Abstract
When Saccharomyces cerevisiae divides, a structure composed of different septin proteins arranged according to a certain rule is formed at the cell division site. The structure undergoes multiple remodeling stages during the cell cycle, thus guiding the yeast cells to complete the entire division process. Although the higher-order structure of septins can be determined using electron microscopy, the septin’s dynamic processes are poorly understood because of limitations in living cell super-resolution imaging technology. Herein, we describe a high lateral resolution and temporal resolution technique, known as fast fluctuation-enhanced structured illumination microscopy (fFE-SIM), which more than doubles the SIM resolution at a frame rate of 38 Hz in living cells. This allows a highly dynamic and sparse septin structure to be observed in Saccharomyces cerevisiae.
Highlights
Septins exist in most eukaryotes and are conserved GTP-binding proteins that were first discovered to play a role in the cell cycle of budding yeast, S. cerevisiae [1,2]
Using the proposed fast fluctuation-enhanced structured illumination microscopy (fFE-SIM) method to image live budding yeast, we found Cdc12-related structures in the middle portion of the splitting septin ring, and determined that these structures are highly dynamic during cytokinesis
To verify the lateral resolution, we fixed BS-C-1 cells transfected with EGFP-alphaTubulin and imaged them with lattice SIM to obtain a sequence of time-series images and them processed with the Super-resolution radial fluctuations (SRRF) algorithm after processing with the SIM algorithm
Summary
Septins exist in most eukaryotes and are conserved GTP-binding proteins that were first discovered to play a role in the cell cycle of budding yeast, S. cerevisiae [1,2]. Lattice SIM is the most suitable super-resolution microscopy technology for imaging living cells, its spatial resolution, at only 120 nm, is far less than that of STED (~50 nm) and SMLM (~20 nm) [25]. To overcome such limited resolution, many researchers have combined different superresolution techniques. We improved the processing method to enhance the temporal resolution of living-cell imaging and named the proposed method fluctuation-enhanced structured illumination microscopy (fFE-SIM). Using the proposed fFE-SIM method to image live budding yeast, we found Cdc12-related structures in the middle portion of the splitting septin ring, and determined that these structures are highly dynamic during cytokinesis
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