Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing

In this paper, the Rapid Transfer System (RTS), an attachment to the Leica EMPACT2 high‐pressure freezer, is described as a new tool for special applications within the cryofixation field. The RTS is an automated system that allows for fast processing of samples (<5 s) that make it possible for the first time to use high‐pressure freezing in combination with high time resolution correlative light and electron microscopy. In addition, with a working cycle of 30 s this rapid turn over time allows one to acquire more samples of biopsy material before it deteriorates than with other HPF machines with longer cycle times. With the use of the RTS it was possible to obtain three samples each of four different tissues in 6 min. Together with the finding that 90% of samples of cells grown on sapphire discs were well frozen, the RTS was both fast and reliable. Most important, together with other newly developed accessories, the RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, to cryofix that sample/event within 4 s, and then to analyze that event at high resolution in the electron microscope with excellent preservation of ultra‐structure. These developments should give us the tools to unravel intracellular processes that can be observed by live cell imaging but are too rare or fast to be picked up by routine EM methods or where the history of a structure is necessary to be able to discern its nature.     

Identification of Receptor‐Interacting Regions of Vitellogenin within Evolutionarily Conserved β‐Sheet Structures by Using a Peptide Array

Vitellogenesis, a key process in oviparous animals, is characterized by enhanced synthesis of the lipoprotein vitellogenin, which serves as the major yolk‐protein precursor. In most oviparous animals, and specifically in crustaceans, vitellogenin is mainly synthesized in the hepatopancreas, secreted to the hemolymph, and taken up into the ovary by receptor‐mediated endocytosis. In the present study, localization of the vitellogenin receptor and its interaction with vitellogenin were investigated in the freshwater prawn Macrobrachium rosenbergii. The receptor was immuno‐histochemically localized to the cell periphery and around yolk vesicles. A receptor blot assay revealed that the vitellogenin receptor interacts with most known vitellogenin subunits, the most prominent being the 79 kDa subunit. The receptor was, moreover, able to interact with trypsin‐digested vitellogenin peptides. By combining a novel peptide‐array approach with tandem mass spectrometry, eleven vitellogenin‐derived peptides that interacted with the receptor were identified. A 3D model of vitellogenin indicated that four of the identified peptides are N‐terminally localized. One of the peptides is homologous to the receptor‐recognized site of vertebrate vitellogenin, and assumes a conserved β‐sheet structure. These findings suggest that this specific β‐sheet region in the vitellogenin N‐terminal lipoprotein domain is the receptor‐interacting site, with the rest of the protein serving to enhance affinity for the receptor. The conservation of the receptor recognition site in invertebrate and vertebrate vitellogenin might have vast implications for oviparous species reproduction, development, immunity, and pest management.     

Absence of See1p,a widely conserved Saccharomyces cerevisiae protein,confers both deficient heterologous protein production and endocytosis

The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein, recognized by sequence similarity as a putative S‐adenosyl‐L ‐methionine methyltransferase. A micro‐scale screening performed in our laboratory with the EUROSCARF S. cerevisiae BY4741 deletion mutant collection identified YIL064w deletion as negatively affecting secretory production of reporter α‐amylase. The work presented here corroborates the later observations of the yil064w mutant in a larger‐scale assay and shows that Yil064p is necessary for the efficient secretory production of two reporter proteins, murine α‐amylase and fungal polygalacturonase. Further, we analysed endocytosis in the yil064w mutant strain and observed defects at both very early and later stages of endocytic transport in cells in the late logarithmic phase. The defects at very early stages may decisively account for the low transfection (DNA uptake by endocytosis) efficiency that we also observed in the yil064w mutant. These are the first in vivo data reporting a functional role for the protein encoded by ORF YIL064w and identify Yil064p, named here s ecretion and e arly e ndocytosis 1 protein (See1p), as a novel component of intracellular transport. Copyright © 2008 John Wiley & Sons, Ltd.     

Membrane Trafficking of Death Receptors: Implications on Signalling

Death receptors were initially recognised as potent inducers of apoptotic cell death and soon ambitious attempts were made to exploit selective ignition of controlled cellular suicide as therapeutic strategy in malignant diseases. However, the complexity of death receptor signalling has increased substantially during recent years. Beyond activation of the apoptotic cascade, involvement in a variety of cellular processes including inflammation, proliferation and immune response was recognised. Mechanistically, these findings raised the question how multipurpose receptors can ensure selective activation of a particular pathway. A growing body of evidence points to an elegant spatiotemporal regulation of composition and assembly of the receptor-associated signalling complex. Upon ligand binding, receptor recruitment in specialized membrane compartments, formation of receptor-ligand clusters and internalisation processes constitute key regulatory elements. In this review, we will summarise the current concepts of death receptor trafficking and its implications on receptor-associated signalling events.     

FRET analysis of transmembrane flipping of FM4-64 in plant cells: is FM4-64 a robust marker for endocytosis?

Although the styryl dye FM4–64 is now used routinely to monitor endocytosis in plants, the argument about its potential to cytoplasmically and non-endocytically relocate into a selective set of vesicular compartments persists. To address this question, we determined whether fluorescence resonance energy transfer (FRET) could occur between a cytoplasmically expressed, short-wavelength excitation green fluorescent protein (GFP) and FM4–64 in Nicotiana benthaminana . After exposure to FM4–64, the root hair plasma membrane and internal organelles became labelled. Under these conditions, no FRET with cytoplasmic GFP was seen. However, if the cells were treated with a low concentration of quillajasaponin, a membrane permeabilization agent, the cells continued to stream and FRET was detected. Thereby, we demonstrate that under conditions that do not severely compromise cell viability, the FM4–64 dye becomes a suitable FRET partner for the cytoplasmically localized GFP. Under normal conditions, FM4–64 does not significantly enter the cytosolic side of the membrane, but remains at the plasma membrane or trapped in the organelles of the endocytic pathway. Hence, when the structure or permeability of the plasma membrane is unaltered, FM4–64 dye is a robust marker for endocytosis.     

Liposomal Treatment of Cancer Cells Modulates Uptake Pathway of Polymeric Nanoparticles by Altering Membrane Stiffness

Nanomedicines can be taken up by cells via nonspecific and dynamin‐dependent (energy‐dependent) clathrin and caveolae‐mediated endocytosis. While significant effort has focused on targeting pathway‐specific transporters, the role of nanobiophysics in the cell lipid bilayer nanoparticle uptake pathway remains largely unexplored. In this study, it is demonstrated that stiffness of lipid bilayer is a key determinant of uptake of liposomes by mammalian cells. Dynamin‐mediated endocytosis (DME) of liposomes is found to correlate with its phase behavior, with transition toward solid phase promoting DME, and transition toward fluidic phase resulting in dynamin‐independent endocytosis. Since liposomes can transfer lipids to cell membrane, it is sought to engineer the biophysical properties of the membrane of breast epithelial tumor cells (MD‐MBA‐231) by treatment with phosphatidylcholine liposomes, and elucidate its effect on the uptake of polymeric nanoparticles. Analysis of the giant plasma membrane vesicles derived from treated cells using flicker spectroscopy reveals that liposome treatment alters membrane stiffness and DME of nanoparticles. Since liposomes have a history of use in drug delivery, localized priming of tumors with liposomes may present a hitherto unexploited means of targeting tumors based on biophysical interactions.     

Carbon Nanotubes in the Biological Interphase: The Relevance of Noncovalence

With the increasing interest in the biological applications of carbon nanotubes, their interactions in the biological interphase and their general cytotoxicity have become major issues. In spite of their salient properties, major hurdles still exist for their use in biological applications, due to their main characteristics, including their hydrophobic surfaces and tendency to aggregate, as well as their unknown interactions in the cellular interphase. In this Research News, these characteristics of carbon nanotubes, a model nanomaterial, are investigated. Thus, the cytotoxicity of carbon nanotubes, the influence of functionalization, as well as their interactions with different mammalian cell lines are studied. Moreover, suggestions for the improvement of their biocompatibility and the design of biocompatible carbon nanotube‐based systems are provided.     

Role of Soil and Foliar-Applied Carbon Dots in Plant Iron Biofortification and Cadmium Mitigation by Triggering Opposite Iron Signaling in Roots

In China, iron (Fe) availability is low in most soils but cadmium (Cd) generally exceeds regulatory soil pollution limits. Thus, biofortification of Fe along with mitigation of Cd in edible plant parts is important for human nutrition and health. Carbon dots (CDs) are considered as potential nanomaterials for agricultural applications. Here, Salvia miltiorrhiza-derived CDs are an efficient modulator of Fe, manganese (Mn), zinc (Zn), and Cd accumulation in plants. CDs irrigation (1 mg mL⁻¹, performed every week starting at the jointing stage for 12 weeks) increased Fe content by 18% but mitigated Cd accumulation by 20% in wheat grains. This finding was associated with the Fe³⁺-mobilizing properties of CDs from the soil and root cell wall, as well as endocytosis-dependent internalization in roots. The resulting excess Fe signaling mitigated Cd uptake via inhibiting TaNRAMP5 expression. Foliar spraying of CDs enhanced Fe (44%), Mn (30%), and Zn (19%) content with an unchanged Cd accumulation in wheat grains. This result is attributed to CDs-enhanced light signaling, which triggered shoot-to-root Fe deficiency response. This study not only reveals the molecular mechanism underlying CDs modulation of Fe signaling in plants but also provides useful strategies for concurrent Fe biofortification and Cd mitigation in plant-based foods.