Contribution of the LIM domain and nebulin-repeats to the interaction of Lasp-2 with actin filaments and focal adhesions

Abstract

Lasp-2 binds to actin filaments and concentrates in the actin bundles of filopodia and lamellipodia in neural cells and focal adhesions in fibroblastic cells. Lasp-2 has three structural regions: a LIM domain, a nebulin-repeat region, and an SH3 domain; however, the region(s) responsible for its interactions with actin filaments and focal adhesions are still unclear. In this study, we revealed that the N-terminal fragment from the LIM domain to the first nebulin-repeat module (LIM-n1) retained actin-binding activity and showed a similar subcellular localization to full-length lasp-2 in neural cells. The LIM domain fragment did not interact with actin filaments or localize to actin filament bundles. In contrast, LIM-n1 showed a clear subcellular localization to filopodial actin bundles. Although truncation of the LIM domain caused the loss of F-actin binding activity and the accumulation of filopodial actin bundles, these truncated fragments localized to focal adhesions. These results suggest that lasp-2 interactions with actin filaments are mediated through the cooperation of the LIM domain and the first nebulin-repeat module in vitro and in vivo. Actin filament binding activity may be a major contributor to the subcellular localization of lasp-2 to filopodia but is not crucial for lasp-2 recruitment to focal adhesions.

Figure 1. A schematic representation of the domain structure of avian lasp-2 and its fragments.

LIM and SH3 indicate the LIM and Src homology 3 domains, respectively. The number of nebulin-repeats is shown by n1 to n3. The LIM-n1 fragment contains the LIM domain and first nebulin-repeat. ΔLIM is a deletion fragment of the LIM domain. The LIM fragment contains only the LIM domain. The numbers indicate the positions of the corresponding amino acid residues.

Figure 2. F-actin-binding activity of recombinant lasp-2 and its fragments in the co-sedimentation assay.

0.2 mg/ml G-actin was polymerized with GST-lasp-2 (L2), GST-LIM-n1 (LIM-n1), GST-ΔLIM (ΔLIM), or GST-LIM (LIM). The molar ratio of GST-fused fragment to G-actin was set at 1/5. GST-lasp-2 (arrowhead in A) and GST-LIM-n1 (double-arrowhead in A) were co-precipitated with actin filaments, but GST-ΔLIM (arrowhead in B) and GST-LIM (double-arrowhead in B) were not. FA indicates a control experiment using F-actin without the recombinant peptides. The precipitants (p) were separated from the supernatant (s) by ultracentrifugation. The arrowheads and double-arrowheads indicate bands of recombinant GST fused proteins with the predicted molecular mass. The mobilities of molecular mass markers are listed on the right of the gel images in kilodaltons.

Figure 3. Localization of EGFP-tagged lasp-2 fragments in NG108-15 cells.

Cells transfected with EGFP-lasp-2 (A), EGFP-LIM-n1 (B), EGFP-ΔLIM (C), or EGFP-LIM (D) are shown as indicated. The inset in C shows widely distributed lamellipodial extension. Actin filaments were visualized by staining with rhodamine-phalloidin (F-actin in A). The bars in A and D represent 5 and 10 µm, respectively.

Figure 4. Localization of EGFP-tagged lasp-2 fragments in C2C12 cells.

Cells transfected with EGFP-actin (A), EGFP-lasp-2 (B), EGFP-ΔLIM (C), or EGFP-LIM-n1 (D) are shown as indicated. Corresponding images of interference refraction microscopy (IRM) are shown as A′ to D′. The block arrows indicate the position of focal contacts visualized by IRM, and the corresponding areas in the fluorescence images are indicated by white arrows. The bar in D′ represents 10 µm.

Figure 5. The predicted functions of structural domains in lasp-2.

The results of this study are summarized in A. (+) indicates interaction with actin filaments, localization to actin bundles in NG108-15 cells, or focal adhesion in C2C12 cells. (−) indicates negative results compared to (+). The identities of the amino acid sequences of the LIM domain, first nebulin-repeat region, and SH3 domain between lasp-1 and lasp-2 are shown in B. The grey double-headed arrow above lasp-1 indicates the region containing actin-binding activity as reported by Schreiber et al. (1998). The black double-headed arrows under lasp-2 show the regions responsible for localization to focal adhesions and interaction with F-actin in vitro and in vivo.