Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line‐derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin‐associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin‐mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.      

Light-induced exocytosis in cell development and differentiation

Calcium-dependent exocytosis of fluorescently labeled single secretory vesicles in PC12 cells and primary embryonic telencephalon cells can be triggered by illumination with visible light and imaged by TIRF or epifluorescence microscopy. Opsin 3 was identified by quantitative PCR expression analysis as the putative light receptor molecule for light-induced exocytosis. In primary chicken telencephalon cells, light-induced exocytosis is restricted to a specific period during embryonic development, and involves fusion of rather large vesicles. Strictly calcium-dependent exocytosis starts after a delay of a few seconds of illumination and lasts for up to 2 min. We analyzed the frequency, time course and spatial distribution of exocytotic events. Exocytosis in PC12 cells and telencephalon cells occurs at the periphery or the interface between dividing cells, and the duration of single secretion events varies considerably. Our observation strongly supports the idea that light induced exocytosis is most likely a mechanism for building plasma membrane during differentiation, development and proliferation rather than for calcium-dependent neurotransmitter release.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

The crystal structure of C176A mutated [Fe]-hydrogenase suggests an acyl-iron ligation in the active site iron complex

[Fe]-hydrogenase is one of three types of enzymes known to activate H₂. Crystal structure analysis recently revealed that its active site iron is ligated square-pyramidally by Cys176-sulfur, two CO, an “unknown” ligand and the sp²-hybridized nitrogen of a unique iron-guanylylpyridinol-cofactor. We report here on the structure of the C176A mutated enzyme crystallized in the presence of dithiothreitol (DTT). It suggests an iron center octahedrally coordinated by one DTT-sulfur and one DTT-oxygen, two CO, the 2-pyridinol’s nitrogen and the 2-pyridinol’s 6-formylmethyl group in an acyl-iron ligation. This result led to a re-interpretation of the iron ligation in the wild-type.     

Infrared spectroscopic study of the structural and functional properties of the Na/H antiporter MjNhaP1 from Methanococcus jannaschii

In this study, structural, functional, and mechanistic properties of the Na⁺/H⁺ antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na⁺/H⁺ antiporters are generally responsible for the regulation of cytoplasmic pH and Na⁺ concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive.The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to α-helical segments. In contrast, the temperature-induced structural changes for β-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.     

Imaging clathrin dynamics in Drosophila melanogaster hemocytes reveals a role for actin in vesicle fission

Clathrin-mediated endocytosis (CME) is essential for maintaining many basic cellular processes. We monitored the dynamics of clathrin in live Drosophila melanogaster hemocytes overexpressing clathrin light chain fused to enhanced green fluorescent protein (EGFP) using evanescent wave microscopy. Membrane-associated clathrin-coated structures (CCS) constitutively appeared at the peripheral filopodial membrane, moved centripetally while growing in intensity, before being eventually endocytosed within a few tens of seconds. This directed CCS traffic was independent of microtubules but could be blocked by latrunculin A. Taking advantage of available mutants of Drosophila, we expressed clathrin-EGFP in wasp and shibire mutant backgrounds to study the role of actin and dynamin in CCS dynamics and CME in hemocytes. We show that actin plays an essential role in CME in these cells, and that actin and dynamin act at the same stage, but independent of each other. Drosophila melanogaster hemocytes proved to be a promising model system to uncover the molecular events during CME in combining live-cell imaging and genetic analysis.     

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

Ligand-induced assembling of the type I interferon receptor on supported lipid bilayers

Type I interferons (IFNs) elicit antiviral, antiproliferative and immuno-modulatory responses through binding to a shared receptor consisting of the transmembrane proteins ifnar1 and ifnar2. Differential signaling by different interferons, in particular IFNalphas and IFNbeta, suggests different modes of receptor engagement. Using reflectometric interference spectroscopy (RIfS), we studied kinetics and affinities of the interactions between IFNs and the extracellular receptor domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC). For IFNalpha2, we determined a K(D) value of 3 nM and 5 microM for the interaction with ifnar2-EC and ifnar1-EC, respectively. As compared to IFNalpha2, IFNbeta formed complexes with ifnar2-EC as well as ifnar1-EC with substantially higher affinity. For neither IFNalpha2 nor IFNbeta was stabilization of the complex with ifnar1-EC in the presence of soluble ifnar2-EC observed. We investigated ligand-induced complex formation with ifnar1-EC and ifnar2-EC being tethered onto solid-supported, fluid lipid bilayers by RIfS and total internal reflection fluorescence spectroscopy. We observed very stable binding of IFNalpha2 at high receptor surface concentrations with an apparent k(d) value approximately 200 times lower than that for ifnar2-EC alone. The apparent k(d) value was strongly dependent on the surface concentration of the receptor components, suggesting kinetic stabilization. This was corroborated by the fast exchange of labeled IFNalpha2 bound to the receptor by unlabeled IFNalpha2. Taken together, our results indicate that IFN first binds to ifnar2 and subsequently recruits ifnar1 in a transient fashion. In particular, this second step is much more efficient for IFNbeta than for IFNalpha2, which could explain differential activities observed for these IFNs.     

Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of approximately 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.     

Characterization of hypoglycemiant plants by total reflection X-ray fluorescence spectrometry

In this work, synchrotron radiation total reflection X-ray fluorescence spectrometry (SRTXRF) was used to determine trace elements in eight hypoglycemiant plants (Trigonella foenum graecum, Panax ginseng, Pfaffia paniculata, Myrcia speciosa, Zea mays, Harpagophytum procumbens, Syzygium jambolona, and Bauhinia forficate). The elements P, K, Ca, Ti, Mn, Fe, Cu, Zn, Rb, and Sr were detected in all medicinal plants investigated, whereas Si, S, Sc, V, Cr, Co, Ni, Se, Nb, Mo, Sn, Sb, Ba, Hg, and Pb were detected only in some of the samples. The concentration of elements in hypoglycemiant plants varied from 0.15 μg/g of Co to 3.0×10⁴ μg/g of K and the mean of experimental limit of detection for these elements were 0.14 and 3.6 μg/g, respectively.