Establishing super-resolution imaging for proteins in diatom biosilica

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.

Single-molecule fluorescence microscopy review: shedding new light on old problems

“…Photoconvertible fluorescent proteins, Dendra2, mEos3.2 and Dronpa, were fused to biosilica-associated protein Silaffin-3 of model diatom Thalassiosira pseudonana [153]. …”

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection

“…Problems arise when combining peptides with incompatible fusion preferences, as was found to be the case in creating a series of diatom biosilica-targeted constructs encoding sdAbs against the B. anthracis S-layer protein EA1. In particular, both the Sil3 T8 (e.g., [12,15,16,32]) and sdAb EA1 (e.g., [33,34]) peptides previously have been made only as N-terminal fusions to their partners. One reason for this order of fusion partners is the requirement of many signal peptides' proximity to the protein terminus, rather than just their amino acid sequence.…”

Understanding Diatom Cell Wall Silicification—Moving Forward

“…A current limitation in linking molecular entities with ultrastructure is the insufficient resolution of confocal or other optical sectioning microscopy to image fluorescent proteins. The first application of super-resolution fluorescence microscopy on TpSil3 (Gröger et al, 2016) suggests a direction to move toward. In this study, three different fluorescent proteins fused to TpSil3 displayed fluorescence of the protein embedded in silica, with resolution on the order of 25 nm.…”

“…Despite the challenges, multiple aspects of cell wall formation have been described through a combination of experimental approaches. We expect that the recent breakthroughs in identifying a new class of SDV associated proteins (Kotzsch et al, 2017;Tesson et al, 2017), the presence of transcriptomic datasets to probe for candidate silicification genes (Shrestha et al, 2012;Keeling et al, 2014;Brembu et al, 2017), dynamic and higher resolution imaging (Gröger et al, 2016;Kotzsch et al, 2017) and genetic manipulation approaches (Tesson et al, 2017) will complement the previously established characterization approaches to allow the field to move forward with a better understanding of the dynamic spatial and temporal protein interactions that are responsible for generating silica structure in a coordinated and controlled manner.…”