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Liquid–liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.

We review the principles and applications of statistical filtering in multichannel fluorescence microscopy. This alternative approach to separation of signals from individual fluorophore populations has many important advantages, especially when spectral and/or temporal overlap, or the complicated nature of those signals, makes their discrimination or sorting impossible by means of hardware. This situation is typically encountered for biological samples. This review of well established statistical filtering techniques and of emerging, very promising new methods of analysis reveals remarkable progress in bioanalytical applications of fluorescence microscopy.

Fluorescence light-up aptamers (FLAPs) are tools for RNA imaging, wherein the RNA of interest is appended with a FLAP sequence that can bind to a corresponding small-molecule fluorogen and enhance its fluorescence. The fluorescence properties of FLAPs have mostly been analyzed in bulk and described as the average of a large number of RNA–fluorogen complexes. In this study, we evaluated the feasibility of fluorescence correlation spectroscopy (FCS)- and fluorescence cross-correlation spectroscopy (FCCS)-based quantifications of FLAPs in a solution using Broccoli, a common FLAP, and its corresponding fluorogen, DFHBI-1T. We investigated the folding efficiency, photostability, and photophysical properties of the Broccoli–DFHBI-1T complex using their FCS/FCCS characteristics. With FCS, we observed that the fluorescence was affected by the affinity between Broccoli and DFHBI-1T and the folding (maturation) state of Broccoli RNA. Moreover, the FCCS measurement of ATTO647N-labeled Broccoli and its complex with DFHBI-1T revealed the proportion of the mature Broccoli–DFHBI-1T complex. The current FCS/FCCS-based study of Broccoli–DFHBI-1T provides a model for analyzing FLAPs and their fluorogen pairs at the single-molecule level.

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

Fluctuation correlation spectroscopy (FCS) is a single-molecule/particle detection technique based on measuring signal fluctuations in a highly focused detection volume. Multiple-parameter information can be obtained from the FCS measurement including the amplitude, characteristic diffusion time of correlation curve, and brightness of the adopted probes. The multiple-parameter change is related with physical or chemical change occurring in the probes. Meanwhile, the detection method has advantages such as short sample time in seconds, sample volume with low limit in femtoliters, and mixing to detection without any separations. These advantages make the FCS technique suitable for homogeneous analysis. In this review, we summarized recent novel applications of FCS and its variants in homogeneous analysis including nucleic acid analysis, protein analysis, enzyme activity assay, direct characterization of nanoparticles in solution, and others.

Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with$K_{{\\rm d}}>400-500\\ {\\rm nM}$. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation.

Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA.

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca ²⁺ triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca ²⁺-dependent and -independent cis -binding and trans -tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca ²⁺-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca ²⁺-dependent cis -binding prevents trans -interactions if the cis -membrane contains 12–20% anionic phospholipids. Similarly, no trans -interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans -interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis - and trans -binding, which may play an important modulatory role in synaptic transmission.

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods.