Abstract
Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.
Highlights
Cells interact with their environment through a complex set of biochemical networks that transmit information across the plasma membrane
Disordered domains were marked with a short transmembrane peptide (TM) and a peptide anchored to the inner leaflet through a polybasic sequence and geranylgeranyl modification (GG) (Pyenta et al, 2001; Baumgart et al, 2007; Levental et al, 2010)
Ordered membrane domains were marked with a minimal lipidated peptide anchored to the inner leaflet through saturated palmitoyl and myristol modifications (PM) (Pyenta et al, 2001; Baumgart et al, 2007), or with cholera toxin subunit B (CTxB), which binds to the ganglioside GM1 on the outer leaflet of the plasma membrane
Summary
Cells interact with their environment through a complex set of biochemical networks that transmit information across the plasma membrane. Pair cross-correlation functions indicate that TM clusters are enriched in GG and depleted of PM on average compared to the membrane as a whole (Figure 1c, left panel), consistent with the partitioning of these probes into disordered domains in GPMVs. In separate experiments we stabilized an ordered domain by clustering biotinylated CTxB bound to endogenous GM1 with streptavidin, and imaged these clusters in combination with PM or TM peptides (Figure 1b, right panel).
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