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. 2019 May 9:10:1058.
doi: 10.3389/fimmu.2019.01058. eCollection 2019.

Myo1f, an Unconventional Long-Tailed Myosin, Is a New Partner for the Adaptor 3BP2 Involved in Mast Cell Migration

Arnau Navinés-Ferrer  1   2 , Erola Ainsua-Enrich  1   2 , Eva Serrano-Candelas  1   2 , Joan Sayós  3 , Margarita Martin  1   2
Affiliations

Myo1f, an Unconventional Long-Tailed Myosin, Is a New Partner for the Adaptor 3BP2 Involved in Mast Cell Migration

Arnau Navinés-Ferrer et al. Front Immunol. .

Abstract

Mast cell chemotaxis is essential for cell recruitment to target tissues, where these cells play an important role in adaptive and innate immunity. Stem cell factor (SCF) is a major chemoattractant for mast cells. SCF binds to the KIT receptor, thereby triggering tyrosine phosphorylation in the cytoplasmic domain and resulting in docking sites for SH2 domain-containing molecules, such as Lyn and Fyn, and the subsequent activation of the small GTPases Rac that are responsible for cytoskeletal reorganization and mast cell migration. In previous works we have reported the role of 3BP2, an adaptor molecule, in mast cells. 3BP2 silencing reduces FcεRI-dependent degranulation, by targeting Lyn and Syk phosphorylation, as well as SCF-dependent cell survival. This study examines its role in SCF-dependent migration and reveals that 3BP2 silencing in human mast cell line (LAD2) impairs cell migration due to SCF and IgE. In that context we found that 3BP2 silencing decreases Rac-2 and Cdc42 GTPase activity. Furthermore, we identified Myo1f, an unconventional type-I myosin, as a new partner for 3BP2. This protein, whose functions have been described as critical for neutrophil migration, remained elusive in mast cells. Myo1f is expressed in mast cells and colocalizes with cortical actin ring. Interestingly, Myo1f-3BP2 interaction is modulated by KIT signaling. Moreover, SCF dependent adhesion and migration through fibronectin is decreased after Myo1f silencing. Furthermore, Myo1f silencing leads to downregulation of β1 and β7 integrins on the mast cell membrane. Overall, Myo1f is a new 3BP2 ligand that connects the adaptor to actin cytoskeleton and both molecules are involved in SCF dependent mast cell migration.

Keywords: KIT signaling; adaptor molecules; cell migration and adhesion; cytoske leton; mast cells; unconventional myosins.

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Figures

Figure 1
Figure 1
3BP2 knockdown impairs SCF-dependent mast cell migration. (A) Western blot showing the 3BP2 shRNA knockdown in LAD2 cells. (B) Percentage of migrated non-target control (NT-shRNA) o 3BP2-knockdown (3BP2-shRNA) LAD2 mast cells toward SCF gradient (100 ng/mL). (C) Western blot determination of the reconstitution of both groups with 3BP2-GFP or GFP alone. (D) GFP and KIT receptor expression determined by flow cytometry. NT-shRNA is represented by an empty black curve and 3BP2-shRNA is represented by a filled gray curve. Mean intensity of fluorescence (MIF) of each group is represented in parentheses. (E) Percentage of cell migration after 4 h chemotaxis with SCF (100 ng/mL) of silenced and reconstituted cells, as indicated in the figure. Viability was tested in the upper wells after the assay. The one-way ANOVA test was used for the statistical analysis (*p < 0.05, ***p < 0.001). Migration data are the mean of three independent experiments.
Figure 2
Figure 2
3BP2 knockdown decreases migration of mast cells harboring the KITD816V mutation. (A) Western blot showing the 3BP2 shRNAs knockdown in HMC-1 cells. (B) Percentage of migrated HMC-1 mast cells silenced for 3BP2 (3BP2-shRNA) or non-target control (NT-shRNA) toward an SCF gradient (100 ng/mL). The student's t-test was used for the statistical analysis (*p < 0.05, ****p < 0.0001). In all migration assays, viability was tested in the upper wells after the assay. Data are the mean of three independent experiments.
Figure 3
Figure 3
3BP2 also reduces significantly IgE-dependent cell migration. (A) FcεRI expression in NT and 3BP2-silenced LAD2 cells. (B) Percentage of cell migration after 4 h chemotaxis with IgE+ Streptavidin in NT and 3BP2-silenced cells. Viability was tested in the upper wells after the assay. The one-way ANOVA test was used for the statistical analysis (***p < 0.001). Migration data are the mean of three independent experiments.
Figure 4
Figure 4
3BP2 knockdown impairs Cdc42 and Rac2 activation in mast cells. G-Lisa quantification of GTP-bound (active) small GTPases Rho (A) Cdc42; (B) Rac2, (C) Rac1, and (D) RhoA were performed before and after activation by IgE-stv plus SCF for 30 min in LAD2 cells. (E) Western blot showing GTPases levels in NT and 3BP2-silenced cells. The one-way ANOVA test was used for the statistical analysis (**p < 0.01, ****p < 0.0001). Data are the mean of three independent experiments.
Figure 5
Figure 5
Myo1f, a new ligand for 3BP2. (A) Representation of Myo1f. The clone identified through the three-hybrid assay was the SH3 region of this protein (aa901-1098). (B) Immunoprecipitation of the 3BP2 protein in COS-7 cells transfected with 3BP2 and Myo1f-flag, with or without the presence of Fyn kinase. Membrane was blotted with α-pTyr, α-flag, and α-3BP2 antibodies. (C) Immunoprecipitation of 3BP2 in COS-7 cells transfected with the SH2 domain of 3BP2 (SH2-GFP) and Myo1f-flag, with or without the presence of Fyn kinase. Membrane was blotted with α-flag and α-GFP antibodies.
Figure 6
Figure 6
3BP2 and Myo1f colocalization is modulated by KIT signaling in mast cells. (A) Expression of Myo1f in the mast cell lines HMC-1 and LAD2 and in CD34+-derived human mast cells (huMCs). (B) Immunofluorescence of LAD2 mast cells showing Myo1f (green), actin staining (phalloidin; red) and the nucleus (Hoechst; blue) after 5 min of stimulation with SCF (100 ng/mL) or resting (0 min). (C) Immunofluorescence of LAD2 mast cells showing Myo1f (green), 3BP2 (red) and the nucleus (Hoechst; blue) after stimulation for different times with SCF (100 ng/mL) or resting (0 min). (D) Immunofluorescence of HMC-1 cells treated with sunitinib (10 μM) or untreated showing Myo1f (green), 3BP2 (red) and the nucleus (Hoechst; blue). (E) Immunoprecipitation of 3BP2 in HMC-1 cells treated with sunitinib (10 μM) or control (DMSO). Membrane was blotted with α-Myo1f, α-3BP2, and α-KIT/α-pKIT antibodies to check KIT inhibition by sunitinib.
Figure 7
Figure 7
Myo1f knockdown does not affect SCF-dependent migration. (A) Western blot showing the Myo1f shRNAs tested. (B) Western blot showing Myo1f knockdown and (C) membrane surface expression of KIT receptor in LAD2 mast cells silenced for Myo1f (Myo1f-shRNA) or non-target control (NT-shRNA) after doxycycline induction (0.5 μg/mL). (D) Percentage of migrated LAD2 mast cells silenced for Myo1f (Myo1f-shRNA) or non-target control (NT-shRNA) toward an SCF gradient (100 ng/mL) with doxycycline induction (0.5 μg/mL). The student's t-test was used for the statistical analysis. In all migration assays, viability was tested in the upper wells after the assay. Data are the mean of three independent experiments.
Figure 8
Figure 8
β1 and β7 integrin expression are reduced as well as adhesion and migration to fibronectin in Myo1f knockdown mast cells. (A) Flow cytometry analysis of β1 (CD29) expression on NT and Myo1f shRNA LAD2 cells, (B) β7 integrin expression in NT shRNA and Myo1f shRNA. NT shRNA is represented by an empty black curve and Myo1f shRNA is represented by a filled gray curve. The mean of each group is represented in parentheses. (C) NT shRNA cells and Myo1f shRNA LAD2 cells were assayed for adhesion in fibronectin-coated wells, stimulated with SCF (100 ng/mL) for 30 min, and the percentage of adhesion was determined. (D) NT shRNA and Myo1f shRNA LAD2 mast cells were assessed for migration for 4 h in fibronectin-coated Transwell chambers and then counted under optic microscopy. Viability was tested in the upper wells after the assay. The student's t-test (A,B) or ordinary one-way ANOVA test (C,D) was used for the statistical analysis (*p < 0.05, **p < 0.01). The bar chart data correspond to the mean of at least three independent experiments.
Figure 9
Figure 9
3BP2 silencing decreases β1 expression and significantly impairs adhesion to fibronectin. (A) Flow cytometry analysis of β1 (CD29) expression on NT and 3BP2 shRNA LAD2 cells, (B) β7 integrin expression in NT shRNA and 3BP2 shRNA. NT shRNA is represented by an empty black curve and 3BP2 shRNA is represented by a filled gray curve. The mean of each group is represented in parentheses. (C) NT shRNA cells and 3BP2 shRNA LAD2 cells were assayed for adhesion in fibronectin-coated wells, stimulated with SCF (100 ng/mL) for 30 min, and the percentage of adhesion was determined. The student's t-test (A,B) or ordinary one-way ANOVA test (C) was used for the statistical analysis (**p < 0.01). The bar chart data correspond to the mean of at least three independent experiments.
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References

    1. Halova I, Draberova L, Draber P. Mast cell chemotaxis - chemoattractants and signaling pathways. Front Immunol. (2012) 3:119. 10.3389/fimmu.2012.00119 - DOI - PMC - PubMed
    1. Kim MS, Radinger M, Gilfillan AM. The multiple roles of phosphoinositide 3-kinase in mast cell biology. Trends Immunol. (2008) 29:493–501. 10.1016/j.it.2008.07.004 - DOI - PMC - PubMed
    1. Draber P, Halova I, Polakovicova I, Kawakami T. Signal transduction and chemotaxis in mast cells. Eur J Pharmacol. (2016) 778:11–23. 10.1016/j.ejphar.2015.02.057 - DOI - PMC - PubMed
    1. Ren R, Mayer BJ, Cicchetti P, Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. (1993) 259:1157–61. 10.1126/science.8438166 - DOI - PubMed
    1. Ainsua-Enrich E, Alvarez-Errico D, Gilfillan AM, Picado C, Sayos J, Rivera J, et al. . The adaptor 3BP2 is required for early and late events in FcepsilonRI signaling in human mast cells. J Immunol. (2012) 189:2727–34. 10.4049/jimmunol.1200380 - DOI - PMC - PubMed

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