Abstract
Although the distinct distribution of certain molecules along the anterior or posterior edge is essential for directed cell migration, the mechanisms to maintain asymmetric protein localization have not yet been fully elucidated. Here, we studied a mechanism for the distinct localizations of two Dictyostelium talin homologues, talin A and talin B, both of which play important roles in cell migration and adhesion. Using GFP fusion, we found that talin B, as well as its C-terminal actin-binding region, which consists of an I/LWEQ domain and a villin headpiece domain, was restricted to the leading edge of migrating cells. This is in sharp contrast to talin A and its C-terminal actin-binding domain, which co-localized with myosin II along the cell posterior cortex, as reported previously. Intriguingly, even in myosin II-null cells, talin A and its actin-binding domain displayed a specific distribution, co-localizing with stretched actin filaments. In contrast, talin B was excluded from regions rich in stretched actin filaments, although a certain amount of its actin-binding region alone was present in those areas. When cells were sucked by a micro-pipette, talin B was not detected in the retracting aspirated lobe where acto-myosin, talin A, and the actin-binding regions of talin A and talin B accumulated. Based on these results, we suggest that talin A predominantly interacts with actin filaments stretched by myosin II through its C-terminal actin-binding region, while the actin-binding region of talin B does not make such distinctions. Furthermore, talin B appears to have an additional, unidentified mechanism that excludes it from the region rich in stretched actin filaments. We propose that these actin-binding properties play important roles in the anterior and posterior enrichment of talin B and talin A, respectively, during directed cell migration.
Highlights
Directed cell migration underlies diverse biological processes including development, wound healing, cancer metastasis, and the immune response [1]
To generate the constructs to express GFP-I/LWEQ(talA), GFP-I/LWEQ(talB), GFP-I/ LWEQ(talB)-proline-rich region (PRR)-villin headpiece (VHP), and GFP-PRR-VHP, the cDNA fragments coding for amino acid residues 2255 to 2492 of talin A, 2225 to 2457 of talin B, 2225 to 2614 of talin B, and 2458 to 2614 of talin B were amplified by PCR using the talin A-GFP construct [28] or the GFP-talin B construct as a template, and were subcloned into pTX-GFP as SacI/XbaI fragments
To generate the constructs to express GST-I/LWEQ, GST-I/LWEQ(talB), and GST-I/LWEQ(talB)-PRR-VHP in E. coli, the cDNA fragments coding for amino acid residues 2255 to 2492 of talin A, 2225 to 2457 of talin B, and 2225 to 2614 of talin B were amplified by PCR using the talin A-GFP construct [28] or the GFP-talin B construct as a template, and were subcloned into pGEX-6-P3 vector as a BamHI/EcoRI fragment for I/LWEQ(talA), a BamHI/XhoI fragment for I/LWEQ(talB), and a BamHI/XhoI fragment for I/LWEQ(talB)-PRR-VHP
Summary
Directed cell migration underlies diverse biological processes including development, wound healing, cancer metastasis, and the immune response [1]. Focal adhesions disassemble near the lagging edge, and the contraction of bundles composed of actin and myosin II filaments leads to retraction of the rear [3, 4]. The cytoskeletal cortex, which is mainly composed of actin filaments and associated proteins such as myosin II, flows backward during directed cell migration, and transports cell surface markers linked to the cortex toward the rear end [7, 8]. As examples of directed cell migration, TIAM1, which enhances pseudopod extension via Rac activation, is recruited along the leading edge by binding to Par, a member of the Par protein family [10]. Another PAR protein member, binds aPKC along the leading edge, and the complex recruits Smurf, an E3 ubiquitin ligase, to the leading edge to degrade RhoA, which mediates acto-myosin contraction [11]
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