The plasma membranes and intracellular membranes of live cells and the artificial membranes of liposomes represent a significant area of application for fluorescent probes. Membrane probes include fluorescent analogs of natural lipids, as well as lipophilic organic dyes that have little structural resemblance to natural biomolecules (Figure 13.2.1 in Fatty Acid Analogs and Phospholipids—Section 13.2). We offer a wide range of both types of membrane probes. These probes are used for structural and biophysical analysis of membranes, for following lipid transport and metabolism in live cells () and for investigating synaptosome recycling (Probes for Following Receptor Binding and Phagocytosis—Section 16.1) and lipid-mediated signal transduction processes (Probes for Signal Transduction—Chapter 17). Due to their low toxicity and stable retention, some lipid probes are particularly useful for long-term cell tracing (Tracers for Membrane Labeling—Section 14.4). Other, slightly less lipophilic probes are used as membrane markers of endocytosis and exocytosis (Probes for Following Receptor Binding and Phagocytosis—Section 16.1).
Fluorescent Analogs of Natural Lipids
We offer fluorescent and, in a few cases, biotinylated analogs of five naturally occurring lipid classes—phospholipids, sphingolipids (including ceramides), fatty acids, triglycerides and steroids. Phospholipids are the principal building blocks of cell membranes. Most phospholipids are derivatives of glycerol comprising two fatty acyl residues (nonpolar tails) and a single phosphate ester substituent (polar head group). Despite their overall structural similarity (Figure 13.1.1), natural phospholipids exhibit subtle differences in their fatty acid compositions, degree of acyl chain unsaturation and type of polar head group. These differences can produce significant variations in membrane physical properties, in the location of phospholipids in a lipid bilayer and in their biological activity. Fluorescent phospholipid analogs (Fatty Acid Analogs and Phospholipids—Section 13.2) can be classified according to where the fluorophore is attached. The fluorophore can be attached to one (or both) of the fatty acyl chains or to the polar head group. The attachment position of the fluorophore determines whether it is located in the nonpolar interior or at the water/lipid interface when the phospholipid analog is incorporated into a lipid bilayer membrane (Figure 13.2.1 in Fatty Acid Analogs and Phospholipids—Section 13.2).
Fatty acids are the building blocks for a diverse set of biomolecules. Some fatty acids (e.g., arachidonic acid) are important in cell signaling. Fatty acids are liberated by the enzymatic action of phospholipase A on phospholipids (Probes for Lipid Metabolism and Signaling—Section 17.4) and also by various other lipases. Fluorescent fatty acids can often be used interchangeably with the corresponding phospholipids as membrane probes; however, fatty acids transfer more readily between aqueous and lipid phases. Although fatty acids are ionized at neutral pH in water (pKa ~5), their pKa is typically ~7 in membranes, and thus a significant fraction of membrane-bound fatty acids are neutral species. Certain fluorescent fatty acids (Fatty Acid Analogs and Phospholipids—Section 13.2) are readily metabolized by live cells to phospholipids, mono-, di- and triacylglycerols, cholesteryl esters and other lipid derivatives.
Sphingolipids play critical roles in processes such as proliferation, apoptosis, signal transduction and molecular recognition at cell surfaces. Defects in the lysosomal breakdown of sphingolipids are the underlying cause of lipid storage disorders such as Niemann–Pick, Tay–Sachs, Krabbe and Gaucher diseases. The sphingolipids described in Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3 include ceramides, sphingomyelins, glycosylceramides (cerebrosides) and gangliosides. The structural backbone of sphingolipids is the lipophilic amino–dialcohol sphingosine (2-amino-4-octadecen-1,3-diol, Figure 13.1.1) to which a single fatty acid residue is attached via an amide linkage. Our fluorescent analogs of sphingolipids are prepared by replacing the natural amide-linked fatty acid with a fluorescent analog. Sphingolipids with an unmodified hydroxyl group in the 1-position are classified as ceramides. As part of the lipid-sorting process in cells, ceramides are glycosylated to cerebrosides (Figure 13.1.1) or converted to sphingomyelins (Figure 13.1.1) in the Golgi complex. Glycosylated sphingolipids (cerebrosides and gangliosides) occur in the plasma membranes of all eukaryotic cells and are involved in cell recognition, signal transduction and modulation of receptor function. Gangliosides have complex oligosaccharide head groups containing at least one sialic acid residue in place of the single galactose or glucose residues of cerebrosides.
Fluorescent cholesteryl esters and triglycerides (Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3) can be used as structural probes and transport markers for these important lipid constituents of membranes and lipoproteins. They may also serve as fluorescent substrates for lipases and lipid-transfer proteins and can be incorporated into low-density lipoproteins (LDL, Probes for Following Receptor Binding and Phagocytosis—Section 16.1).
Figure 13.1.1 A) Phosphatidylcholines, phosphatidylinositols and phosphatidic acids are examples of glycerolipids derived from glycerol. B) Sphingomyelins, ceramides and cerebrosides are examples of sphingolipids derived from sphingosine. In all the structures shown, R represents the hydrocarbon tail portion of a fatty acid residue.
Other Lipophilic and Amphiphilic Fluorescent Probes
The probes described in Dialkylcarbocyanine and Dialkylaminostyryl Probes—Section 13.4 and Other Nonpolar and Amphiphilic Probes—Section 13.5 are not analogs of any particular biological lipid class, but they have a general structural resemblance that facilitates labeling of membranes, lipoproteins and other lipid-based molecular assemblies. Particularly notable members of this group are the lipophilic carbocyanines DiI (), DiO, DiD and DiR, the lipid fluidity probes DPH and TMA-DPH and the membrane-surface probes ANS and laurdan. These probes generally have limited water solubility and exhibit substantially enhanced fluorescence upon partition into lipid environments. They can be classified as either amphiphilic (having both polar and nonpolar structural elements) or neutral (lacking charges and most soluble in very nonpolar environments). We use similar neutral lipophilic dyes for internal staining of our fluorescent polystyrene microspheres (Microspheres—Section 6.5).
In addition to the lipophilic probes described in this chapter, we have available the following products for studying the properties and functions of cell membranes:
- Moderately lipophilic stains for the endoplasmic reticulum and Golgi apparatus (Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4)
- FM dyes—amphiphilic probes for cell membrane labeling (Tracers for Membrane Labeling—Section 14.4, Probes for Following Receptor Binding and Phagocytosis—Section 16.1)
- CellLight Plasma Membrane-CFP, CellLight Plasma Membrane-GFP and CellLight Plasma Membrane-RFP, which are BacMam 2.0 vectors encoding fluorescent proteins targeted to the plasma membrane (C10606, C10607, C10608; Tracers for Membrane Labeling—Section 14.4)
- Alexa Fluor dye–labeled cholera toxin subunit B conjugates for labeling lipid rafts (Protein Conjugates—Section 14.7)
- Annexin V conjugates for detection of phosphatidylserine exposure in apoptotic cell membranes (Assays for Apoptosis—Section 15.5)
- Fluorescent and fluorogenic phospholipase A substrates (Probes for Lipid Metabolism and Signaling—Section 17.4)
- Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit and Amplex Red Phospholipase D Assay Kit (A12218, A12219; Probes for Lipid Metabolism and Signaling—Section 17.4)
- Antibodies to phosphatidylinositol phosphates (Probes for Lipid Metabolism and Signaling—Section 17.4)
- Lipophilic pH indicators (pH Indicator Conjugates—Section 20.4)
- Membrane potential–sensitive probes (Fast-Response Probes—Section 22.2, Slow-Response Probes—Section 22.3)