Lipid droplets lighting up: Insights from live microscopy

Lipid droplets emerge as important intracellular organelles relevant for lipid homeostasis and the pathophysiology of metabolic diseases. Here, we present a personal view on the current knowledge about the biogenesis of mammalian cytoplasmic lipid droplets, with a focus on microscopy and especially live imaging. We also discuss difficulties related to the lipid droplet proteome, contentious views on lipid droplet growth, and last but not least the evidence for the heterogeneity of lipid droplets within a single cell. We conclude with an outline of the most important future challenges.     

Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes

Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)₃ using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; K_Tel22 ≈ <K_dsDNA) and for highly selective quadruplex-specific ligands (Phen-DC3, 360A-Br; K_Tel22 > K_dsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation.     

Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells

In the early events of human immunodeficiency virus type 1 (HIV-1) infection, immature dendritic cells (DCs) expressing the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptor capture small amounts of HIV-1 on mucosal surfaces and spread viral infection to CD4(+) T cells in lymph nodes (22, 34, 45). RNA interference has emerged as a powerful tool to gain insight into gene function. For this purpose, lentiviral vectors that express short hairpin RNA (shRNA) for the delivery of small interfering RNA (siRNA) into mammalian cells represent a powerful tool to achieve stable gene silencing. In order to interfere with DC-SIGN function, we developed shRNA-expressing lentiviral vectors capable of conditionally suppressing DC-SIGN expression. Selectivity of inhibition of human DC-SIGN and L-SIGN and chimpanzee and rhesus macaque DC-SIGN was obtained by using distinct siRNAs. Suppression of DC-SIGN expression inhibited the attachment of the gp120 envelope glycoprotein of HIV-1 to DC-SIGN transfectants, as well as transfer of HIV-1 to target cells in trans. Furthermore, shRNA-expressing lentiviral vectors were capable of efficiently suppressing DC-SIGN expression in primary human DCs. DC-SIGN-negative DCs were unable to enhance transfer of HIV-1 infectivity to T cells in trans, demonstrating an essential role for the DC-SIGN receptor in transferring infectious viral particles from DCs to T cells. The present system should have broad applications for studying the function of DC-SIGN in the pathogenesis of HIV as well as other pathogens also recognized by this receptor.     

Detecting RNA viruses in living mammalian cells by fluorescence microscopy

Traditional methods that rely on viral isolation and culture techniques continue to be the gold standards used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells.     

Imaging dynamics of endogenous mitochondrial RNA in single living cells

We developed genetically encoded RNA probes for characterizing localization and dynamics of mitochondrial RNA (mtRNA) in single living cells. The probes consist of two RNA-binding domains of PUMILIO1, each connected with split fragments of a fluorescent protein capable of reconstituting upon binding to a target RNA. We designed the probes to specifically recognize a 16-base sequence of mtRNA encoding NADH dehydrogenase subunit 6 (ND6) and to be targeted into the mitochondrial matrix, which allowed real-time imaging of ND6 mtRNA localization in living cells. We showed that ND6 mtRNA is localized within mitochondria and concentrated particularly on mitochondrial DNA (mtDNA). Movement of the ND6 mtRNA is restricted but oxidative stress induces the mtRNA to disperse in the mitochondria and gradually decompose. These probes provide a means to study spatial and temporal mRNA dynamics in intracellular compartments in living mammalian cells.     

Imaging proteolysis by living human glioma cells

Degradation of basement membrane is an essential step for tumor invasion. In order to study degradation in real time as well as localize the site of proteolysis, we have established an assay with living human cancer cells in which we image cleavage of quenched-fluorescent basement membrane type IV collagen (DQ-collagen IV). Accumulation of fluorescent products is imaged with a confocal microscope and localized by optically sectioning both the cells and the matrix on which they are growing. For the studies described here, we seeded U87 human glioma cells as either monolayers or spheroids on a 3-dimensional gelatin matrix in which DQ-collagen IV had been embedded. As early as 24 hours after plating as monolayers, U87 cells were present throughout the 3-dimensional matrix. Cells at all levels had accumulated fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Similar observations were made for U87 spheroids and the individual cells migrating from the spheroids into the gelatin matrix. Both the migrating cells and those within the spheroid contained fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Thus, glioma cells like breast cancer cells are able to degrade type IV collagen intracellularly, suggesting that this is an important pathway for matrix degradation.     

Remodelling epithelial tubes through cell rearrangements: from cells to molecules

Epithelial cell movements, such as those that occur during cell intercalation, largely contribute to the formation of epithelial structures during the morphogenesis of multicellular organisms. As the architecture of epithelial tissues relies on strong adhesion between cells at adherens junctions (AJs), the intercalation or rearrangements of epithelial cells might be controlled by modulating the adhesion dynamics of the AJs by internal or external forces. In this review, we describe recent progress in understanding cell rearrangements during epithelial tube remodelling and discuss several models that might account for the developmental control of the spatial dynamics of AJs.     

Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.     

Hexokinase binding to polypropylene test tubes: Artifactural activity losses from protein binding to disposable plastics

In the course of our studies with rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), we have frequently used disposable polypropylene centrifuge tubes. As long as relatively crude systems with substantial amounts of extraneous protein were used, no difficulties were encountered. However, with more purified preparations, substantial apparent activity losses were incurred. Investigation of the situation disclosed that the reason for these apparent losses was adsorption of the hexokinase to polypropylene vessels, an unusual adsorption that appeared to be particularly effective at the air-water interface. Subsequently, we found similar effects with other enzymes. Although binding of some proteins, such as ribonuclease (1,2), to glassware has been reported, we do not know of a similar phenomenon being described for plastic ware. The increased use of disposable plastic laboratory ware necessitates an awareness of this potential problem. We present here the results of our study of the adsorption of hexokinase to polypropylene.     

RNA interference in human cells is restricted to the cytoplasm

RNA interference (RNAi) is an evolutionarily conserved eukaryotic adaptive response that leads to the specific degradation of target mRNA species in response to cellular exposure to homologous double-stranded RNA molecules. Here, we have analyzed the subcellular location at which RNA degradation occurs in human cells exposed to double-stranded short interfering RNAs. To unequivocally determine whether a given mRNA is subject to degradation in the cytoplasm, the nucleus, or both, we have used the retroviral Rev/RRE system to control whether target mRNAs remain sequestered in the nucleus or are exported to the cytoplasm. In the absence of export, we found that the nuclear level of the RRE-containing target mRNA was not affected by activation of RNAi. In contrast, when nuclear export was induced by expression of Rev, cytoplasmic target mRNAs were effectively and specifically degraded by RNAi. Curiously, when the target mRNA molecule was undergoing active export from the nucleus, induction of RNAi also resulted in a reproducible approximately twofold drop in the level of target mRNA present In the nuclear RNA fraction. As this same mRNA was entirely resistant to RNAi when sequestered in the nucleus, this result suggests that RNAi is able to induce degradation of target mRNAs not only in the cytoplasm but also during the process of nuclear mRNA export. Truly nucleoplasmic mRNAs or pre-mRNAs are, in contrast, resistant to RNAi.     

Small RNA flow from tapetum cells to germ cells in plants

In plants and animals,male gamete differentiates and ferti-lizes an egg cell,which develops into different types of specialized cells,tissues,and ultimately an ...     

Microspectrofluorometry of autofluorescence emission from human leukemic living cells under oxidative stress.

Image cytometry was applied to study the intracellular localization of autofluorescence and the influence of an oxidative stress on this emission. K562 erythroleukemia cancer cells were analyzed with a microspectrofluorometer, coupled with a Argon laser (Ar+) (363 nm). From each cell, 15 x 15 emission spectra were recorded in the 400-600 nm spectral range to generate a spectral image of autofluorescence. The intracellular locations of the autofluorescence emission and of the specific mitochondrial probe rhodamine 123 (R123) were matched. Under a 363 nm excitation, all spectra from K562 cells show equivalent profiles with a 455 nm maximum emission, near of reduced nicotinamide adenine dinucleotide-(Phosphate) solution (NAD(P)H) (465 nm maximum emission). The spatial distribution of autofluorescence is homogeneous and different from the one of R123. Hydrogen peroxide (H2O2) (200 microM) and menadione (Men) (5 microM) induce a weak spectral change and a decrease in autofluorescence intensity, down to 40% of the initial emission. Doxorubicin (Dox) induces a dose-dependent decrease in autofluorescence emission and a release of intracellular free radicals. When cells were pre-treated 1 h with 1 mM glutathione (GSH), Dox induces a lower free radicals release, no significant variation of autofluorescence intensity and a lower growth inhibitory effect. Images cytometry of autofluorescence suggest that the intracellular NAD(P)H would not be restricted to mitochondrial compartments. The release of free radicals was associated with a decrease in autofluorescence intensity, mainly attributed to NAD(P)H oxidation both inside and outside mitochondria.     

Magnetic detection to position human nasogastric tubes

Placement of nasogastric tubes is one of the most commonly performed diagnostic and therapeutic medical procedures. Proper placement of the tube in the digestive tract below the diaphragm is crucial for efficacy and safety. This study evaluates a magnet detection system that allows percutaneous non-radiographic localization of the nasogastric tube tip. Each volunteer subject had the magnet detector placed over the abdomen, and was then intubated with a magnet-tagged nasogastric tube. Eighty-eight nasogastric tube placements were performed in 22 volunteers. The detection system located the nasogastric tube tip below the diaphragm in all 88 placements. Location in all attempts was confirmed by fluoroscopy. This method of correctly locating the tip of nasogastric tubes may obviate the need for radiographic imaging in most cases.