LASP1, CERS6, and Actin Form a Ternary Complex That Promotes Cancer Cell Migration

Figures

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  Figure 1. LASP1 is a CERS6 binding partner. (A) CERS6-binding proteins identified by IP with the anti-CERS6 antibody and LC-MS/MS analysis. The top ten candidates based on abundance ratio (anti-CERS6 antibody-bound samples vs. those from anti-IgG antibody) are listed. (B) CLIP-WB with anti-CERS6 or anti-HA to show LASP1 interaction with CERS6. A549 cells with overexpression of CERS6-HA were incubated with 1% formaldehyde in RPMI1640, then the reaction was quenched using 125 mM of a glycine solution. Cells were harvested, lysed, and sonicated, then IP was performed using a Dynabeads co-IP kit, after which co-precipitated protein complexes were eluted, and subjected to SDS-PAGE and WB analyses. (C) IP-WB with anti-LASP1 to show CERS6 interaction with LASP1. After A549 cells were harvested and lysed, IP-WB was performed using a Dynabeads co-IP kit (see Materials and Methods). (D) Immunofluorescent staining of LASP1 and CERS6 at lamellipodia of A549, LNM35, and CERS6-overexpressed BEAS-2B cells. The Pearson’s coefficient values for A549, LNM35, and CERS6-overexpressed BEAS-2B were 0.702, 0.789, and 0.812, respectively. Original western blots are presented in File S1.

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  Figure 2. LIM domain of LASP1 required for CERS6 interaction. (A) Schematic illustration of C-terminal FLAG-tagged LASP1. The full-length (FL) and four truncated mutants (mut1-4) are shown. Predicted molecular weights are indicated. (B) IP-WB with an anti-FLAG antibody to precipitate LASP1-FLAG and demonstrate CERS6 interaction. EV, empty pCMV vector. Arrowheads: predicted positions for full-length and truncated LASP1. * Nonspecific band. (C) CERS6-HA was overexpressed in A549 cells and each truncated LASP1-FLAG mutant, then IP was performed using an anti-HA antibody. EV, empty pCMV vector. Arrowheads: predicted positions for full-length and truncated LASP1. Original western blots are presented in File S1.

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  Figure 3. LASP1 and CERS6 in the same axis, with a possible contribution to the promotion of cell migration. (A) WB analyses of LNM35 cells treated with siCTRL, siLASP1-1, and siLASP1-2 were performed. Relative values to histone H3 are indicated. (B) LNM35 cells were treated with siCTRL, siLASP1-1, or siLASP1-2; then, cell migration activity was determined in the presence or absence of 1 μM of C16 ceramide (C16 cer). At least 100 cells were counted and the results are shown as the mean ± SD value relative to that of siCTRLs. * p < 0.05, six independent experiments. (C) LNM35 cells were treated with siCTRL, siLASP1-1, or siCERS6-1, then fixed and stained with phalloidin. Scale bar = 40 μm. Arrowhead: lamellipodia. (D) Quantitative results are shown as values relative to siCTRL. At least 200 cells were counted and the results are shown as the mean ± SD. * p < 0.05, three independent experiments. Original western blots are presented in File S1.

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  Figure 4. LASP1 and CERS6 modulated counterpart protein levels. (A) WB analysis of LNM35 cells was performed under knock-down conditions, as indicated. Relative values to histone H3 are shown. (B) Using LC-MS/MS, ceramide amounts were determined in siCTRL-treated (black) and siLASP1-1-treated (gray) LNM35 cells (n = 3). Quantitative values are shown as relative to siCTRL. * p < 0.05. Original western blots are presented in File S1.

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  Figure 5. Association of β actin with LASP1 and CERS6. (A) Immunofluorescent staining of phalloidin and LASP1 or CERS6 at lamellipodia of LNM35 cells treated with siCTRL, siCERS6-1, or siLASP1-1. (B) IP-WB analysis was performed with the anti-LASP1 antibody to evaluate the interaction of LASP1–β actin. * Long exposure. (C) IP-WB analysis was performed with the anti-LASP1 antibody in the presence or absence of C16 ceramide (C16 cer) to evaluate interactions with LASP1–β actin. * Long exposure. (D) IP-WB analysis was performed with the anti-CERS6 antibody. Original western blots are presented in File S1.

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  Figure 6. Schematic illustration showing a ternary complex, lamellipodia formation, and cell migration.