A general method to fine-tune fluorophores for live-cell and in vivo imaging
From Nature Methods (September 2017)
The development of cutting-edge fluorescence microscopy methods goes along with the search for dyes with optimal chemical and spectral properties. These optimal dyes must have a large extinction coefficient and quantum yield, they should be cell permeable for in vivo imaging, and the spectrum of their absorption and emission wavelengths should allow different dye combinations for multicolour imaging. Unfortunately, such dyes are rare. The Lavis laboratory first increased the brightness and photostability of rhodamine with the insertion of four-membered azetidine rings. Then, they developed an elegant set of rules to rationally fine-tune the spectral and chemical properties of their rhodamine dyes by the incorporation of 3-substituted azetidine groups. The series of dyes that they designed goes from λabs/λem = 503 nm/529 nm to λabs/λem = 646 nm/ 664 nm and can reach a quantum yield of 0.91 or an extinction coefficient as good as 11.3×104 M-1cm-1 (for comparison the rhodamine 110 quantum yield is 0.88 and its extinction coefficient is 7.6x 104 M-1 cm-1 while EGFP quantum yield is 0.6 and its extinction coefficient is 5.5×104 M-1 cm-1). They conclude their work by testing some of their dyes with two-photons and light sheet microscopy in vivo imaging experiments. The dyes are available for free on demand.
The structure of the COPI coat determined within the cell
From eLife (November 2017)
“We believe that the ability to determine structure directly within the cellular environment can transform cell and structural biology”, is how the authors rightfully conclude their paper. In this work, Bykov et al. pioneered the application of cryo-focused ion beam milling, cryo-electron tomography and reference-free subtomogram averaging to determine the structure of a protein complex, COPI, directly in its native environment, in C. reinhardtii cells. COPI is a conserved protein complex that coats vesicles which mediate the trafficking within the Golgi apparatus and from the Golgi to the endoplasmatic reticulum. The COPI coat structure that the authors find in situ is remarkably similar to the COPI coat modelled from in vitro studies using mouse proteins, which suggests that the COPI architecture is highly conserved in distantly related species. In situ, they observed that vesicles are only incompletely coated, with each vesicle containing a gap in the coat at the site of scission. They also inferred the relative dynamics of vesicle uncoating from the distribution of COPI coat completeness in the proximity of the Golgi: the coat disassembly starts after budding but it is not catastrophic and it is not related to structural changes in the COPI coat. Finally, they could identify densities in the luminal side of the membrane that are probably cargos bound by ß’-COP.
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