Cellulose Synthase-Like D (CSLD) proteins, important for tip growth and cell division, are known to generate β-1,4-glucan. However, whether they are propelled in the membrane as the glucan chains they produce assemble into microfibrils is unknown. To address this, we endogenously tagged all eight CSLDs in Physcomitrium patens and discovered that they all localize to the apex of tip-growing cells and to the cell plate during cytokinesis. Actin is required to target CSLD to cell tips concomitant with cell expansion, but not to cell plates, which depend on actin and CSLD for structural support. Like Cellulose Synthase (CESA), CSLD requires catalytic activity to move in the plasma membrane. We discovered that CSLD moves significantly faster, with shorter duration and less linear trajectories than CESA. In contrast to CESA, CSLD movement was insensitive to the cellulose synthesis inhibitor isoxaben, suggesting that CSLD and CESA function within different complexes possibly producing structurally distinct cellulose microfibrils.

mEGFP-CesA10 and mEGFP-CSLD6 particles were imaged with VAEM every 2s for 10-20 minutes. Images were processed before tracking using subtract background and enhance contrast in Fiji. Particle tracking was performed with the Fiji plugin TrackMate (Tinevez et al., 2017). For CesA10, stacks were reduced by 10, resulting in 20 s time intervals between frames. For CSLD6, stacks were reduced by 6, resulting in 12 s time intervals between frames. LoG detector (estimate blob diameter 0.3 µm) and LAP tracker (frame to frame linking max distance 0.5 µm; Penalties: Quality=1; no gap closing allowed) were selected. The filters on tracks were set as follows: duration >60 s; track displacement >0.5 µm. Mean speed and the confinement ratio of tracks were obtained and plotted in Figure 3. The confinement ratio is a ratio of the straight-line distance between the start and end points of the trajectory divided by the actual trajectory distance. The closer the confinement ratio value is to one, the more linear the trajectory.