Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 × 10^− 9 cm² s^− 1 independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.     

Ultrapotent miniproteins targeting the SARS-CoV-2 receptor-binding domain protect against infection and disease

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Localization of synthetic glycolipids in the cell and the dynamics of their insertion/loss

Modification of the cell surface with synthetic glycolipids opens up a wide range of possibilities for studying the function of glycolipids. Synthetic glycolipids called Function-Spacer-Lipids (FSL; where F is a glycan or label, S is a spacer, and L is dioleoylphosphatidyl ethanolamine) easily and controllably modify the membrane of a living cells. This current study investigates the dynamics and mechanism of the FSL insertion and release/loss. FSL insert into the cell membrane (~1 million molecules per cell) within tens of minutes, almost regardless of the nature of the cells (including the thickness of their glycocalyx) and the size of the FSL glycan. FSLs do not accumulate uniformly, but instead form patches >300 nm in size either entrapped in the glycocalyx, or integrated in the plane of the plasma membrane, but always outside the cell rafts. The natural release (loss) of FSL from the modified cell was two orders of magnitude slower than attachment/insertion and occurred mainly in the form of released microvesicles with a size of 140 ± 5 nm. The accumulation of FSL as patches in the cell membrane is similar to the coalescence of natural glycosphingolipids and supports (along with their long residence time in the membrane) the use of FSL as probes for the study of glycosphingolipid-protein interactions.     

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures

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Comparison of methods used in zooplankton sampling and counting in the joint Russian-Finnish evaluation of the trophic state of Lake Ladoga

The results of a joint Russian-Finnish investigation on zooplankton in Lake Ladoga are presented, and a comparison is made between two sampling techniques, tube sampler and plankton net, and between two counting methods. The precision of subsampling and sampling is further discussed on the basis of zooplankton data gathered in Lake Saimaa, Finland. The comparison clearly indicates that a tube sampler is required for reliable sampling of small-sized animals, while a plankton net saves time and is a more economical sampler for large, rare or active animals. The comparison between the results obtained by Finnish and Russian workers, using different counting procedures, shows that the main groups of crustacean zooplankton are similarly counted and identified in the two laboratories.     

COORDINATED CAROTENOID AND LIPID SYNTHESES INDUCED IN PARIETOCHLORIS INCISA (CHLOROPHYTA,TREBOUXIOPHYCEAE) MUTANT DEFICIENT IN Δ5 DESATURASE BY NITROGEN STARVATION AND HIGH LIGHT1

The responses to PAR intensity and nitrogen deficiency have been investigated in the Δ5‐desaturase‐deficient mutant (P127) of the microalga Parietochloris incisa (Reisigl) Shin Watan. (Chlorophyta, Trebouxiophyceae). The mutant accumulates dihomo‐γ‐linolenic acid (DGLA, C20:3 ω6) instead of arachidonic acid (C20:4 ω6) characteristic of the wildtype. The growth, fatty acid and pigment composition, and light absorption by P127 cell suspensions were studied for the first time during cultivation on complete and N‐free BG‐11 medium at 35, 130, and 270 μE · m^?2 · s^?1. On complete medium under high irradiance, an increase in biomass was observed, and total fatty acid (TFA) and DGLA contents were higher than in N‐starving cultures. A distinct irradiance‐dependent rise in carotenoid‐to‐chl ratio was recorded in P127 due to an increase in carotenoids (on complete medium) or by a decline in chl (on N‐free medium). Cultivation under high and medium irradiances caused a decline in light‐harvesting xanthophylls and an increase in β‐carotene, localized predominantly in cytoplasmic oil bodies (OB). The P127 mutant, similar to wildtype, responded to the stresses by coordinated induction of fatty acid and carotenoid syntheses, but displayed the same magnitude of the response as was observed in wildtype under 30% lower irradiance. The changes in optical properties of the P127 cultures tightly correlated with their pigment composition, and hence with fatty acid content, making it possible to develop a nondestructive technique for the assay of TFA and DGLA. The peculiarities of the stress responses in the wildtype and the mutant are discussed.