Distribution and Dynamics of Rat Basophilic Leukemia Immunoglobulin E Receptors (FcɛRI) on Planar Ligand-Presenting Surfaces

Abstract

There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcepsilonRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (+/-50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D approximately 10(-1) microm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.

Single‐particle tracking of immunoglobulin E receptors (FcεRI) in micron‐sized clusters and receptor patches

“…The IgE-FceRI diffusivity in clusters is similar to that of the monomeric, uncrosslinked IgE-FceRI [14,24]. The average cluster radius hRi was 206 nm (Â/1.36), which is consistent with the microscopically observable cluster size and previous measurements [8]. IgE-FceRI receptor complexes confined within central patches of apparent average radius hRi = 503 nm (Â/1.45) diffused faster than expected, with an average diffusivity of hDi = 0.17 lm 2 /s (Â/1.7).…”

“…2 shows the average diffusion coefficient (solid black line) for IgE-FceRI receptor complexes diffusing within central patches (circles) and clusters (squares). The average diffusivity of clusters (triangles) was hDi = 3.3 Â 10 À3 lm 2 /s (Â/2.1), significantly smaller than IgE-FceRI diffusivity and within previously reported observations [8]. Clusters tracked in this experiment did not show evidence of directed motion (as has been previously reported [8]); however, the tracking time was shorter in this study, making detection of directed motion more difficult.…”

“…The average diffusivity of clusters (triangles) was hDi = 3.3 Â 10 À3 lm 2 /s (Â/2.1), significantly smaller than IgE-FceRI diffusivity and within previously reported observations [8]. Clusters tracked in this experiment did not show evidence of directed motion (as has been previously reported [8]); however, the tracking time was shorter in this study, making detection of directed motion more difficult. For each population, the dashed line above and below the mean represents one standard deviation in the lognormal distribution.…”

Controlled initiation and quantitative visualization of cell interaction dynamics

“…It is complex to separate these processes in the data analysis. Therefore, Spendier et al (2010) use a micropipette to initiate cell contact with a functionalized substrate resulting in a large initial contact site. A micropipette can exert high enough forces to press the cell against the substrate (Hocde et al, 2009).…”

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“…Studies include effects of D 2 O on tobacco seed growth (Lewis, 1934), IgE-Mediated histamine release from human leukocytes (Gillespie and Lichtenstein, 1972), actin filament velocities (Chaen et al, 2001), protein flexibility (Cioni and Strambini, 2002), human pancreatic tumor cells (Hartmann et al, 2005), phospholipid membranes (Beranova et al, 2012), kinesin-1 gliding assay (Maloney et al, 2014), and stabilization of tubulin as observed previously in microtubule gliding assays (Panda et al, 2000) and in biochemical experiments on isolated tubulin proteins from beef brain (Houston et al, 1974) and goat brain (Das et al, 2008). Microtubules are polarized polymers of α/β tubulin heterodimers and undergo alternating phases of growth and shrinkage with sudden transitions between the two (Bartolini et al, 2005 In this paper we use the RBL-2H3 cell line, a typical mast cell model system (Thomas et al, 1992;Posner et al, 1995;Carroll-Portillo et al, 2010;Spendier et al, 2010), to start investigating this question. Mast cells are immune cells that originate from the bone marrow and circulate in an immature form in the body until they settle in tissue and mucosal surfaces, where they mature.…”