Lipids are characteristically different from proteins, making in vivo identification and labeling more difficult. Additionally, the complexity of lipid biosynthesis prevents the use of techniques analogous to protein research, such as overexpression and radiolabeling, as many different lipid species may be affected due to their similar pathways. One of the first notable techniques for tagging lipids originated in the early 1980’s when one of the fatty acids in phospholipid and glycosphingolipids-like analogues were replaced by 6 carbon tethers that were also connected to a fluorescent probe [1,2,3,4]. Lipid labeling offers an alternative to destructive methods that use detergents to induce phase separation or separate membranes, allowing for measurement made in native environments without immobilization .
Fluorescent Lipid Probes
Fluorescent probes are molecules associated with lipid systems that can be excited to emit energy that can be measured to identify and track specific lipids. They can be divided into three categories: probes that target lipid components (such as gangliosides), probes that flexibly associate with lipids despite their phase, and probes that have ordered or disordered phase preferentiality . The first of these more specific groups consists mainly of fluorescent proteins that target lipid components, like cholesterol, through inactivation mechanisms; this is the smallest group of lipid probes [6,7]. These offer bright and photostable characteristics that are not very sensitive to environmental polarity or viscosity and are often the optimum dye to work with fluorescence correlation spectroscopy, single molecule imaging and other super-resolution techniques [8,9,10,11].
The second group includes lipid-like dyes that associate with lipid membranes due to labeling of lipids or lipophilic structures of non-lipid molecules that can demonstrate similar phases of biological membranes, such as the ordered and disordered phases seen with lipid rafts . Fluorescent lipid probes are chemical markers that can bind to certain functional groups that belong to one of the two amphiphilic structures found in lipids: the hydrophilic headgroup or the hydrophobic tail. The function of study for the lipid will depend on the choice of dye, as the additional probe will chemically affect the local functionalization by changing hydrophilic/phobic balance while also sterically impeding lipid packing in membranes. Head group-associated probes are useful for research where lipid head groups play a significant role, such as lipid sorting, while chain-associated probes are useful for cases when chain regions are important, like studying membrane fluidity [13, 14]. The insertion of these marked lipids into the membrane or system of study is then facilitated by the lipid base of the probe. These tagged lipids have preferential association with certain membrane phases and thus will show varying fluorescence depending on the environment, as well as affecting membrane stability [15, 16, 17]. Additionally, lipophilic probes that consist of non-lipid structures can take the form of long-chain hydrocarbons (LCH dyes) or poly-cyclic aromatic hydrocarbons (PAH dyes) can also be used . LCH dyes include alkylated cyanines and rhodamines, which have more similar structures to lipids than PAH dyes that consist mainly of neutral aromatic compounds that lack alkyl chains . These probes are often used with fluorescence correlation spectroscopy and single molecule imaging [18, 19].
The third group of dyes, commonly referred to as environment-sensitive dyes, respond spectroscopically to local environment properties like polarity, hydration, viscosity and pH while also being able to distinguish between ordered and disordered membrane phases [20, 21]. These can be further categorized into solvatochromic probes that have fluorescence that responds to the environmental polarity and viscosity-sensitive, molecular rotor probes that vary their fluorescence intensity and duration with local viscosity. Solvatochromic dyes rely on changes in dipole-dipole or hydrogen bonding interactions with their environment that affect the maxima of their excitation/emission spectra to give optical responses. These probes are used most often with ratiometric or lifetime measurements due to the change in their emission spectra and lifetime as a response to their local membrane phase [14, 22]. Molecular rotors rely on local lipid interactions, which are directly related to membrane viscosity to affect the intramolecular rotation of the fluorophore and thus the fluorescence quantum yield (or the ratio of number of photons emitted to the number of photons absorbed) [23, 15]. These probes are also used with fluorescence lifetime measurements due to the relationship between their fluorescence lifetime and local viscosity [24, 25].
|Name||Absorbance Max (nm)||Fluorescence Max (nm)||Partitioning in giant unicellular vesicles||Partitioning in giant plasma membrane vesicles|
|TF-Chol||495||507||Lo (A, 80%)||Lo (66%)|
|PE head group labeled|
|Texas Red-DPPE||595||614||Ld (A)||–|
|PC acyl chain labeled|
|SM acyl chain labeled|
|5-BODIPY-SM||505||512||Ld (A, 78%)||–|
|12-BODIPY-SM||505||512||Ld (A, 69%)||Lo (66%)|
|6-NBD-SM||470||538||Ld (A, 88%)||Ld (54%)|
|12-NBD-SM||470||538||Ld (A, 95%)||Ld (65%)|
|4-Atto647N-SM||644||669||Ld (A, 97%)||Ld (82%)|
|4-Atto532-SM||532||552||Ld (A, 90%)||Ld (53%)|
|SM head group labeled|
|SM-Atto647N||644||669||Ld (A, 97%)||Ld (85%)|
|SM-Atto532||532||552||Ld (A, 88%)||Ld (62%)|
|DiI-C18||550||568||Ld (A), Lo (B)||–|
|FAST DiO||490||505||Ld (A)||–|
|DPH||350||452||Lo/Ld (A, B)||–|
|5-BODIPY-GM1||505||512||Ld (A, 79%)||Lo (65%)|
|6-NBD GM1||470||538||Ld (A, 75%)||Lo (67%)|
* demonstrate some preference for the lipid disordered phase. Partioning of probes in giant unicellular vesicles (GUVs) for the following lipid mixtures: A, SM/DOPC/cholesterol; B, DSPC/DOPC/cholesetrol; C, DPPC/DQPC/cholesterol; D, DPPC/PhyPC/cholesterol.
A spin probe is molecule with an unpaired electron that can associated to another molecule and can be used with electron paramagnetic resonance (EPR) spectroscopy to identify lipid membrane dynamics. Common species include nitroxide radicals associated with either the hydrophilic headgroup or the hydrophobic chains of the lipid molecules that can then be identified by EPR to determine membrane fluidity through correlation with line-width and line-shape, which can describe local environmental influences on the labeled lipids . Other available spin probes applicable to lipids include TEMPO and DTBN, which are notably water-soluble and can interact with the aqueous-hydrocarbon interface . Spin labels are used in low concentrations in lipid systems to avoid spin-spin interactions and prevent membrane disruption. Common experiments involve measuring the response of chain-labeled lipids as the labelling group moves down the acyl chain of lipid segments away from the headgroup, which demonstrates an increase in spin the further away with increasing distance from the headgroup [28, 29, 30].
2H, 13C, 15N isotopes are introduced into specific metabolic pathways in biological systems and are used as labels for both lipid intermediate and products [31, 32, 33]. They replace the more commonly found atom (1H, 12C, 14N) in certain molecules generally without significantly affecting any biological processes while providing information for how those molecules are synthesized and transported. For plants and bacterial lipids, 34S provides another source of isotopic labelling due to the prevalence of sulfolipids . These isotopic labeled molecules can then be identified using nuclear magnetic resonance (NMR) or mass spectrometry (MS), which take advantage of non-zero net spins characteristic of deuterium and carbon-13 isotopes or the difference in mass/charge ratio attributable to the excess neutron in the isotope, respectively. A major benefit of using MS is being able to couple the use of several isotopic labels with chromatography methods to label the same molecule through comparison of mass/charge ratios and chromatographic retention times, which may be difficult with a single method due to the similarity in lipid structure and synthesis pathways .
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