Through the looking-glass: Structured illumination microscopy reveals new dynamic behaviors of cellulose synthase

Cellulose is the most abundant biopolymer on Earth and accounts for ∼40% of total plant biomass. Understanding cellulose biosynthesis is fundamental for efficient utilization of this renewable resource, promoting the transition to a greener economy. Cellulose Synthase Complexes (CSCs) are thought to comprise rosette-shaped hexamers of CELLULOSE SYNTHASE (CESA) trimers, producing 18-chain cellulose microfibrils (Hill et al., 2014; Purushotham et al., 2020). Although significant advances in deciphering the structure of CSCs have been made in recent years, many questions remain concerning CSC behaviors in living plant cells, including how the movements of CSCs are regulated through the plasma membrane as they spin cellulose into the wall and how they are affected by the cytoskeleton and pre-existing wall patterning.

Live-cell imaging by confocal microscopy has been used to monitor the dynamics of CESAs and their interactions with microtubules and other molecular partners during cellulose biosynthesis. The resolution limit of light microscopy, ∼200 nm in most cases, is much larger than the size of CSCs (∼25 nm), hindering the precise recording of CSC movements. In a new study, Sydney Duncombe and coauthors (Duncombe et al., 2021) reveal new dynamic behaviors of CESA in living plant cells through structured illumination microscopy (SIM), a super-resolution microscopy technique that achieves a resolution of ∼100 nm.

SIM generates a series of images illuminated by a grating pattern with shifted phases. Using Fourier transformation, information about the spatial frequency of the illuminated features is collected and the series of Fourier-transformed images are computationally combined to generate a new image with improved resolution (Gustafsson, 2000). Sydney Duncombe and coauthors (Duncombe et al., 2021) performed SIM using epidermal cells at the midvein of the cotyledon petioles in Arabidopsis seedlings expressing GFP-CESA3. Cells were pressed gently against the coverslip to maintain sample stability so that consistent images of CESA particles were collected over time. SIM can generate time-lapse movies of up to 20 min with intervals as short as 5 s. The dynamic behaviors of CESA particles were monitored effectively by SIM, whereas faster-moving Golgi and CESA-containing vesicles left streaking artifacts in the reconstructed images. GFP proved bright and photostable enough for time-lapse imaging of CESA particles.

Unresolved CSCs that are tracked as single particles may result in erroneous analyses and interpretations of how CSCs produce cellulose. To compare precision in tracking, SIM and confocal data were analyzed by Imaris software. CESA particles tracked from the SIM data did not deviate extensively from their trajectories, whereas they meandered from linear trajectories in confocal images (see figure). Improved tracking from the SIM data also revealed that individual particle speeds remain relatively steady. The density of CESA particles observed by SIM (∼2 particles/µm²) was more than twice that of confocal microscopy, but still well below the density of cellulose microfibrils, as seen by atomic force microscopy or electron microscopy. Given the resolution of SIM (∼100 nm) and the size of CSCs (∼25 nm), these data raise the possibility that there are perhaps still unresolved groups of CSCs moving coordinately at relatively stable speeds, generating larger cellulose microfibril bundles. By contrast, particles from different regions within a cell were observed to move at different speeds. Closer examination of the relationships between particle speeds and microtubule organization and dynamics as well as wall patterning might provide additional clues for how CESA motility is regulated. A small number of CESA particles were also observed making U-turns without changing speeds. Further investigations are merited to probe the mechanisms underlying this U-turn behavior, and how this new behavior of CSCs might influence cell wall patterning.

Figure.

SIM improves tracking precision of CESA particles in living plant cells. New dynamic behaviors of CESA particles such as U-turns were also revealed by SIM. Scale bar = 2 µm. Figure adapted from Duncombe et al. (2021).