Subcellular imaging of lipids and sugars using genetically encoded proximity sensors.

Live cell imaging of lipids and other metabolites is a long-standing challenge in cell biology. Bioorthogonal labeling tools allow for the conjugation of fluorophores to several phospholipid classes, but cannot discern their trafficking between adjacent organelles or asymmetry across individual membrane leaflets. Here we present fluorogen-activating coincidence sensing (FACES), a chemogenetic tool capable of quantitatively imaging subcellular lipid pools and reporting their transbilayer orientation in living cells.

Probing Glycerolipid Metabolism using a Caged Clickable Glycerol-3-Phosphate Probe.

In this study, we present the probe SATE-G3P-N as a novel tool for metabolic labeling of glycerolipids (GLs) to investigate lipid metabolism in yeast cells. By introducing a clickable azide handle onto the glycerol backbone, this probe enables general labeling of glycerolipids. Additionally, this probe contains a caged phosphate moiety at the glycerol sn-3 position to not only facilitate probe uptake by masking negative charge but also to bypass the phosphorylation step crucial for initiating phospholipid synthesis, thereby enhancing phospholipid labeling.

The small molecule CBR-5884 inhibits the phosphatidylserine synthase.

Unlabelled: Systemic infections by spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens.

Harnessing Clickable Acylated Glycerol Probes as Chemical Tools for Tracking Glycerolipid Metabolism.

We report the use of clickable monoacylglycerol (MAG) analogs as probes for the labeling of glycerolipids during lipid metabolism. Incorporation of azide tags onto the glycerol region was pursued to develop probes that would label glycerolipids, in which the click tag would not be removed through processes including acyl chain and headgroup remodeling. Analysis of clickable MAG probes containing acyl chains of different length resulted in widely variable cell imaging and cytotoxicity profiles.

Development of GTP-responsive liposomes by exchanging the metal-DPA binding site in a synthetic lipid switch.

We report stimuli-responsive liposomes that selectively release encapsulated contents upon treatment with guanosine triphosphate (GTP) over a wide variety of phosphorylated metabolites, validated by fluorescence-based leakage assays. Significant changes in liposome self-assembly properties were also observed. Our results showcase the potential of this platform for triggered release applications.

Cellular Labeling of Phosphatidylserine Using Clickable Serine Probes.

Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amino (l) or carbonyl (l) groups. The conservative click tag modification enabled these compounds to infiltrate normal lipid biosynthetic pathways, thereby producing tagged PS molecules as supported by mass spectrometry studies, thin-layer chromatography (TLC) analysis, and further derivatization with fluorescent reporters via click chemistry to enable imaging in yeast cells.

Metabolic labeling of glycerophospholipids via clickable analogs derivatized at the lipid headgroup.

Metabolic labeling, in which substrate analogs containing diminutive tags can infiltrate biosynthetic pathways and generate labeled products in cells, has led to dramatic advancements in the means by which complex biomolecules can be detected and biological processes can be elucidated. Within this realm, metabolic labeling of lipid products, particularly in a manner that is headgroup-specific, brings about a number of technical challenges including the complexity of lipid metabolic pathways as well as the simplicity of biosynthetic precursors to headgroup functionality. As such, only a handful of strategies for metabolic labeling of lipids have thus far been reported.