ABSTRACT
The ability of cells to assemble, adhere to and dynamically sense the extracellular matrix (ECM) is essential for
multicellular life. Integrins, a family of heterodimeric aß transmembrane adhesion receptors, bind specific
ECM ligands via their ectodomains and permit bidirectional communication vital for cell adhesion, migration,
differentiation, and survival. Proper integrin function is paramount for tissue morphogenesis, and is perturbed
in cancer, skin disorders, musculoskeletal, cardiovascular and inflammatory diseases. A major site of integrin-
matrix engagement is in dynamic micron-sized signaling platforms, called focal adhesions (FA). Integrins can
laterally exchange into and out of FA, but also traffic to and from the cell surface and this trafficking influences
cell migration, invasion and cancer metastasis. However, despite its importance, understanding at the cellular
and mechanistic level of precisely where and how integrins undergo exocytic and endocytic traffic has
been challenging due to difficulties directly visualizing integrin exo-endocytosis in live cells. In response to this
challenge we recently generated `ecto-tagged' integrins containing the pH-sensitive fluorophore pHluorin or a
chemical-genetic Halo-tag inserted into an extracellular loop. These ecto-tagged integrins provided the first
direct views of integrin exocytosis and revealed that, contrary to initial expectations, integrin
exocytosis occurs at a subset of FA. Drawing on our expertise in integrins (Calderwood) and live-cell
imaging of membrane trafficking (Toomre), this multi-investigator R01 proposal seeks to test major new
hypotheses arising from these results. In Aim 1 we test the hypothesis that integrins are selectively delivered to
growing FA in a subunit-specific, ECM-regulated process. In Aim 2 we test the hypotheses that integrin
endocytosis occurs at a distinct set of FA that are turning over and that endocytosed integrins are recycled to
growing FA. Furthermore, we probe molecular mechanisms involved in recycling to FA. Finally, in Aim 3 we
test the hypothesis that integrin-dependent fibronectin (FN) exocytosis also occurs at growing FA, while FN
endocytosis occurs at FA that are turning over. Our experimental approaches combine novel ecto-tagged
integrins and matrix constructs with new cleavable Halo dyes and pH-switching to follow exo-endocytosis in
live cells so as to test new hypothesis about where integrin is delivered and the underlying mechanisms.