Formation of a Mast Cell Synapse: FcεRI Membrane Dynamics upon Binding Mobile or Immobilized Ligands on Surfaces

Abstract

High affinity IgE receptors (FcεRI) on mast cells form a “synapse” when presented with mobile, bilayer incorporated antigen. Here, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells (RBL-2H3) primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand) or chemically crosslinked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of FcεRI to other cellular components at the synapse, such as actin, cholesterol and LAT, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that FcεRI crosslinking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low level degranulation upon ligand recognition.

Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence

“…Our data extend data reported by Wu et al who focused on localized endosomal recycling and not on mast cell degranulation 30 . In agreement with this and other reports [30][31][32] , we show that FcR and the associated signalling component LAT cluster in the proximity of the budding granules. These results, together with the observation of a localized clearance of actin cytoskeleton at the degranulatory synapse, provide a mechanistic link between localized signalling and polarized secretion.…”

“…Silverman et al showed that FceRI and the associated signalling components such as SLP76, Syk and LAT selectively cluster at the site of stimulation 31 . Carroll-Portillo et al showed that FcR clusters are detectable at the mast cell/lipid bilayer contact area 1 min after stimulation and then, they coalesce leading to the formation of a mast cell synapse 32 . Using patterned lipid bilayers, Wu et al reported that secretion is directed towards the substrate where the stimuli are located, but do not strictly co-localize with the signalling clusters 30 .…”

“…Studies using supported lipid bilayers showed that localized FceRI stimulation leads to receptor clustering and signalling and to granule exocytosis [29][30][31][32] . Although surrogate stimulatory surfaces are instrumental to study the dynamics of receptors and associated signalling components, they do not mimic the complex molecular interactions occurring at the cell-cell contact site.…”

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane

“…Flat sheets of plasma membranes from the apical cellular surface were prepared from virus-infected or transfected cells and processed for electron microscopy by a variation of the "membrane rip" technique (49,50). This type of analysis has been used successfully to examine the membrane distribution of a single protein, such as the IgE receptor FcR1 and CC chemokine receptor 5 (51)(52)(53). These analyses has also been extended to compare two protein populations to each other, showing whether or not they specifically cocluster with each other, for example, prion protein, AP2, transferrin receptor (54), FcR1, tyrosine kinase Lyn (55), lipid raft markers (56), Ras proteins (57), and a preliminary examination of influenza virus M1, M2, and HA (58).…”

Effects of deuterium oxide on cell growth and vesicle speed in RBL-2H3 cells

“…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.…”