Function and dynamics of microtubules and plus-end-tracking proteins during 3d epithelial remodeling

Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays essential roles in development and disease of multicellular organisms. How cytoskeleton dynamics, especially microtubule dynamics, are controlled and contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We optimize confocal live-cell imaging to analyze microtubule function and dynamics during 3D epithelial remodeling of polarized Madin-Darby kidney epithelial cells that undergo a partial epithelial-to-mesenchymal transition (pEMT) in response to hepatocyte growth factor (HGF). We found that cellular extensions at the basal surface of HGF-treated cysts are densely packed with microtubules. Computational tracking of EB1-2xEGFP showed large numbers of microtubules growing persistently from the apical domain into these extensions and an increase in microtubule growth rate in response to HGF. We tested the role of microtubule plus-end-tracking protein (+TIP) complexes in 3D epithelial remodeling by depleting cells of EB1, an adaptor protein that mediates recruitment of other +TIP proteins to growing microtubule plus ends. In EB1-depleted cells, microtubules displayed rapid lateral and retrograde movements demonstrating that EB1 is required to stabilize and organize microtubules in HGF-induced extensions. EB1-depleted cysts formed shorter, more branched, extensions suggesting that EB1 is required for productive HGF-induced extension outgrowth. Analysis of cell-matrix interactions and F-actin dynamics revealed that control extensions progressively pulled on and deformed the extracellular matrix (ECM) typically with one F-actin-rich protrusion near the cell tip with cell-matrix adhesions that turned over in a coordinated fashion. In contrast, EB1-depleted cells produced multiple highly dynamic protrusions with nascent adhesions that were uncoordinated, mislocalized and did not productively engage the matrix. As a result EB1-depleted extensions rapidly protruded, retracted and changed direction. Finally we show that trafficking of VAMP3-positive vesicles to the protrusion tip is disrupted in EB1-depleted cells. Together these data suggest that EB1-mediated organization of the MT cytoskeleton and associated vesicle delivery to the tip of HGF-induced extensions are likely required to coordinate cell-matrix adhesion and protrusion dynamics during 3D epithelial remodeling. It will be important in the future to investigate a broader role for EB1-mediated +TIP complexes in other normal and disease states of epithelial remodeling.