Comparison of clustering approaches with application to dual colour protein data.

Cells communicate with their environment via proteins, located at the plasma membrane separating the interior of a cell from its surroundings. The spatial distribution of these proteins in the plasma membrane under different physiological conditions is of importance, since this may influence their signal transmission properties. In this study, the authors compare different methods such as hierarchical clustering, extensible Markov models and the gammics method for analysing such a spatial distribution.

Single Particle Tracking Reveals that EGFR Signaling Activity Is Amplified in Clathrin-Coated Pits.

Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity.

Site-specific protection and dual labeling of human epidermal growth factor (hEGF) for targeting, imaging, and cargo delivery.

Well-defined human epidermal growth factor (hEGF) constructs featuring selectively addressable labels are urgently needed to address outstanding questions regarding hEGF biology. A protein-engineering approach was developed to provide access to hEGF constructs that carry two cysteine-based site-specific orthogonal labeling sites in multi-milligram quantities. Also, a site-selective (de)protection and labeling approach was devised, which allows selective modification of these hEGF constructs.

Coordinate-based colocalization analysis of single-molecule localization microscopy data.

Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis.

Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy.

We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching.

FRET in cell biology: still shining in the age of super-resolution?

Interest in imaging of Förster resonance energy transfer (FRET) in biological systems has been steadily increasing in the last 30 years. The ability to transduce a near-field interaction into a far-field signal has provided a unique optical tool to assess biological phenomena well below the resolution of standard optical microscopy. In recent years, sub-diffraction microscopy techniques have achieved maturation and are increasingly used in biological applications.

Cytohesins are cytoplasmic ErbB receptor activators.

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors.

Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers.

Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Neurotrophins interact with dimers of the p75 neurotrophin receptor (p75(NTR)), but the mechanism of receptor activation has remained elusive. Here, we show that p75(NTR) forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys(257) in its transmembrane domain.

Global analysis of time correlated single photon counting FRET-FLIM data.

Fluorescence lifetime imaging microscopy (FLIM) can be used to quantify molecular reactions in cells by detecting fluorescence resonance energy transfer (FRET). Confocal FLIM systems based on time correlated single photon counting (TCSPC) methods provide high spatial resolution and high sensitivity, but suffer from poor signal to noise ratios (SNR) that complicate quantitative analysis. We extend a global analysis method, originally developed for single frequency domain FLIM data, with a new filtering method optimized for FRET-FLIM data and apply it to TCSPC data.

Quantitative microscopy and systems biology: seeing the whole picture.

Understanding cellular function requires studying the spatially resolved dynamics of protein networks. From the isolated proteins we can only learn about their individual properties, but by investigating their behavior in their natural environment, the cell, we obtain information about the overall response properties of the network module in which they operate. Fluorescence microscopy methods provide currently the only tools to study the dynamics of molecular processes in living cells with high temporal and spatial resolution.

High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy.

We report that single (or selective) plane illumination microscopy (SPIM), combined with a new deconvolution algorithm, provides a three-dimensional spatial resolution exceeding that of confocal fluorescence microscopy in large samples. We demonstrate this by imaging large living multicellular specimens obtained in a three-dimensional cell culture. The ability to rapidly image large samples at high resolution with minimal photodamage provides new opportunities especially for the study of subcellular processes in large living specimens.

Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin.

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication.

Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate.

The mitogen-activated protein kinase (MAPK) network is a conserved signalling module that regulates cell fate by transducing a myriad of growth-factor signals. The ability of this network to coordinate and process a variety of inputs from different growth-factor receptors into specific biological responses is, however, still not understood. We investigated how the MAPK network brings about signal specificity in PC-12 cells, a model for neuronal differentiation.

Imaging protein interactions by FRET microscopy: cell preparation for FRET analysis.

The following protocol describes the preparation of cells for FRET analysis on live and fixed cells. The reagents used have been optimized to minimize the quenching of GFP mutants and fluorescent dyes.

Imaging protein interactions by FRET microscopy: labeling proteins with fluorescent dyes.

This protocol provides a method for labeling proteins, such as antibodies and purified recombinant proteins, with succinimide esters of sulfoindocyanine (Cy) dyes. Cy dyes covalently bind to free amino groups (the α-amino-terminal or ε-amino groups on lysine side chains).

Imaging protein interactions by FRET microscopy: FLIM measurements.

This image acquisition protocol is a basic plan for taking a fluorescence lifetime imaging (FLIM) series. FLIM makes live-cell FRET measurements based only on donor fluorescence more feasible, because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.

Imaging protein interactions by FRET microscopy: FRET measurements by acceptor photobleaching.

This protocol describes the detection of fluorescence resonance energy transfer (FRET) by measuring the quenching of donor emission alone. As opposed to sensitized emission measurements, photobleaching can be performed with high selectivity of the acceptor because absorption spectra are steep at their red edge, allowing the acceptor to be bleached without excitation of the donor. When using acceptor photobleaching FRET measurements, care should be taken that the photochemical product of the bleached acceptor does not have residual absorption at the donor emission and, more importantly, that it does not fluoresce in the donor spectral region.

Imaging protein interactions by FRET microscopy: FRET measurements by sensitized emission.

This protocol describes a method for measuring fluorescence resonance energy transfer (FRET) by the detection of acceptor-sensitized emission. This approach is useful in situations where donor intensities are low and/or there is contamination with high background (auto) fluorescence in the donor channel. However, absorption spectra characteristically exhibit long tails in the higher-energy, shorter-wavelength (blue) region, which may result in the direct excitation of the acceptor molecule in addition to that of the donor, thus resulting in mixing of direct and sensitized emission.

An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization.