Biology subjects

Kurz, A.

Publications and source records attributed to Kurz, A..

2 recordsLinked to original sources

bioRxiv2019

Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy

The synaptonemal complex (SC) is a meiosis-specific nuclear multiprotein complex that is essential for proper synapsis, recombination and segregation of homologous chromosomes. We combined structured illumination microscopy (SIM) with different ExM protocols including U-ExM, proExM, and magnified analysis of the proteome (MAP) to investigate the molecular organization of the SC. Comparison with structural data obtained by single-molecule localization microscopy of unexpanded SCs allowed us to investigate ultrastructure preservation of expanded SCs. For image analysis, we developed an automatic image processing software that enabled unbiased expansion factor determination. Here, MAP-SIM provided the best results and enabled reliable three-color super-resolution microscopy of the SCs of a whole set of chromosomes in a spermatocyte with 20-30 nm spatial resolution. Our data demonstrate that post-expansion labeling by MAP-SIM improves immunolabeling efficiency and allowed us thus to unravel previously hidden details of the molecular organization of SCs.

biophysics↗

bioRxiv2018

Bioorthogonal labeling with tetrazine-dyes forsuper-resolution microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

biochemistry↗