Confocal microscopy has allowed for numerous advancements in the characterization of cellulose synthases (CESAs) and their behaviors in plants, providing insights into how plant cell walls are synthesized. However, the resolution limit of confocal microscopy restricts the amount of information we can gain from live cell imaging. Structured Illumination Microscopy (SIM) is a super-resolution microscopy technique that can surpass the diffraction limit of light while still imaging live cells expressing fluorescent tags. With a two-fold increase in resolution, we have observed a higher density of cellulose synthase particles at the cell surface in seedlings of Arabidopsis thaliana. By imaging Arabidopsis seedlings expressing both GFP-CESA3 and mCherry-TUA5 with SIM to simultaneously observe CESAs and microtubules, we can detect more nuanced CESA behaviors and how they relate to the microtubules that guide their trajectories as they polymerize cellulose. Cells in the petioles of cotyledons exhibit several different patterns of CESA trajectories, varying from transverse patterning to longitudinal patterning to more random, isotropic track orientations. Time-lapse imaging in these cells reveals a rare yet recurring CESA behavior in which a CESA particle leaves its microtubule track, only to turn 180 degrees and join a new microtubule track heading in the opposite direction, similar to a “U turn” maneuver. The mechanism behind this phenomenon is still unknown, but might involve a bias in CESA particle motility toward one end of a microtubule. Using SIM, these nanoscale CESA particle behaviors can be tracked more accurately, especially in relation to their movements and interactions with microtubules, allowing for detailed analysis of the deposition of the most abundant biopolymer on Earth.
Coauthors: Charles Anderson – The Pennsylvania State University