EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration

Abstract

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. We investigated whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functioned as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevented microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, was sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibited fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration.

Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules

“…Detyrosination occurs exclusively on stable MTs, which are capped at their plus ends by a complex which includes ϩTIPs and mDia (Wen et al, 2004). Consistent with these results, we find that Ad infection induces the formation of stable detyrosinated MTs (Glu-MTs) in a RhoA-dependent pathway.…”

“…Treatment of A549 cells with the Rac1-inhibitor NSC23766 blocked EB1 binding at MT plus ends (our unpublished data). Inhibition of RhoA would also be incompatible with an EB1-based nucleation assay due to EB1 functioning downstream of RhoA (Wen et al, 2004).…”

“…The accumulation of Glu-MTs in serum-starved fibroblasts is regulated by the activation of the RhoGTPase RhoA and its downstream effector mDia, which associates with MT plus-end binding proteins to "cap" the MT plus end and stabilize MT dynamics (Infante et al, 2000;Palazzo et al, 2001;Wen et al, 2004). The molecular mechanisms regulating acetylation of MTs is less clearly understood, although activation of RhoA has been implicated in the HHV-8 -induced hyperacetylation of MTs (Naranatt et al, 2005).…”

Generation of noncentrosomal microtubule arrays

“…For example, EB1, APC and MACF/ACF7 mediate MT capture in fibroblasts and endodermal cells (Kodama et al, 2003;Wen et al, 2004). EB1, APC and Shortstop (Drosophila MACF/ACF7) are found at the myotendenous junction in Drosophila and are necessary for the arrangement of the noncentrosomal MTs found there (Subramanian et al, 2003).…”

Poleward Tubulin Flux in Spindles: Regulation and Function in Mitotic Cells

“…Microtubule polymerization and stability is also promoted by the plus-end tracking protein (ϩTIP), EB1 (Rogers et al, 2002;Tirnauer et al, 2002;Wen et al, 2004). The specific changes to microtubule dynamics vary between experimental systems, but generally EB1 stabilizes microtubules by increasing the rescue frequency and decreasing the time spent in a paused state.…”