Establishing super-resolution imaging for proteins in diatom biosilica.

Philip Gröger,Michael Schlierf,Jennifer Klemm,Nils Kröger,Nicole Poulsen

doi:10.1038/srep36824

Abstract

The intricate, genetically controlled biosilica nano- and micropatterns produced by diatoms are a testimony for biology’s ability to control mineral formation (biomineralization) at the nanoscale and regarded as paradigm for nanotechnology. Previously, several protein families involved in diatom biosilica formation have been identified, and many of them remain tightly associated with the final biosilica structure. Determining the locations of biosilica-associated proteins with high precision is, therefore expected to provide clues to their roles in biosilica morphogenesis. To achieve this, we introduce here single-molecule localization microscopy to diatoms based on photo-activated light microscopy (PALM) to overcome the diffraction limit. We identified six photo-convertible fluorescent proteins (FPs) that can be utilized for PALM in the cytoplasm of model diatom Thalassiosira pseudonana. However, only three FPs were also functional when embedded in diatom biosilica. These were employed for PALM-based localization of the diatom biosilica-associated protein Silaffin-3 (tpSil3) with a mean precision of 25 nm. This allowed for the identification of distinct accumulation areas of Sil3 in the biosilica, which cannot be resolved by confocal fluorescence microscopy. The enhanced microscopy technique introduced here for diatoms will aid in elucidating the molecular mechanism of silica biomineralization as well as other aspects of diatom cell biology.

Highlights

Diatoms are one of the most abundant unicellular microalgae found in all aquatic ecosystems across the world[1]
Dronpa is a photo-switchable fluorescent proteins (FPs) that can be repeatedly switched between a non-fluorescent and a fluorescent state
We focused on a range of different emission wavelengths, since diatom chloroplasts exhibit a broad autofluorescent background that could interfere with imaging of the FPs

Summary

Introduction

Diatoms are one of the most abundant unicellular microalgae found in all aquatic ecosystems across the world[1]. The silaffin tpSil[3] is associated with the so called valve region whereas the cingulins are associated with the girdle band region (Fig. 1)[7] These studies have not been able to determine the precise localization of the GFP-fusion proteins as confocal imaging is limited by the diffraction limit of light (≈250 nm) (Fig. 1B). We have screened six different fluorescent proteins (FPs) to identify and evaluate suitable candidates To this end, we compared the photo-conversion abilities of cytosolic FPs with that of biosilica-embedded FPs. For the biosilica embedding we created fusion proteins with Silaffin-3 (tpSil3), which is thought to be involved in biosilica formation and remains permanently entrapped inside the biosilica of the valve region[19,20]. Reconstruction microscopy on the single-molecule level allowed for localization of Dendra[2], mEOS3.2 and Dronpa fusion-proteins embedded in the silica with an average precision of 28 nm, 25 nm and 25 nm, respectively

Methods

Results

Conclusion