Visualizing the molecular timing of a physiological decision at the nanoscale.

Mast cells are critical players in numerous diseases, including allergy, asthma, infectious disease, cancer, and even many central nervous system disorders such as autism, anxiety, and multiple sclerosis (1,2). In conjunction with these numerous roles, mast cells are found in almost every tissue in the human body. Many of the molecular elements crucial to mast cell function, such as cytoskeletal involvement and calcium signaling, are also essential to signaling in a variety of cell types, including neurons and T cells. The article by Shelby et al. (3) in this issue of the Biophysical Journal demonstrates what we believe to be the first superresolution imaging of living mast cells, where an important cellular decision (related to a physiological response) is made at the molecular level through the lateral organization of the high-affinity IgE receptor FceRI. This molecular decision determines whether the cell will degranulate, releasing histamine, serotonin, and other effectors that lead to a host of downstream consequences. Upon antigen-induced aggregation of IgE-bound FceRI receptors, mast cell signaling results in a tyrosine phosphorylation cascade, leading to the activation of phospholipase C. In turn, inositol 1,4,5-triphosphate is produced and binds to its receptor in the endoplasmic reticulum, which activates Ca2+ influx into the cytosol, which then leads to degranulation (4). Shelby et al. (3) show ground-breaking superresolution imaging of IgE receptors in living cells using stochastic optical reconstruction microscopy (5,6), and find a dramatic reorganization of receptor molecules visualized with nanometer resolution as a function of time after stimulation. Their work is an excellent application of superresolution to membrane lateral organization: it includes careful quantification of membrane receptor clustering and diffusion, and it highlights how both spatial and dynamic information can be obtained with single-molecule localization-based superresolution methods in live cells. Furthermore, Shelby et al. (3) demonstrate an advance in our understanding of an important biological system. Importantly, this work demonstrates not just the properties of membrane clustering, but the relationships among molecular dynamics, nanoscale clustering (at length scales not previously accessible), and (crucially) cellular function. The authors are able to separate the effects of stimulation on both receptor mobility and clustering. Somewhat surprisingly, reduction in receptor mobility and assembly of receptors into clusters do not occur simultaneously. Rather, on short timescales (within 2 min of stimulation), receptor mobility reduces without strong clustering, and this occurs before the functional response of Ca2+ mobilization. Then, at ∼5 min after stimulation, the receptors reorganize into ∼70 nm clusters with ∼100 receptors each. It would be extremely difficult to carry out these studies without the use of localization microscopy: electron microscopy provides outstanding resolution, but is generally incompatible with living cells, and conventional fluorescence imaging would not reach the necessary resolution (i.e., <70 nm) or be able to count molecules. Thus, this work is a substantial biological advance that highlights the advantages of localization-based superresolution microscopy for addressing certain kinds of biological questions. Furthermore, the ability of these methods to obtain high-resolution images and to quantify molecular dynamics is a powerful combination (7–9). For example, the authors were able to determine that single receptors showed slower, more confined diffusion in regions of high receptor density—a connection between molecular mobility and spatial distribution even for receptors that are not yet strongly clustered. This finding is in contrast to the properties of hemagglutinin from influenza, another membrane-associated protein, which shows lateral confinement and clustering but only weak dependence of mobility on HA density (10). The ability to observe such differences between cellular and viral membrane proteins is only possible because of the ability to obtain both spatial and dynamic information at the molecular scale. These detailed observations are crucial to our development of a more general understanding of cell membrane organization. Several models of membrane organization now incorporate actin and/or tubulin in their explanations of membrane clustering and dynamics (11,12). Interestingly, the authors’ evidence for lateral confinement of receptors is reminiscent of the lateral confinement of a number of membrane-associated molecules, and prior evidence of actin’s role in membrane biology during mast cell signaling (13) has been obtained. These findings together strongly motivate further investigation of the nanoscale role of the cytoskeleton (14) in the observed phenomena. These illuminating findings set the stage for future superresolution investigations into the molecular details of signaling in this important biological system. It will be interesting to observe interactions among these receptors, their associated kinases, actin, and other important players in the plasma membrane with superresolution microscopy, and Shelby et al. (3) demonstrate that it can be done successfully. The powerful capabilities of live-cell super-resolution microscopy provide a promising path to a unified molecular model of membrane proteins, lipids, signaling, and the cytoskeleton.