Super-resolved imaging and Bayesian analysis of single exocytosis events reveals molecular-scale patterning by...

The microtubule cytoskeleton organizes exocytosis to enable cellular morphogenesis, but how non-centrosomal arrays control exocytotic site positioning remains poorly understood. Using elongating plant 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 enrichment zone widths exceeding the ∼50 nm reach of known tethering proteins, challenges a direct vesicle capture mechanism. Instead, our data support a membrane domain mechanism where microtubules organize lipid/protein composition to create preferred exocytosis territories. These findings establish quantitative spatial rules for how non-centrosomal microtubule arrays organize secretion.