Migrating Human Neutrophils Exhibit Dynamic Spatiotemporal Variation in Membrane Lipid Organization

Abstract

Highly ordered sphingolipid-enriched lipid raft microdomains (LRMs) within plasma membranes purportedly function as specialized signaling platforms. Leukocyte migration is believed to entail LRM redistribution, but progress in studying LRMs in situ during cell movement has been limited. By using an improved method for imaging the spectral shift of the environmentally sensitive probe, laurdan (expressed as a generalized polarization function), the plasma membrane order (i.e., tight packing of membrane bilayer lipids) of human polymorphonuclear neutrophils (PMNs) was mapped in real time during migration. Morphologically polarized PMNs exhibited prominent LRM clusters at the uropod, where in every instance membrane order was found to oscillate with mean periodicities of 37.0 6 1.46 and 149.9 6 9.0 seconds (P , 0.01). LRM aggregates were also demonstrated in punctate and clustered distributions of nonpolarized cells and transiently at the lamellipodia of polarized PMNs. Cellular polarization was not accompanied by an overall increase in membrane order. LRM disorganization with methyl-b-cyclodextrin had small negative effects on cell velocity, but it abrogated directionally biased migration toward chemotactic gradients of FMLP or leukotriene B 4 . LRMs disruption also caused redistribution of Rac 1/2 GTPase and GM3 ganglioside away from the lamellipodium, as well as extension of multiple pseudopods simultaneously or in rapid succession, rather than formation of a defined leading edge. Thus, we demonstrate that the plasma membrane order of migrating PMNs changes dynamically, with prominent oscillations consistently seen at the uropod. These findings solidify the existence of rapidly reorganizing LRMs in situ and support a role for LRMs in chemotaxin responsiveness.Keywords: neutrophils; chemotaxis; lipid rafts; inflammation Inflammation mediated by polymorphonuclear neutrophils (PMNs) requires the cells to recognize and migrate toward chemotactic signals. Coupled with morphologic polarization, there must be complex spatial redistribution of signaling molecules, cytoskeletal structures, and membrane constituents for directionally biased movement (1-4). Although the distribution of proteins has been examined extensively, there is growing appreciation for the importance of compartmentalized membrane lipids and lipid-mediated signaling (2, 4-6). Earlier models have depicted a uniform lipid bilayer in the plasma membrane, but considerable evidence indicates that it is highly heterogeneous, containing small phase-separated regions often designated as lipid raft microdomains (LRMs) (reviewed in Refs. 2, 7-9). These microdomains are rigid, gel-like regions enriched in tightly packed sphingolipids, resulting in a liquidordered (Lo) state that is stabilized by cholesterol. It is believed that LRMs act as platforms where multiple constituents colocalize to form competent signaling complexes. Cell fractionation experiments have been used to isolate detergent-resistant/lowdensity membrane fractions containing an array of receptor ...

Microfluidic platform for the quantitative analysis of leukocyte migration signatures

“…The trend for reduced directional persistence and slower speed with decreasing mechanical confinement is further supported by the evaluation of neutrophil migration measurements in 2D surface microfluidic devices and traditional Dunn chambers ( Fig 1F ). In these assays, neutrophil migration takes place on flat surfaces, guided exclusively by chemical gradients and no mechanical restrictions, resulting in low migratory persistence (0.11-0.42 on flat surfaces vs. 0.99 in 6μm channels) and speed (8.1-10.9 μm min −1 vs. 20.2 μm min −1 in 6μm channels) 18 , 27 , 28 . Thus, confinement of cells within narrow channels which have cross sections comparable to extracellular matrix 29 is similar to that of leukocytes following paths of least resistance in tissues 30 and is associated with a fast and persistent migratory response.…”

“…F) Effect of migration channel width on migratory persistence and speed of cells migrating towards fMLP. Comparison between the Bi-Directional microfluidic device and established 2D microfluidic devices and Dunn chambers 18 , 27 , 28 . A total of 878 cells were analyzed in 6μm channels (672 migrated); 320 (264 migrated) in 50μm channels, N=3.…”

An Obligate Role for Membrane-Associated Neutral Sphingomyelinase Activity in Orienting Chemotactic Migration of Human Neutrophils

“…The polarized distribution of membrane sphingolipids is a common characteristic of migrating leukocytes (1,25). Previous work showed that disrupting sphingolipid-enriched microdomains may slow or immobilize PMNs (20,26) or selectively interfere with their orientation toward a chemotaxin (27), but important questions remain unanswered regarding whether sphingolipids were responsible, and by what mechanisms they may have functioned. Here, we examined the effects of N-SMase and its proximate product, ceramide, on PMN chemotaxis.…”

pH sensing by lipids in membranes: The fundamentals of pH-driven migration, polarization and deformations of lipid bilayer assemblies

“…However, an interplay of local juxtamembrane pH effects and ganglioside topography may also be involved. In-cidentally, polarized segregation of raft and/or ganglioside microdomains does occur in migrating cells [56][57][58] and differences in lipid rafts and ganglioside distribution between the apical and basal membranes have also been established for epithelial cells. Besides, NHE1 partially colocalizes with GM1 ganglioside-containing rafts [59].…”