Structural model and functional significance of pH-dependent talin–actin binding for focal adhesion remodeling

Abstract

Actin filament binding by the focal adhesion (FA)-associated protein talin stabilizes cell-substrate adhesions and is thought to be rate-limiting in cell migration. Although F-actin binding by talin is known to be pH-sensitive in vitro, with lower affinity at higher pH, the functional significance of this pH dependence is unknown. Because increased intracellular pH (pHi) promotes cell migration and is a hallmark of metastatic carcinomas, we asked whether it increases FA remodeling through lower-affinity talin-actin binding. Talin contains several actin binding sites, but we found that only the COOH-terminal USH-I/LWEQ module showed pH-dependent actin binding, with lower affinity and decreased maximal binding at higher pH. Molecular dynamics simulations and NMR of this module revealed a structural mechanism for pH-dependent actin binding. A cluster of titratable amino acids with upshifted pKa values, including His-2418, was identified at one end of the five-helix bundle distal from the actin binding site. Protonation of His-2418 induces changes in the conformation and dynamics of the remote actin binding site. Structural analyses of a mutant talin-H2418F at pH 6.0 and 8.0 suggested changes different from the WT protein, and we confirmed that actin binding by talin-H2418F was relatively pH-insensitive. In motile fibroblasts, increasing pHi decreased FA lifetime and increased the migratory rate. However, expression of talin-H2418F increased lifetime 2-fold and decreased the migratory rate. These data identify a molecular mechanism for pH-sensitive actin binding by talin and suggest that FA turnover is pH-dependent and in part mediated by pH-dependent affinity of talin for binding actin. intracellular pH ͉ NHE1 ͉ migration F ocal adhesion (FA) remodeling is a rate-limiting determinant in haptokinetic migration of adherent cells. At the leading edge of migrating cells, FAs undergo rapid cycles of assembly and turnover, creating and disrupting, respectively, sites of traction necessary for forward movement of the cell body. Force generation for traction requires linkage among the extracellular matrix, integrin receptors, and actin filaments. Actin filaments do not directly bind to the cytoplasmic domain of integrins but bind to integrin-associated FA proteins such as talin and vinculin. Although several mechanisms contribute to FA remodeling in migrating cells (1), emerging evidence indicates that talin plays a central role in the dynamic linkage between integrins and actin filaments necessary for cell migration (2, 3). Talin functions in distinct albeit complementary mechanisms that promote FA turnover. First is cleavage of talin by the protease calpain, which also modulates adhesion complex composition and likely signaling functions of talin (4). Second is regulated talin binding to actin filaments, which is proposed to act as a clutch to control FA turnover and membrane protrusion dynamics (3, 5-7). How actin binding by talin is dynamically regulated during cell migration, however, remains undetermined.Previous stu...

Regulation of Early Neurite Morphogenesis by the Na+/H+ Exchanger NHE1

“…Finally, netrin-1 may regulate adhesive complexes to promote neurite outgrowth (Li et al, 2004;Liu et al, 2004;Ren et al, 2004). Although NHE1 may not be structurally associated with adhesion molecules, it's activity promotes the proper assembly and turnover of focal adhesion complexes at the leading edge of migrating cells (Meima et al, 2007;Srivastava et al, 2008), in which NHE1-dependent H ϩ efflux also facilitates adhesion to the extracellular matrix (Stock et al, 2008). Together, these observations provide another possible link between NHE1 and the stimulation of early neurite morphogenesis by netrin-1.…”

“…A role for pH i is also suggested by the facts that NHE1-null neurons exhibit a lower resting pH i (Yao et al, 1999) and reduced neurite elaboration and elongation (present study) than their WT counterparts, with NHE1 ϩ/Ϫ neurons exhibiting intermediate levels of NHE1-dependent H ϩ extrusion (Luo et al, 2005) and neurite outgrowth. NHE1-dependent elevations in growth cone pH i could affect actin cytoskeletal remodeling by altering the state of actin polymerization directly and/or by modulating the activities of actin-binding and other proteins that together regulate the dynamic reorganization of the actin cytoskeleton (Srivastava et al, 2007(Srivastava et al, , 2008. For example, increases in pH i promote not only the activation of actin depolymerizing factor and cofilin, which enhance filopodial dynamics and neurite outgrowth, but also their translocation to the leading edge of migrating cells (Bernstein et al, 2000;Frantz et al, 2008).…”

Electrostatics Control Actin Filament Nucleation and Elongation Kinetics

“…The physiological role of increasing pH in actin polymerization has already been observed in early studies on fertilization of sea urchin eggs (6) and on the acrosomal reaction of echinoderm sperms (7). More recently, molecular mechanisms contributing to pH regulation of actin turnover (8) and force generation (9) have been identified. In this respect, the major actin severing and depolymerizing factor cofilin has been shown to be inhibited by low pH (8).…”

“…In this respect, the major actin severing and depolymerizing factor cofilin has been shown to be inhibited by low pH (8). Similarly, activity of talin, which links actin to integrins in focal adhesion and helps traction force generation, has been shown to be sensitive to local pH (9). The major regulator of intracellular pH (pH i ) in mammalian cells, the Na/H ϩ exchanger 1 (NHE1), 4 is localized at the leading edge, where continuous polymerization and turn-over of actin occurs during cell migration (10).…”

Cancer cell behaviors mediated by dysregulated pH dynamics at a glance

“…During normal cell migration, dynamic changes in pH i enable both cytoskeletal and focal adhesion remodeling, with increased pH i decreasing the stability of focal adhesions (Srivastava et al, 2008) and increasing overall cell migratory rates (Choi et al, 2010) (see poster). Importantly, decreasing pH i or increasing pH e inhibits cell migration (Parks and Pouyssegur, 2015;Cong et al, 2014;Frantz et al, 2008;Denker and Barber, 2002).…”