Plasma membrane fluidity gradients of human peripheral blood leukocytes

Collins, James M.; Grogan, W. McLean; Scott, Robert B.

doi:10.1002/jcp.1041440107

Cited by 11 publications

(4 citation statements)

References 55 publications

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“…These were, expressed as percentages of the 2AF value, 100% (2AF), 96% (3AF), 7 8 8 (6AF), 70% (7AF), 61% (9AF), 49% (12AF), and 47% (16AF). This curious correlation of decreasing amount of uptake with increasing n-number has been noted previously (7)(8)(9)14). Figure 1B shows the same data expressed as percentage of quenching and indicates that the order of quenching is, with respect to n, 2 2 3 > 6 2 7 > 9 > 12 > 16.…”

Section: Resultssupporting

confidence: 79%

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Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Collins

Grogan

1991

Cytometry

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The transverse location normal to the bilayer surface of a series of n-(g-anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cuc2, was 2 2 3 > 6 2 7 > 9 > 12 > 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells. Key terms: Anthroyloxy fatty acids, membrane fluidity, HeLaThe introduction by Waggoner and Stryer (22) of a fluorescent fatty acid, 12-(9-anthroyloxy) stearate, as a membrane probe able to locate a t a defined depth, offered a potentially useful approach to understanding the dynamics of the outer hemi-leaflet of the membrane bilayer; other members of the n-AF, or n-(9-anthroyloxy) fatty acid, series quickly followed. At the outset these probes were expected to tether their negatively charged carboxyl groups a t the surface in contact with the aqueous phase, with the anthroyloxy moieties thus locating a t different depths. There is presently little doubt that the n-AF probes indeed locate at different depths in model lipid bilayers. In dipalmitoylphosphatidyl lecithin vesicles, NMR spectra detected a chemical shift near the first and second methylene groups of the lipid acyl chains with 2-(9-anthroyloxy) palmitate, thus placing the absolute position of this anthroyloxy group level with the acyl chain's ester linkage, near the surface (19). With 12-(9-anthroyloxy) stearate, the shift occurred for the terminal methylene groups of the acyl chains, thus placing that, anthroyloxy group nearer the bilayer center (19). In dipalmitoylphosphatidyl choline bilayers, quenching agents of fluorescence, which locate at the surface or a t the bilayer center, revealed that 2-AF had portions of the anthroyloxy moiety in both the aqueous and the lipid phases, whereas 6-AF was entirely in the lipid phase (21). In model lipid bilayers, with spin-labeled fatty acids as paramagnetic quenchers, the order of quenching with respect to alkyl chain position, n, was always 2 > 6 > 9 > 12 > 16 (1). The aqueous quenching of anthroyloxy fluorescence in isolated beef mitochondria by C u f " was in the order, 2 > 6 > 9 > 12 > 16, whereas with dimethylaniline, a quencher that partitions into the lipid phase near the bilayer center, the order was reversed (5).Therefore, the most reasonable interpretation of these studies is that the n-AF probes appear to locate in a graded series of depths from the surface to the center of the lipid bilayer, at least in model systems. However, this has never been demonstrated with intact, living cells. The recent adaptation of commercial flow cytometers for pol...

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Section: Resultssupporting

confidence: 79%

“…Attempts to use a quencher that preferentially partitions into the lipid phase, N,N-dimethyl aniline (21), were unsuccessful as the amounts necessary to obtain quenching, 20-200 pM, produced immediate membrane perturbation as evidenced by the rapid loss of cellular light scatter properties (7)(8)(9).…”

Section: Resultsmentioning

confidence: 99%

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Collins

Grogan

1991

Cytometry

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“…Fatty acids are known to play diverse roles for immune cell function acting as a source of carbon for oxidation to CO 2 (Newsholme et al, 1987), as structural components of cell membrane phospholipids (Collins et al, 1990), as signaling molecules and as precursors for the synthesis of eicosanoids and other mediators (Nieves and Moreno, 2006). High concentration of fatty acids, however, are known to be toxic to cells leading to impairment of their function inhibiting cell activation, cytokine production, and proliferation (Anel et al, 1990;Purasiri et al, 1997).…”

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confidence: 98%

Activation of survival and apoptotic signaling pathways in lymphocytes exposed to palmitic acid

Takahashi

Cambiaghi

Luchessi

et al. 2011

Journal Cellular Physiology

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The toxicity of palmitic acid (PA) towards a human T-lymphocyte cell line (Jurkat) has been previously investigated but the mechanism(s) of PA action were unknown. In the current study, Jurkat cells were treated with sub-lethal concentrations of PA (50-150µM) and the activity of various signaling proteins was investigated. PA-induced apoptosis and mitochondrial dysfunction in a dose-dependent manner as evaluated by DNA fragmentation assay and depolarization of the mitochondrial membrane, respectively. PA treatment provoked release of cytochrome c from the inner mitochondrial membrane to the cytosol, activated members of the MAPK protein family JNK, p38, ERK, activated caspases 3/9, and increased oxidative/nitrosative stress. Exposure of cells to PA for 12 h increased insulin receptor (IR) and GLUT-4 levels in the plasma membrane. Insulin treatment (10 mU/ml/30 min) increased the phosphorylation of the IR β-subunit and Akt. A correlation was found between DNA fragmentation and expression levels of both IR and GLUT-4. Similar results were obtained for PA-treated lymphocytes from healthy human donors and from mesenteric lymph nodes of 48-h starved rats. PA stimulated glucose uptake by Jurkat cells (in the absence of insulin), stimulated accumulation of neutral lipids (triglyceride), and other lipid classes (phospholipids and cholesterol ester) but reduced glucose oxidation. Our results suggest that parameters of insulin signaling and non-oxidative glucose metabolism are stimulated as part of a coordinated response to prompt survival in lymphocytes exposed to PA but at higher concentrations, apoptosis prevails. These findings may explain aspects of lymphocyte dysfunction associated with diabetes.

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“…Later (3), they used the method to verify the occurrence of energy transfer between labeled cell‐surface receptors. Other investigators have used similar methods to measure the fluorescence polarization of intracellular fluorescein (4–7) and of various DNA‐ and membrane‐bound dyes (8–15), because polarization provides information on the cellular context of these molecules. Measurements of fluorescence polarization of peripheral blood cells have been reported to be useful for the study and diagnosis of cancer (16) and Alzheimer's disease (17).…”

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Polarization of scatter and fluorescence signals in flow cytometry

Asbury

Engh

2000

Cytometry

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Background The pulses of light scatter and fluorescence measured in flow cytometers exhibit varying degrees of polarization. Flow cytometers are heterogeneously sensitive to this polarization, depending on the light source(s), the optical layout, and the types of mirrors and filters used. Therefore, fluorescence polarization can affect apparent intensity ratios between particles and interfere with schemes for interlaboratory standardization. Methods We investigate the degree to which polarization affects common flow cytometry measurements. Our technique for determining polarization differs from previous methods because complete distributions of intensity versus polarization angle are measured, rather than intensities at just two orthogonal polarization angles. Theoretical models for scatter and fluorescence are presented and verified by making polarization measurements of calibration beads. Results Measurements of cells stained with a variety of dyes illustrate that fluorescence polarization occurs frequently in flow cytometry. Conclusions Consequences for quantitative cytometry are discussed, and the use of the “magic angle” to make a flow cytometer insensitive to fluorescence polarization is proposed. Cytometry 40:88–101, 2000 © 2000 Wiley‐Liss, Inc.

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Plasma membrane fluidity gradients of human peripheral blood leukocytes

Cited by 11 publications

References 55 publications

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

Activation of survival and apoptotic signaling pathways in lymphocytes exposed to palmitic acid

Polarization of scatter and fluorescence signals in flow cytometry

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