Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.

Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.

The distinctive distributions of proteins within subcellular compartments both at steady state and during signaling events have essential roles in cell function. Here we describe a method for delineating the complex arrangement of proteins within subcellular structures visualized using point-localization superresolution (PL-SR) imaging. The approach, called pair correlation photoactivated localization microscopy (PC-PALM), uses a pair-correlation algorithm to precisely identify single molecules in PL-SR imaging data sets, and it is used to decipher quantitative features of protein organization within subcellular compartments, including the existence of protein clusters and the size, density and number of proteins in these clusters. We provide a step-by-step protocol for PC-PALM, illustrating its analysis capability for four plasma membrane proteins tagged with photoactivatable GFP (PAGFP). The experimental steps for PC-PALM can be carried out in 3 d and the analysis can be done in ∼6-8 h. Researchers need to have substantial experience in single-molecule imaging and statistical analysis to conduct the experiments and carry out this analysis.

Pointillistic based super-resolution techniques, such as photoactivated localization microscopy (PALM), involve multiple cycles of sequential activation, imaging, and precise localization of single fluorescent molecules. A super-resolution image, having nanoscopic structural information, is then constructed by compiling all the image sequences. Because the final image resolution is determined by the localization precision of detected single molecules and their density, accurate image reconstruction requires imaging of biological structures labeled with fluorescent molecules at high density. In such image datasets, stochastic variations in photon emission and intervening dark states lead to uncertainties in identification of single molecules. This, in turn, prevents the proper utilization of the wealth of information on molecular distribution and quantity. A recent strategy for overcoming this problem is pair-correlation analysis applied to PALM. Using rigorous statistical algorithms to estimate the number of detected proteins, this approach allows the spatial organization of molecules to be quantitatively described.

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

The glucose transporter, GLUT4, redistributes to the plasma membrane (PM) upon insulin stimulation, but also recycles through endosomal compartments. Different Rab proteins control these transport itineraries of GLUT4. However, the specific roles played by different Rab proteins in GLUT4 trafficking has been difficult to assess, primarily due to the complexity of endomembrane organization and trafficking. To address this problem, we recently performed advanced live cell imaging using total internal reflection fluorescence (TIRF) microscopy, which images objects ~150 nm from the PM, directly visualizing GLUT4 trafficking in response to insulin stimulation. Using IRAP-pHluorin to selectively label GSVs undergoing PM fusion in response to insulin, we identified Rab10 as the only Rab protein that binds this compartment. Rab14 was found to label transferrin-positive, endosomal compartments containing GLUT4. These also could fuse with the PM in response to insulin, albeit more slowly. Several other Rab proteins, including Rab4A, 4B and 8A, were found to mediate GLUT4 intra-endosomal recycling, serving to internalize surface-bound GLUT4 into endosomal compartments for ultimate delivery to GSVs. Thus, multiple Rab proteins regulate the circulation of GLUT4 molecules within the endomembrane system, maintaining optimal insulin responsiveness within cells.

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.