Selective chemical imaging of static actin in live cells.

We have characterized rationally designed and optimized analogues of the actin-stabilizing natural products jasplakinolide and chondramide C. Efficient actin staining was achieved in fixed permeabilized and non-permeabilized cells using different combinations of dye and linker length, thus highlighting the degree of molecular flexibility of the natural product scaffold. Investigations into synthetically accessible, non-toxic analogues have led to the characterization of a powerful cell-permeable probe to selectively image static, long-lived actin filaments against dynamic F-actin and monomeric G-actin populations in live cells, with negligible disruption of rapid actin dynamics.

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. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.

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. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.

Structure of the p53 C-terminus bound to 14-3-3: implications for stabilization of the p53 tetramer.

The adaptor protein 14-3-3 binds to and stabilizes the tumor suppressor p53 and enhances its anti-tumour activity. In the regulatory C-terminal domain of p53 several 14-3-3 binding motifs have been identified. Here, we report the crystal structure of the extreme C-terminus (residues 385-393, p53pT387) of p53 in complex with 14-3-3sigma at a resolution of 1.28A. p53pT387 is accommodated by 14-3-3 in a yet unrecognized fashion implying a rationale for 14-3-3 binding to the active p53 tetramer. The structure exhibits a potential binding site for small molecules that could stabilize the p53/14-3-3 protein complex suggesting the possibility for therapeutic intervention.