Seipin regulates ER–lipid droplet contacts and cargo delivery

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.     

Use of BODIPY‐Cholesterol (TF‐Chol) for Visualizing Lysosomal Cholesterol Accumulation

Dipyrromethene difluoride‐cholesterol (TopFluor‐Cholesterol, TF‐Chol) is a widely used cholesterol analogue due to its excellent fluorescence properties and considerable similarity with natural cholesterol in terms of membrane partitioning. However, the suitability of TF‐Chol for detecting lysosomal cholesterol deposition has recently been questioned. Here, we highlight the fact that the method of lipid delivery and the analysis of time‐point both affect the membrane distribution and labeling pattern of TF‐Chol, similarly as with radiolabeled cholesterol. Lysosomal sterol accumulation characteristic to a lysosomal storage disease is most readily detected when the probe is introduced via the physiological route, i.e. as a sterol fatty acid ester in low‐density lipoprotein particles. When administered to cells from solvent, lysosomal sterol sequestration becomes evident after an overnight equilibration between membranes.      

Abiotic stress modifies the synthesis of alpha‐tocopherol and beta‐carotene in phytoplankton species

We performed laboratory experiments to investi‐gate whether the synthesis of the antioxidants α‐tocopherol (vitamin E) and β‐carotene in phytoplankton depends on changes in abiotic factors. Cultures of Nodularia spumigena, Phaeodactylum tricornutum, Skeletonema costatum, Dunaliella tertiolecta, Prorocentrum cordatum, and Rhodomonas salina were incubated at different tempe‐ratures, photon flux densities and salinities for 48 h. We found that abiotic stress, within natural ecological ranges, affects the synthesis of the two antioxidants in different ways in different species. In most cases antioxidant production was stimulated by increased abiotic stress. In P. tricornutum KAC 37 and D. tertiolecta SCCAP K‐0591, both good producers of this compound, α‐tocopherol accumulation was negatively affected by environmentally induced higher photosystem II efficiency (F_v/F_m). On the other hand, β‐carotene accumulation was positively affected by higher F_v/F_m in N. spumigena KAC 7, P. tricornutum KAC 37, D. tertiolecta SCCAP K‐0591 and R. salina SCCAP K‐0294. These different patterns in the synthesis of the two compounds may be explained by their different locations and functions in the cell. While α‐tocopherol is heavily involved in the protection of prevention of lipid peroxidation in membranes, β‐carotene performs immediate photo‐oxidative protection in the antennae complex of photosystem II. Overall, our results suggest a high variability in the antioxidant pool of natural aquatic ecosystems, which can be subject to short‐term temperature, photon flux density and salinity fluctuations. The antioxidant levels in natural phytoplankton communities depend on species composition, the physiological condition of the species, and their respective strategies to deal with reactive oxygen species. Since α‐tocopherol and β‐carotene, as well as many other nonenzymatic antioxidants, are exclusively produced by photo‐synthetic organisms, and are required by higher trophic levels through dietary intake, regime shifts in the phytoplankton as a result of large‐scale environmental changes, such as climate change, may have serious consequences for aquatic food webs.     

HIF-1α and iNOS levels in crucian carp gills during hypoxia-induced transformation

Hypoxia inducible factor 1 alpha (HIF-1α) initiates expression of a wide variety of genes, some of which are involved in apoptosis and cell cycle arrest. We have previously shown that crucian carp increases its respiratory surface area 7.5-fold in response to hypoxia. This change is due to apoptosis and cell cycle arrest in specific parts of its gills. Here we have characterized crucian carp HIF-1α, and measured mRNA, protein and DNA binding levels during hypoxia exposure in crucian carp gills. We have also measured an HIF-1α-induced gene, the inducible nitric oxide synthase (iNOS), which has the ability to initiate apoptosis and cell cycle arrest. Crucian carp HIF-1α was found to have all critical domains known to be important for function. Comparison of the peptide sequence with other species indicated high similarity with other cyprinid fish, but a pronounced variation compared to the salmonid, rainbow trout. Further, we found HIF-1α protein to be stabilized during hypoxia. Further, HIF-1α was often present in normoxia, and showed marked individual weight-dependent variation. We found no alteration of iNOS mRNA levels during hypoxia exposure. These findings suggest HIF-1α involvement in hypoxia-induced change of respiratory surface area in crucian carp gills. However, its activity does not seem to be mediated through iNOS.     

3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.     

The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.

Focal adhesion kinase (FAK) is a tyrosine kinase found in focal adhesions, intracellular signaling complexes that are formed following engagement of the extracellular matrix by integrins. The C-terminal 'focal adhesion targeting' (FAT) region is necessary and sufficient for localizing FAK to focal adhesions. We have determined the crystal structure of FAT and show that it forms a four-helix bundle that resembles those found in two other proteins involved in cell adhesion, alpha-catenin and vinculin. The binding of FAT to the focal adhesion protein, paxillin, requires the integrity of the helical bundle, whereas binding to another focal adhesion protein, talin, does not. We show by mutagenesis that paxillin binding involves two hydrophobic patches on opposite faces of the bundle and propose a model in which two LD motifs of paxillin adopt amphipathic helices that augment the hydrophobic core of FAT, creating a six-helix bundle.     

Cell adhesion regulates the interaction between the docking protein p130(Cas) and the 14-3-3 proteins

Integrin ligand binding induces a signaling complex formation via the direct association of the docking protein p130(Cas) (Cas) with diverse molecules. We report here that the 14-3-3zeta protein interacts with Cas in the yeast two-hybrid assay. We also found that the two proteins associate in mammalian cells and that this interaction takes place in a phosphoserine-dependent manner, because treatment of Cas with a serine phosphatase greatly reduced its ability to bind 14-3-3zeta. Furthermore, the Cas-14-3-3zeta interaction was found to be regulated by integrin-mediated cell adhesion. Thus, when cells are detached from the extracellular matrix, the binding of Cas to 14-3-3zeta is greatly diminished, whereas replating the cells onto fibronectin rapidly induces the association. Consistent with these results, we found that the subcellular localization of Cas and 14-3-3 is also regulated by integrin ligand binding and that the two proteins display a significant co-localization during cell attachment to the extracellular matrix. In conclusion, our results demonstrate that 14-3-3 proteins participate in integrin-activated signaling pathways through their interaction with Cas, which, in turn, may contribute to important biological responses regulated by cell adhesion to the extracellular matrix.     

Negative regulation of FAK signaling by SOCS proteins

Focal adhesion kinase (FAK) becomes activated upon integrin-mediated cell adhesion and controls cellular responses to the engagement of integrins, including cell migration and survival. We show here that a coordinated signaling by integrins and growth factor receptors induces expression of suppressor of cytokine signaling-3 (SOCS-3) and subsequent interaction between endogenous FAK and SOCS-3 proteins in 3T3 fibroblasts. Cotransfection studies demonstrated that SOCS-3, and also SOCS-1, interact with FAK in a FAK-Y397-dependent manner, and that both the Src homology 2 (SH2) and the kinase inhibitory region (KIR) domains of the SOCS proteins contribute to FAK binding. SOCS-1 and SOCS-3 were found to inhibit FAK-associated kinase activity in vitro and tyrosine phosphorylation of FAK in cells. The SOCS proteins also promoted polyubiquitination and degradation of FAK in a SOCS box-dependent manner and inhibited FAK-dependent signaling events, such as cell motility on fibronectin. These studies suggest a negative role of SOCS proteins in FAK signaling, and for a previously unidentified regulatory mechanism for FAK function.     

GEF what? Dock180 and related proteins help Rac to polarize cells in new ways

Rho GTPase activation, which is mediated by guanine nucleotide exchange factors (GEFs), is tightly regulated in time and space. Although Rho GTPases have a significant role in many biological events, they are best known for their ability to restructure the actin cytoskeleton profoundly through the activation of specific downstream effectors. Two distinct families of GEFs for Rho GTPases have been reported so far, based on the features of their catalytic domains: firstly, the classical GEFs, which contain a Dbl homology-pleckstrin homology domain module with GEF activity, and secondly, the Dock180-related GEFs, which contain a Dock homology region-2 domain that catalyzes guanine nucleotide exchange on Rho GTPases. Recent exciting data suggest key roles for the DHR-2 domain-containing GEFs in a wide variety of fundamentally important biological functions, including cell migration, phagocytosis of apoptotic cells, myoblast fusion and neuronal polarization.