Matthew B. Stone

Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes

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. DOI: http://dx.doi.org/10.7554/eLife.19891.001

Ezrin Tunes the Magnitude of Humoral Immunity

Ezrin is a member of the ezrin–radixin–moesin family of membrane-actin cytoskeleton cross-linkers that participate in a variety of cellular processes. In B cells, phosphorylation of ezrin at different sites regulates multiple processes, such as lipid raft coalescence, BCR diffusion, microclustering, and endosomal JNK activation. In this study, we generated mice with conditional deletion of ezrin in the B cell lineage to investigate the physiological significance of ezrin’s function in Ag receptor–mediated B cell activation and humoral immunity. B cell development, as well as the proportion and numbers of major B cell subsets in peripheral lymphoid organs, was unaffected by the loss of ezrin. Using superresolution imaging methods, we show that, in the absence of ezrin, BCRs respond to Ag binding by accumulating into larger and more stable signaling microclusters. Loss of ezrin led to delayed BCR capping and accelerated lipid raft coalescence. Although proximal signaling proteins showed stronger activation in the absence of ezrin, components of the distal BCR signaling pathways displayed distinct effects. Ezrin deficiency resulted in increased B cell proliferation and differentiation into Ab-secreting cells ex vivo and stronger T cell–independent and -dependent responses to Ag in vivo. Overall, our data demonstrate that ezrin regulates amplification of BCR signals and tunes the strength of B cell activation and humoral immunity.

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Far-Red Organic Fluorophores Contain a Fluorescent Impurity

Far-red organic fluorophores commonly used in traditional and super-resolution localization microscopy are found to contain a fluorescent impurity with green excitation and near-red emission. This near-red fluorescent impurity can interfere with some multicolor stochastic optical reconstruction microscopy/photoactivated localization microscopy measurements in live cells and produce subtle artifacts in chemically fixed cells. We additionally describe alternatives to avoid artifacts in super-resolution localization microscopy.

Oxygen Depletion Speeds and Simplifies Diffusion in HeLa Cells

Many cell types undergo a hypoxic response in the presence of low oxygen, which can lead to transcriptional, metabolic, and structural changes within the cell. Many biophysical studies to probe the localization and dynamics of single fluorescently labeled molecules in live cells either require or benefit from low-oxygen conditions. In this study, we examine how low-oxygen conditions alter the mobility of a series of plasma membrane proteins with a range of anchoring motifs in HeLa cells at 37°C. Under high-oxygen conditions, diffusion of all proteins is heterogeneous and confined. When oxygen is reduced with an enzymatic oxygen-scavenging system for ≥ 15 min, diffusion rates increase by > 2-fold, motion becomes unconfined on the timescales and distance scales investigated, and distributions of diffusion coefficients are remarkably consistent with those expected from Brownian motion. More subtle changes in protein mobility are observed in several other laboratory cell lines examined under both high- and low-oxygen conditions. Morphological changes and actin remodeling are observed in HeLa cells placed in a low-oxygen environment for 30 min, but changes are less apparent in the other cell types investigated. This suggests that changes in actin structure are responsible for increased diffusion in hypoxic HeLa cells, although superresolution localization measurements in chemically fixed cells indicate that membrane proteins do not colocalize with F-actin under either experimental condition. These studies emphasize the importance of controls in single-molecule imaging measurements, and indicate that acute response to low oxygen in HeLa cells leads to dramatic changes in plasma membrane structure. It is possible that these changes are either a cause or consequence of phenotypic changes in solid tumor cells associated with increased drug resistance and malignancy.

Diffusion of Single B Cell Receptors in Resting and Stimulated B Lymphocytes using Super-Resolution Microscopy

The B cell antigen receptor (BCR) is an integral part of our immune systems that communicates binding of antigen in the extracellular environment through the plasma membrane to the cytoplasm. Antigen binding to the BCR results in phosphorylation of intracellular tyrosine activating motifs (ITAMs), which act as binding partners for SH2 domain containing adapters and Src family kinases. To examine the spatio-temporal dynamics of the BCR during stimulation, we utilize stochastic optical reconstruction microscopy (STORM) in live CH27 cells. Using Alexa-647 labeled anti-IgM Fab fragments, we resolve the BCR down to tens of nanometers with acquisition rates of 40 frames per second. We then examine BCR diffusion by tracking the localized receptors and found that the BCRs in resting cells exhibit a lognormal distribution of diffusion constants centered at 10-1 μm2/s. Upon stimulation, this population is quickly shifted to one centered at 10-2 μm2/s. In separate experiments, we examined the calcium response of CH27 B cells and found that the buffers used for STORM experiments do not alter calcium responses after receptor crosslinking. We also perform an auto correlation analysis of the localized receptors, which shows an increase in BCR clustering on the same timescale as the reduction in diffusion. In addition, we are investigating the dynamics of other proteins involved in BCR signaling, namely the Src family kinase Lyn and Ezrin. We use two color simultaneous emission of a photoswitchable fluorescent protein mEos2 in conjunction with Alexa-647 in fixed and live cells to resolve the behavior of these interaction partners with respect to the BCR.