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Piperazinium Hexachlorodicuprate (PHCC) is shown to be a frustrated quasi-two-dimensional quantum Heisenberg antiferromagnet with a gapped spectrum. Zero-field inelastic neutron scattering and susceptibility and specific heat measurements as a function of applied magnetic field are presented. At T = 1.5 K, the magnetic excitation spectrum is dominated by a single propagating mode with a gap, Delta = 1 meV, and bandwidth of approximately 1.8 meV in the (h0l) plane. The mode has no dispersion along the b* direction indicating that neighboring a-c planes of the triclinic structure are magnetically decoupled. The heat capacity shows a reduction of the gap as a function of applied magnetic field in agreement with a singlet-triplet excitation spectrum. A field-induced ordered phase is observed in heat capacity and magnetic susceptibility measurements for magnetic fields greater than H_c1 approximately equal to 7.5 Tesla. Analysis of the neutron scattering data reveals the important exchange interactions and indicates that some of these are highly frustrated., Comment: 13 pages with 14 figures, 7 pages of text, 6 pages of figures. Submitted to Phys. Rev. B 4/7/2001. email comments to dhr@pha.jhu.edu or mstone@pha.jhu.edu

The B cell receptor (BCR) is responsible for sensing and responding to intact antigen during the immune response and is hypothesized to nucleate a unique composition of lipids surrounding receptor clusters during antigen binding and signaling. This unique lipid composition, sometimes referred to as a “lipid raft,” is predicted to be enriched in glycosphingolipids and cholesterol. An altered lipid composition surrounding the BCR could influence the partitioning and activity of regulatory proteins and lipids, acting as a mechanism for the B cell to regulate signal transduction following stimulus by antigen. However, without direct observations of this phenomenon, theories surrounding lipid compositional heterogeneity in B cells remain controversial. In this work, we utilize two-color super-resolution localization microscopy (STORM and PALM) to directly observe the lipid composition proximal to B cell receptors, using the photoactivatable fluorescent protein mEos3.2 anchored to the plasma membrane through posttranslational lipid modifications. These probes mimic important regulatory proteins involved in BCR signaling by their membrane anchor but lack the protein interaction domains or biological activity found in the native protein. By analyzing the co-distributions of the B cell receptor and lipid probes using correlation functions, we find that the membrane surrounding B cell receptor clusters is depleted of probes bearing unsaturated and branched geranylgeranyl modifications and is enriched in probes bearing saturated palmitoyl modifications. Quantification of lipid probe distributions reveals that B cell compositional heterogeneity influences the partitioning of these lipid modifications with order 1kBT of effective potential, as expected from predications of composition fluctuations in critical systems. These findings provide definitive evidence of membrane compositional heterogeneity in an important biological signaling system. Our results suggest that compositional fluctuations contribute to cellular responses by influencing the spatial distribution of specific components in the plasma membrane.

The B cell antigen receptor (BCR) is an integral part of the adaptive immune system that communicates binding of antigen in the extracellular environment through the plasma membrane. Antigen binding to the BCR results in phosphorylation of intracellular tyrosine activating motifs (ITAMs), which subsequently bind to and activate numerous proteins involved in BCR regulation. Interestingly, many of the proteins regulating the early stages of the BCR signaling pathway are linked to the inner leaflet by saturated lipid anchors which tend to be associated with liquid-ordered membrane phase in model membranes. Also, the BCR becomes transiently detergent resistant following antigen-induced BCR clustering, suggesting that BCR clusters become coupled to membrane order following stimulation. In this work, we aim to characterize how BCR clustering could reorganize plasma membrane lipids by quantifying co-localization of BCR with fluorescent markers of liquid-ordered and liquid-disordered phases. We utilize two-color super-resolution fluorescence localization microscopy (STORM and PALM) in live and chemically fixed CH27 B cells to simultaneously image BCR and membrane anchored proteins, and we quantify their co-clustering using correlation functions. Our results from chemically fixed cells show that proteins anchored to the plasma membrane inner leaflet through saturated acyl-chain lipid modifications exhibit increased co-localization with BCR upon antigen stimulation, whereas those without lipid modifications or those anchored through branched acyl-chain modifications are not significantly co-localized with BCR before or after stimulation. These results are contributing to our long term goal of elucidating the role of lipid mediated interactions in the regulation of BCR signaling.