative contributions of fission and fusion pathways, have remained unclear. To explore this, mitochondrial morphology and Δψ m were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) flow cytometry, respectively, in cultured 143B osteosarcoma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-depleted ρ0) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE fluorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanistically, this threshold is maintained by mechanistic coordination of DRP1-mediated fission and OPA1-mediated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Δψ m, instead maintaining an obligately fused morphology. Collectively, these findings demonstrate a mitochondrial ‘tipping point’ threshold mediated by the interaction of Δψ m with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of fission and fusion pathways. These results suggest that Δψ m below threshold coordinately activates both DRP1-mediated fission and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-016-2421-9) contains supplementary material, which is available to authorized users.

Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling.