Sarah A. Shelby

Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting.

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins.

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

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.

Rab11 Regulates the Mast Cell Exocytic Response

Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell‐mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high‐affinity receptor, FcϵRI, on the cell surface. Here we use the endosomal v‐SNARE VAMP8, and the lysosomal hydrolase β‐hexosaminidase (β‐Hex), each C‐terminally fused to super‐ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin‐tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with total internal reflection (TIRF) microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated β‐Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F‐actin with jasplakinolide inhibits antigen‐stimulated exocytosis but is not additive with S25N Rab11‐mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.

Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling

Spatial targeting of signaling components to activated receptors on the plasma membrane is key for initiating signal transduction. The actin cytoskeleton restricts antigen-stimulated colocalization of IgE-FcεRI with membrane-anchored signaling partner Lyn kinase, and this regulation is mediated by organization of plasma membrane lipids.

Investigating Molecular Mechanisms of IgE-Mediated Signaling at Super Resolution

Antigen-mediated signaling via the IgE-FceRI receptor in mast cells plays a key role in allergic responses and serves as a model system for immune receptor signaling. We utilized recent advances in microscopy in conjunction with structurally defined ligands to study IgE-FceRI signal transduction at a new level of molecular detail.Our defined ligands were built on Y-shaped scaffolds of double-stranded DNA, which allowed for presentation of bivalent or trivalent antigen epitopes at specific spacings, depending on the length of the DNA scaffold used. The ligands, termed Y16 and Y46, had spacings of 5 nm and 13 nm, respectively. Using one-color super-resolution imaging (STORM) on live cells, we found that trivalent Y16 forms clusters with higher receptor densities and smaller areas than trivalent Y46.We performed two-color super-resolution imaging (STORM/PALM) on fixed cells to evaluate the functional implications of these differences in IgE cluster structure. We found that Lyn kinase, responsible for the initial phosphorylation of the receptor, was more effectively recruited to Y16 clusters than Y46 clusters. Additionally, a palmitoyl, myristoyl marker that targets liquid ordered membrane was enriched in Y16 clusters, while a geranylgeranyl marker that targets liquid disordered membrane was not. These results suggest that the higher density of receptors in clusters is essential for the initial stages of signaling, and may act by stabilizing a liquid ordered membrane domain that in turn promotes the recruitment of protein effectors.Our microscopy results are consistent with the predictions of a two-dimensional Ising model for a membrane near a critical point. We additionally explored modeling the membrane with a tricritical Ising model. The tricritical model predicts a larger energetic preference for the recruitment of an ordered kinase to an ordered cluster, as well as a larger dependence on cluster density.

Analysis of Spot Detection and Localization Algorithms for PALM and STORM

Until recently, one of the major pitfalls of using optical microscopy to study cell biology has been the gap between the instrument resolution, constrained by diffraction to λ/NA (∼250 nm), and the size of biological macromolecules, which are roughly two orders of magnitude smaller. With the advent of super-resolution microscopy, optical techniques can now be used to probe structural content that was previously only accessible to electron microscopy with the added benefit of being able to image live specimen. In particular, localization-based methods such as PALM and STORM have been readily adopted by many labs due to their ease of implementation, requiring not much more than a widefield or TIRF system and relatively simple image analysis software. Here, we compare a number of algorithms for image filtering, spot detection and PSF localization, investigating their Type I and II error rates, speed, precision and bias. Computational routines are written in C/C++ and tested on synthetic images and real data recorded from an EMCCD. In addition, we explore the viability of using a solid-state “light engine” (Lumencor, Inc.) for both activation and readout of photo-activatable probes; this novel illumination source allows for computer-controlled millisecond switching and attenuation of up to seven high-power (> 100 mW) spectral bands.