Bioorthogonal probes for imaging sterols in cells.

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.

Biosynthetic labeling and two-color imaging of phospholipids in cells.

Phospholipids with a choline head group are abundant components of all biological membranes, performing critical functions in cellular structure, metabolism, and signaling. In spite of their importance, our ability to visualize choline phospholipids in vivo remains very limited. We present a simple and robust chemical strategy to image choline phospholipids, based on the metabolic incorporation of azidocholine analogues, that accurately reflects the normal biosynthetic incorporation of choline into cellular phospholipids. Azidocholine-labeled phospholipids can be imaged in cells with high sensitivity and resolution, following derivatization with fluorophores, by bio-orthogonal chemical reactions compatible with live-cell imaging. We used this method to visualize the subcellular localization of choline phospholipids. We also demonstrate that double metabolic labeling with azidocholine and propargylcholine allows sensitive two-color imaging of choline phospholipids. Our method represents a powerful approach to directly image phospholipids, and to study their dynamics in cells and tissues.

Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand.

The Hedgehog (Hh) signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp) and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand.

Metabolic labeling and direct imaging of choline phospholipids in vivo.

Choline (Cho)-containing phospholipids are the most abundant phospholipids in cellular membranes and play fundamental structural as well as regulatory roles in cell metabolism and signaling. Although much is known about the biochemistry and metabolism of Cho phospholipids, their cell biology has remained obscure, due to the lack of methods for their direct microscopic visualization in cells. We developed a simple and robust method to label Cho phospholipids in vivo, based on the metabolic incorporation of the Cho analog propargylcholine (propargyl-Cho) into phospholipids. The resulting propargyl-labeled phospholipid molecules can be visualized with high sensitivity and spatial resolution in cells via a Cu(I)-catalyzed cycloaddition reaction between the terminal alkyne group of propargyl-Cho and a labeled azide. Total lipid analysis of labeled cells shows strong incorporation of propargyl-Cho into all classes of Cho phospholipids; furthermore, the fatty acid composition of propargyl-Cho-labeled phospholipids is very similar to that of normal Cho phospholipids. We demonstrate the use of propargyl-Cho in cultured cells, by imaging phospholipid synthesis, turnover, and subcellular localization by both fluorescence and electron microscopy. Finally, we use propargyl-Cho to assay microscopically phospholipid synthesis in vivo in mouse tissues.

Exploring RNA transcription and turnover in vivo by using click chemistry.

We describe a chemical method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddition reaction (often referred to as "click" chemistry) with fluorescent azides, followed by microscopic imaging. We demonstrate the use of this method in cultured cells, in which we examine the turnover of bulk RNA after EU pulses of varying lengths. We also use EU to assay transcription rates of various tissues in whole animals, both on sections and by whole-mount staining. We find that total transcription rates vary greatly among different tissues and among different cell types within organs.

Glycosaminoglycan composition of the vocal fold lamina propria in relation to function.

OBJECTIVES: This study was designed to quantify the specific glycosaminoglycans (GAGs) in the midmembranous vocal fold (VF) lamina propria (LP) and to interpret their presence in relation to the known stresses borne by each LP layer. METHODS: GAGs from normal human LP and from both normal and scarred canine LPs were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). Immunostaining was conducted to give insight into the spatial distribution of each GAG type. RESULTS: Hyaluronan composes roughly 0.64% +/- 0.41% of the human LP as measured relative to tissue total protein. Chondroitin sulfate and/or dermatan sulfate (CS/DS), keratan sulfate, and heparan sulfate chains constitute approximately 23.9% +/- 12.1%, 14.7% +/- 6.1%, and 61.4% +/- 13.6%, respectively, of human LP sulfated GAGs. CONCLUSIONS: Observed CS/DS sulfation patterns imply that versican is a major contributor to human LP CS levels. In addition, examination of LP GAG with respect to gender revealed a significant variation in total levels of CS/DS and a potential difference in the levels of versican relative to decorin and biglycan. In dogs, LP scarring appeared to result in a reduction in hyaluronan and CS/DS. These FACE results were combined with histologic data to update current descriptive models linking LP microstructure with the regional variations in LP loading.

Genetically encoded fluorescent reporters of histone methylation in living cells.

We report the design and characterization of two genetically encoded fluorescent reporters of histone protein methylation. The reporters are four-part chimeric proteins consisting of a substrate peptide from the N-terminus of histone H3 fused to a chromodomain (a natural methyllysine-specific recognition domain), sandwiched between a fluorescence resonance energy transfer (FRET)-capable pair of fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Enzymatic methylation by a methyltransferase induces complexation of the methylated substrate peptide to the chromodomain, changing the FRET level between the flanking CFP and YFP domains. Reporters developed using the chromodomains from HP1 and Polycomb respond to enzymatic methylation at the lysine 9 and lysine 27 positions of histone H3, respectively, giving 60% and 28% YFP/CFP emission ratio increases in vitro or in single living cells. These reporters should be useful for studying gene silencing and X-chromosome inactivation with high spatial and temporal resolution in intact cells and may also aid in the search for conjectured histone demethylase activity.