The sensitivity and specificity afforded by fluorescent labeling makes SRM particularly useful in spatially and compositionally resolving complex biological structures. Recent applications of SRM, discussed below, have been particularly informative in delineating cellular architecture at the nanoscale to complement what is known and not known from EM studies. However, it is important to emphasize a major difference in the information content provided by SRM techniques and the more familiar EM. Unlike in EM where rich ultrastructural context can be gleaned, since membranes and numerous structures are visible, SRM, with its highly specific fluorescent labeling, primarily visualizes the positions of molecules that are labeled. Although many SRM studies have thus far focused on technical demonstrations and generally made use of only a few labeled protein species, for cell biological applications, researchers should probe for as many species as practicable, since the salient features of the structures of interest may not be discernible with only one probe. Furthermore, biological applications of SRM require a clear marker to identify the structure of interest for analysis. While this can be relatively straightforward for well-defined organelles such as the focal adhesions [58
] or neuronal synapses[59
] for which the structures are synonymous with a high density of the protein of interests, for membrane-based organelles or structures with low labeling density, it may be difficult to depend only on SRM for spatial clues. This has spurred the development of correlative SRM-EM[60
] where the high-density molecule-specific SRM data can be directly overlaid with context-rich EM to help identify ultrastructural features of interest.
One very important recent application for SRM has been in prokaryotic cell biology. Since the internal structures of prokaryotes are much more challenging to visualize by CLM due to their small cell sizes, typically only a few microns in length, SRM provides an immediate remedy to this limitation and has been actively employed by several research groups. In one example, Localization Microscopy of FtsZ, the cytoskeletal element involved in cell division in Escherichia coli
, revealed for the first time that this protein forms a compressed helix at the division plane, suggesting that this “Z-ring” is composed of a loose bundle of FtsZ protofilaments [61
]. In another study, PALM was used in conjunction with modeling to map the cellular locations of three proteins central to E. coli
chemotaxis, indicating that chemotactic receptors self-organize into signaling clusters[62
]. Localization Microscopy was also used to determine the cell-cycle dependent changes in organization of HU, a nucleoid-associated protein (NAP) critical to chromosomal architecture in Caulobacter crescentus
]. This showed that HU becomes highly clustered in predivision cells, suggesting the exciting possibility that chromosome condensation may also occur in prokaryotic cells, as it is well known to do in eukaryotes. Altogether, experimental access provided by SRM help to contribute to newfound recognition that prokaryotic cells possess dynamic and intricately organized ultrastructure, in contrast to the old notion that these “simple” cells are mere packets of diffusing biomolecules.
Several SRM applications to eukaryotic cell biology have also been reported recently. For example, 3D-STORM has been used to image the clathrin-dependent endocytic machinery generated in a cell-free system, revealing the roles and organization of specific F-BAR proteins in endocytosis and membrane invagination [64
]. In another example, highlighted in , the molecular architecture of integrin-based focal adhesions (FA) [58
] was dissected by iPALM[25
]. Through their function as dynamic, adhesive organelles that mediate adhesion and force transmission between the actin cytoskeleton and the extracellular matrix, FAs play important roles in cell migration, mechanosensing, and many signaling pathways including those that regulate cell fate and differentiation [65
]. However, despite early recognition of their physiological importance, the underlying ultrastructure of FAs had proven elusive. Because FAs contain a variable ensemble of more than a few hundred protein components [66
] all confined within highly dense and compact plaques measuring <200 nm along the smallest axial dimension [67
], efficient probing by immuno-metal EM methods are hindered. Since the probe density and specificity limitations are largely circumvented by fluorescent protein technologies, with iPALM it was possible to reveal for the first time that FA proteins are distinctly organized along the axial dimension () [58
]. The actin cytoskeleton was observed to be separated from the integrin cytoplasmic domains by a gap of ~30–40 nm, within which contained strata of FA proteins such as focal adhesion kinase (FAK), paxillin, vinculin, and zyxin (). Furthermore, the placement of PA-FP tags at either the N- or C- terminus of a large FA adapter protein talin allowed determination of its orientation, revealing a highly polarized organization, suggestive of the role of talin as a direct integrin-actin linker that could also help organize the FA multilayer architecture (). Interestingly, the interposition of talin and vinculin between actin and integrin () has a parallel in other studies in which these proteins exhibit interdependent dynamics [69
] and localization [70
] as well as interdependence in control of adhesion function [71
]. Taken together, these results corroborate the notion that FAs serve as spatiotemporally regulated molecular clutches during cell migration [58
]. The FA example above also highlight the benefits of using probes with known or well characterized tag locations, which enable determination of molecular orientation within the context of a cell.
Ultrastructural analysis of integrin-based focal adhesions by 3-D super-resolution microscopy
In another recent SRM application to neurobiology, the molecular organization within the neuronal synapses were probed by 3D-STORM[59
]. By immunolabeling the N- and C- terminal domains of the large scaffold proteins Piccolo and Bassoon, SRM imaging revealed their highly directional orientations, reminiscent of talin in FAs, suggesting that the two structures may share a common architectural motif despite different identities of the proteins involved. It remains to be seen whether other dense protein structures at membrane-cytoskeleton interfaces such as the adherens junction or the immunological synapse share similar architecture. Furthermore, these studies suggest that in addition to localizing –N and –C termini of target proteins, SRM analysis should be applicable to other types of fusion constructs containing, for example, domain deletions, rearranged domains, or site-specific mutations.