Sub-resolution imaging and Bayesian analysis of exocytosis events reveals nanoscale patterning by cortical microtubule arrays.

The microtubule cytoskeleton organizes exocytosis to enable cellular morphogenesis, but how non-centrosomal arrays control exocytic site positioning remains poorly understood. Using elongating Arabidopsis thaliana cells as a model, we developed quantitative methods to move beyond coarse correlation and reveal the precise spatial relationship between cortical microtubules and secretion. We identify KEULE, an essential SEC/MUNC protein, as a dynamic exocytosis marker that forms clusters with stereotyped assembly and disassembly kinetics at discrete secretion sites. Combining confocal microscopy with super-resolution analysis and Bayesian inference, we quantified microtubule-exocytosis positioning at nanometer precision. This analysis revealed that microtubules create ∼180-nm enrichment zones flanked by ∼520-nm depletion zones, generating a spatial pattern that replicates the cortical array structure. Unexpectedly, Bayesian inference showed strong evidence for a flat enrichment profile within these zones rather than peaked distributions. This flat profile, combined with the ∼180-nm width, challenges a direct vesicle capture mechanism, suggesting consideration of alternative models such as a mechanism where microtubules organize the local membrane environment to create preferred exocytosis territories. These findings establish quantitative spatial rules for how non-centrosomal microtubule arrays organize secretion.