TLR4 signalling through the MyD88 and TRIF-dependent pathways initiates translocation of the transcription factor NF-κB into the nucleus. In cell population studies using mathematical modeling and ...functional analyses, Cheng et al. suggested that LPS-driven activation of MyD88, in the absence of TRIF, impairs NF-κB translocation. We tested the model proposed by Cheng et al. using real-time single cell analysis in macrophages expressing EGFP-tagged p65 and a TNFα promoter-driven mCherry. Following LPS stimulation, cells lacking TRIF show a pattern of NF-κB dynamics that is unaltered from wild-type cells, but activation of the TNFα promoter is impaired. In macrophages lacking MyD88, there is minimal NF-κB translocation to the nucleus in response to LPS stimulation, and there is no activation of the TNFα promoter. These findings confirm that signalling through MyD88 is the primary driver for LPS-dependent NF-κB translocation to the nucleus. The pattern of NF-κB dynamics in TRIF-deficient cells does not, however, directly reflect the kinetics of TNFα promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.
Disease models of neurodegeneration with brain iron accumulation (NBIA) offer the possibility to explore the relationship between iron dyshomeostasis and neurodegeneration. We analyzed hiPS-derived ...astrocytes from PANK2-associated neurodegeneration (PKAN), an NBIA disease characterized by progressive neurodegeneration and high iron accumulation in the globus pallidus. Previous data indicated that PKAN astrocytes exhibit alterations in iron metabolism, general impairment of constitutive endosomal trafficking, mitochondrial dysfunction and acquired neurotoxic features. Here, we performed a more in-depth analysis of the interactions between endocytic vesicles and mitochondria via superresolution microscopy experiments. A significantly lower number of transferrin-enriched vesicles were in contact with mitochondria in PKAN cells than in control cells, confirming the impaired intracellular fate of cargo endosomes. The investigation of cytosolic and mitochondrial iron parameters indicated that mitochondrial iron availability was substantially lower in PKAN cells compared to that in the controls. In addition, PKAN astrocytes exhibited defects in tubulin acetylation/phosphorylation, which might be responsible for unregulated vesicular dynamics and inappropriate iron delivery to mitochondria. Thus, the impairment of iron incorporation into these organelles seems to be the cause of cell iron delocalization, resulting in cytosolic iron overload and mitochondrial iron deficiency, triggering mitochondrial dysfunction. Overall, the data elucidate the mechanism of iron accumulation in CoA deficiency, highlighting the importance of mitochondrial iron deficiency in the pathogenesis of disease.
Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is ...thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.
Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a relentlessly progressive neurodegenerative disorder caused by mutations in the
gene. C19orf12 has been implicated in playing a ...role in lipid metabolism, mitochondrial function, and autophagy, however, the precise functions remain unknown. To identify new robust cellular targets for small compound treatments, we evaluated reported mitochondrial function alterations, cellular signaling, and autophagy in a large cohort of MPAN patients and control fibroblasts. We found no consistent alteration of mitochondrial functions or cellular signaling messengers in MPAN fibroblasts. In contrast, we found that autophagy initiation is consistently impaired in MPAN fibroblasts and show that
expression correlates with the amount of LC3 puncta, an autophagy marker. Finally, we screened 14 different autophagy modulators to test which can restore this autophagy defect. Amongst these compounds, carbamazepine, ABT-737, LY294002, oridonin, and paroxetine could restore LC3 puncta in the MPAN fibroblasts, identifying them as novel potential therapeutic compounds to treat MPAN. In summary, our study confirms a role for C19orf12 in autophagy, proposes LC3 puncta as a functionally robust and consistent readout for testing compounds, and pinpoints potential therapeutic compounds for MPAN.
The structural plasticity of G-protein coupled receptors (GPCRs) enables the long-range transmission of conformational changes induced by specific orthosteric site ligands and other pleiotropic ...factors. Here, we demonstrate that the ligand binding cavity in the sphingosine 1-phosphate receptor S1PR1, a class A GPCR, is in allosteric communication with both the β-arrestin-binding C-terminal tail, and a receptor surface involved in oligomerization. We show that S1PR1 oligomers are required for full response to different agonists and ligand-specific association with arrestins, dictating the downstream signalling kinetics. We reveal that the active form of the immunomodulatory drug fingolimod, FTY720-P, selectively harnesses both these intramolecular networks to efficiently recruit β-arrestins in a stable interaction with the receptor, promoting deep S1PR1 internalization and simultaneously abrogating ERK1/2 phosphorylation. Our results define a molecular basis for the efficacy of fingolimod for people with multiple sclerosis, and attest that GPCR signalling can be further fine-tuned by the oligomeric state.
Pollen tubes are tip-growing cells that create safe routes to convey sperm cells to the embryo sac for double fertilization. Recent studies have purified and biochemically characterized ...detergent-insoluble membranes from tobacco pollen tubes. These microdomains, called lipid rafts, are rich in sterols and sphingolipids and are involved in cell polarization in organisms evolutionarily distant, such as fungi and mammals. The presence of actin in tobacco pollen tube detergent-insoluble membranes and the preferential distribution of these domains on the apical plasma membrane encouraged us to formulate the intriguing hypothesis that sterols and sphingolipids could be a "trait d'union" between actin dynamics and polarized secretion at the tip. To unravel the role of sterols and sphingolipids in tobacco pollen tube growth, we used squalestatin and myriocin, inhibitors of sterol and sphingolipid biosynthesis, respectively, to determine whether lipid modifications affect actin fringe morphology and dynamics, leading to changes in clear zone organization and cell wall deposition, thus suggesting a role played by these lipids in successful fertilization.
One key step in the activation of inducible transcription factors is their homooligomerization, which can be measured in individual living cells by a fluorescence microscopy technique called Number ...and Brightness analysis (N&B). In this chapter we describe how to acquire and analyze confocal microscopy time-series to provide information about transcription factor oligomerization in living cells using this technique.
Signalling is of particular importance in immune cells, and upstream in the signalling pathway many membrane receptors are functional only as complexes, co-locating with particular lipid species. ...Work over the last 15 years has shown that plasma membrane lipid composition is close to a critical point of phase separation, with evidence that cells adapt their composition in ways that alter the proximity to this thermodynamic point. Macrophage cells are a key component of the innate immune system, are responsive to infections and regulate the local state of inflammation. We investigate changes in the plasma membrane's proximity to the critical point as a response to stimulation by various pro- and anti-inflammatory agents. Pro-inflammatory (interferon
, Kdo 2-Lipid A, lipopolysaccharide) perturbations induce an increase in the transition temperature of giant plasma membrane vesicles; anti-inflammatory interleukin 4 has the opposite effect. These changes recapitulate complex plasma membrane composition changes, and are consistent with lipid criticality playing a master regulatory role: being closer to critical conditions increases membrane protein activity.
Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The ...scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules.
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