 The mycobacterial cell envelope consists of a typical plasma membrane, surrounded by a complex cell wall and a lipid rich outer membrane. The biogenesis of this multilayer structure is a tightly regulated process requiring the coordinated synthesis and assembly of all its constituents. Mycobacteria grow by polar extension and recent studies showed that cell envelope incorporation of mycolic acids, the major constituent of the cell wall and outer membrane, is coordinated with peptidoglycan biosynthesis at the cell poles. However, there is no information regarding the dynamics of incorporation of other families of outer membrane lipids during cell elongation and division. Here, we established that the translocation of non-essential treehalose polyphliates, TPP, occurs at different subcellular locations than that of the essential mycolic acids. Using fluorescence microscopy approaches, we investigated the subcellular localization of MMPL3 and MMPL10 respectively involved in the export of mycolic acids and TPP in growing cells and their collocalization with WAC31A. This article was authored by Laurie Thevenal, Jerome Retch, Christoph Gilhot, and others. We are article.tv, links in the description below.