Super-resolution imaging illuminates new dynamic behaviors of cellulose synthase.

Confocal imaging has shown that CELLULOSE SYNTHASE (CESA) particles move through the plasma membrane as they synthesize cellulose. However, the resolution limit of confocal microscopy circumscribes what can be discovered about these tiny biosynthetic machines. Here, we applied Structured Illumination Microscopy (SIM), which improves resolution two-fold over confocal or widefield imaging, to explore the dynamic behaviors of CESA particles in living plant cells. SIM imaging reveals that Arabidopsis thaliana CESA particles are more than twice as dense in the plasma membrane as previously estimated, helping explain the dense arrangement of cellulose observed in new wall layers. CESA particles tracked by SIM display minimal variation in velocity, suggesting coordinated control of CESA catalytic activity within single complexes and that CESA complexes might move steadily in tandem to generate larger cellulose fibrils or bundles. SIM data also reveal that CESA particles vary in their overlaps with microtubule tracks and can complete U-turns without changing speed. CESA track patterns can vary widely between neighboring cells of similar shape, implying that cellulose patterning is not the sole determinant of cellular growth anisotropy. Together, these findings highlight SIM as a powerful tool to advance CESA imaging beyond the resolution limit of conventional light microscopy.

Imaging the delivery and behavior of cellulose synthases in Arabidopsis thaliana using confocal microscopy.

Confocal microscopy has been a key tool for characterizing the behavior of cellulose synthase (CESA) proteins as they extrude cellulose into the apoplast to help construct plant cell walls. While other microscopy techniques like electron microscopy can achieve higher resolution images of CESAs, confocal microscopy is still the most accessible way to image these proteins in living plants as they are trafficked to and from the cell surface and move through the plasma membrane. Here, we describe a method for imaging fluorescently tagged CESA proteins in seedlings of Arabidopsis thaliana using spinning disk confocal microscopy, with a focus on quantifying the speed, density, and delivery rate of CESA particles. Many of these techniques can be adapted and applied to imaging other membrane-localized proteins and other plant species. In addition to imaging techniques, we describe several options for image analysis that can be optimized for different datasets.