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Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the  Arabidopsis  KNOLLE syntaxin in the plane of cell division is maintained by sterol‐dependent endocytosis involving a clathrin‐ and DYNAMIN‐RELATED PROTEIN1A‐dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis‐localizes to lateral plasma membranes after cell‐plate fusion. Fluorescence‐loss‐in‐photo‐bleaching and fluorescence‐recovery‐after‐photo‐bleaching experiments reveal lateral diffusion of GFP‐KNOLLE from the plane of division to lateral membranes. In an endocytosis‐defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis