Evaluating protein structures in living cells remains a challenge. Here, we investigate Interleukin-4 receptor alpha (IL-4Rα) into which the non-canonical amino acid bicyclo[6.1.0]nonyne-lysine (BCNK) is incorporated by genetic code expansion. Bioorthogonal click labeling is performed with tetrazine-conjugated dyes. To quantify the reaction yield in situ, we develop brightness-calibrated ratiometric imaging, a protocol where fluorescent signals in confocal multi-color images are ascribed to local concentrations. Screening receptor mutants bearing BCNK in the extracellular domain uncovered site-specific variations of both click efficiency and Interleukin-4 binding affinity, indicating subtle well-defined structural perturbations. Molecular dynamics and continuum electrostatics calculations suggest solvent polarization to determine site-specific variations of BCNK reactivity. Strikingly, signatures of differential click efficiency, measured for IL-4Rα in ligand-bound and free form, mirror sub-angstrom deformations of the protein backbone at corresponding locations. Thus, click efficiency by itself represents a remarkably informative readout linked to protein structure and dynamics in the native plasma membrane.

Cells in a developing organism are subjected to particular mechanical forces, which shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is thus an important question, which has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Hence, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing three FRET-based Talin tension sensors reporting different force levels between 1 and 11 pN enabled us to quantify physiologically-relevant, molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension increases. Concomitantly, Talin concentration at attachment sites increases five-fold as quantified by fluorescence correlation spectroscopy, suggesting that only few Talin molecules are mechanically engaged at any given time. We therefore propose that high tissue forces are shared amongst a large excess of adhesion molecules of which less than 15% are experiencing detectable forces at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.

Fluorescent proteins (FPs) feature complex photophysical behavior that must be considered when studying the dynamics of fusion proteins in model systems and live cells. In this work, we characterize mNeonGreen (mNG), a recently introduced FP from the bilaterian Branchiostoma lanceolatum, in comparison to the well-known hydrozoan variants enhanced green fluorescent protein (EGFP) and Aequorea coerulescens GFP by steady-state spectroscopy and fluorescence correlation spectroscopy in solutions of different pH. Blind spectral unmixing of sets of absorption spectra reveals three interconverting electronic states of mNG: a nonfluorescent protonated state, a bright state showing bell-shaped pH dependence, and a similarly bright state dominating at high pH. The gradual population of the acidic form by external protonation is reflected by increased flickering at low pH in fluorescence correlation spectroscopy measurements, albeit with much slower flicker rates and lower amplitudes as compared to Aequorea GFPs. In addition, increased flickering of mNG indicates a second deprotonation step above pH 10 leading to a slight decrease in fluorescence. Thus, mNG is distinguished from Aequorea GFPs by a two-step protonation response with opposite effects that reflects a chemically distinct chromophore environment. Despite the more complex pH dependence, mNG represents a superior FP under a broad range of conditions.

To quantitatively examine the effect of membrane organization on lateral diffusion, we studied fluorescent carbocyanine lipid analogues and EGFP-tagged, single-pass transmembrane proteins in systems of decreasing complexity: (i) the plasma membrane (PM) of living cells, (ii) paraformaldehyde/dithiothreitol-induced giant plasma membrane vesicles (GPMVs), and (iii) giant unilamellar vesicles (GUVs) under physiological buffer conditions. A truncated, signaling-deficient interleukin-4 receptor subunit, showing efficient accumulation in the plasma membrane, served as a model transmembrane protein. Two-dimensional diffusion coefficients (D) were determined by fluorescence correlation spectroscopy (FCS) either at fixed positions (single-point, spFCS) or while scanning a circular orbit (circular scanning, csFCS). Consistent with a different inclusion sizes in the membrane, lipids diffuse slightly faster than the single-spanning membrane proteins in both membrane systems, GUVs and GPMVs. In GPMVs lipids and proteins consistently experienced a fivefold larger viscosity than in GUVs, reflecting the significant fraction of plasma membrane-derived proteins partitioning into GPMVs. Lipid and protein diffusion in the PM was, respectively, 2 times and 4–5 times slower in comparison to GPMVs. This discrepancy was quantitatively confirmed by csFCS. The similarity of diffusion of receptors and lipids in GPMVs and GUVs and its significant difference in the plasma membrane suggest that protein domains as small as EGFP convey sensitivity to the actin cortex on various length scales.

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.

Many important signalling cascades operate through specialized signalling endosomes, but a corresponding mechanism has as yet not been described for hematopoietic cytokine receptors. Based on live-cell affinity measurements, we recently proposed that ligand-induced interleukin-4 receptor (IL-4R) complex formation and thus JAK/STAT pathway activation requires a local subcellular increase in receptor density. Here, we show that this concentration step is provided by the internalization of IL-4R subunits through a constitutive, Rac1-, Pak- and actin-mediated endocytosis route that causes IL-4R subunits to become enriched by about two orders of magnitude within a population of cortical endosomes. Consistently, ligand-induced receptor dimers are preferentially detected within these endosomes. IL-4 signalling can be blocked by pharmacological inhibitors targeting the actin polymerization machinery driving receptor internalization, placing endocytosis unambigously upstream of receptor activation. Taken together, these observations demonstrate a role for endocytosis that is mechanistically distinct from the scaffolding function of signalling endosomes in other pathways.

Extrinsic apoptosis is initiated by recognition and clustering of the single-pass transmembrane proteins Fas ligand and Fas expressed at the surface of closely apposed lymphocytes and target cells, respectively. Since Fas-mediated death response was mainly studied with soluble antibodies, the mobility constraints for receptor activation by a membrane embedded agonist is not well understood. We explored this influence by stimulating apoptosis on functionalized supported lipid bilayers, where we quantified agonist mobility by z-scan fluorescence correlation spectroscopy. Using different lipid compositions, we show that the apoptotic response correlates with increased lateral mobility of the agonist in the lipid bilayer.

Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (Kd,2D) of 180 and 480 receptors per μm2 for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a Kd,2D of ∼1000 receptors per μm2. The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

Scanning fluorescence correlation spectroscopy (SFCS) with a scan path perpendicular to the membrane plane was introduced to measure diffusion and interactions of fluorescent components in free-standing biomembranes. Using a confocal laser scanning microscope (CLSM), the open detection volume is repeatedly scanned through the membrane at a kHz frequency. The fluorescence photons emitted from the detection volume are continuously recorded and stored in a file. While the accessory hardware requirements for a conventional CLSM are minimal, data evaluation can pose a bottleneck. The photon events must be assigned to each scan, in which the maximum signal intensities have to be detected, binned, and aligned between the scans, in order to derive the membrane-related intensity fluctuations of one spot. Finally, this time-dependent signal must be correlated and evaluated by well-known FCS model functions. Here we provide two platform-independent, open source software tools (PyScanFCS and PyCorrFit) that allow to perform all of these steps and to establish perpendicular SFCS in its one- or two-focus as well as its single- or dual-color modality.

Summary: We present a graphical user interface (PyCorrFit) for the fitting of theoretical model functions to experimental data obtained by fluorescence correlation spectroscopy (FCS). The program supports many data file formats and features a set of tools specialized in FCS data evaluation. Availability and implementation: The Python source code is freely available for download from the PyCorrFit web page at http://pycorrfit.craban.de. We offer binaries for Ubuntu Linux, Mac OS X and Microsoft Windows. Contact: paul.mueller@biotec.tu-dresden.de and weidemann@biochem.mpg.de

Fluorescence microscopy provides insight into the subcellular organization of biological functions. However, images are snap shots averaging over a highly dynamic molecular system. Fluorescence fluctuation microscopy, employing similar detection technology, encompasses a powerful arsenal of analysis tools that investigate the molecular heterogeneity in space and time. Analyzing signal fluctuations from small ensembles (several hundred particles) reveals their concentration, the stoichiometry, the stochastic motion, as well as superimposed signatures of the environment such as spatial confinement and binding events. Thus, fluctuation analysis provides access to dynamic molecular properties that can be used to build physical models of cellular processes. In the last decade these methods experienced a remarkable diversification, which we revisit here with a particular focus on live cell applications.

Fluorescence correlation spectroscopy (FCS) can add dynamic molecular information to images of live cells. For example, a confocal laser scanning microscope (CLSM) equipped with an accessory FCS unit provides the possibility to first image the spatial distribution of a fluorescent protein before probing its mobility within defined regions of interest. Whereas specific protein-protein interactions are preferably assayed with a dual-color approach, single-color FCS can still provide valuable information about the size of the diffusing entities and potential interactions with other, nonfluorescent, proteins or subcellular structures. Because number fluctuations are measured, the concentrations of freely diffusing complexes and their state of oligomerization are accessible.