FMNL1 is expressed in diverse hematopoietic lineage-derived cells and shows function-specific polarized localization. A, FMNL1 was visualized in unstimulated and stimulated PBMC subtypes as T cells, B cells, and malignant B cells from patients with CLL by immunofluorescence staining with the rat FMNL1-specific antibody 6F2 followed by Cy3-labeled goat anti-rat antibody. T cells were stimulated with interleukin-2 and OKT3, and B cells as well as CLL cells were activated using soluble CD40-ligand. Scale bars, 2 μm. B, to investigate FMNL1 expression in polarized T cells, the FMNL2-PP2-specific T cell clone SK22 was incubated for 15 min with T2 cells pulsed with the peptide FMNL1-PP2 at 10 μmol. Cells were then fixed and stained with DAPI for nuclear staining (blue), rabbit anti-human CD3 antibody followed by Cy5-labeled goat anti-rabbit antibody (yellow) for T cell identification, and rat FMNL1-specific antibody 6F2 followed by Cy3-labeled goat anti-rat antibody (red). The microtubule-organizing center was stained with mouse anti-human γ-tubulin antibody followed by Alexa Fluor 488-labeled goat anti-mouse antibody (green). Enrichment of FMNL1 and CD3 was observed around the microtubule-organizing center. Scale bar, 2 μm. C, FMNL1 is localized at the phagocytic cup (white arrows) of monocytes during phagocytosis of cells and platelets. FMNL1 (red) was stained as above, F-actin was stained by Alexa 488-conjugated phalloidin (green), and nuclei were stained with DAPI (blue). Scale bars, 2 μm. Fluorescent images were visualized at room temperature through a LEICA TCS SP2/405 confocal microscope using the HCX PL APO objective (63 × 1.4-0.6 OIL λ_BL) and the LEICA DC 300F camera. The LEICA confocal software was used for acquisition and analysis (A–C).

Identification of a novel FMNL1 splice variant (FMNL1γ) containing an intron retention at the C terminus. A, schematic diagrams of FMNL1α, FMNL1β, FMNL1γ, and FMNL1ΔDAD constructs used in this study. B, amino acid sequences of the C termini of FMNL1α and β, corresponding to murine FRLα and β, as well as of the novel isoform FMNL1γ containing a C-terminal intron retention but sharing the final C-terminal amino acids with FMNL1α. The DAD sequence responsible for autoinhibition in the murine homolog is framed, and the identical C-terminal amino acids in FMNL1α and FMNL1γ are underlined. AA, amino acids. C, quantitative mRNA expression of overall FMNL1 (black bars) and the isoform FMNL1γ (gray bars) with specific exon-overlapping primers in wild type (WT) 293T cells and 293T cells transduced with the different FMNL1 isoforms. The relative quantitative expression compared with skeletal muscle was calculated using the Δ-Δ cycle threshold method. D–F, quantitative mRNA expression of general FMNL1 (black bars) and isoform-specific FMNL1γ (gray bars) in healthy tissue (D) and in diverse cell populations isolated from peripheral blood of healthy donors (E) and 20 patients with CLL (F). PBMC subpopulations were isolated by negative (CD4, CD8, CD19, and CD14) and positive (CD34) selection. Dendritic cells (DC) were generated by adherence and cytokine maturation (E).

FMNL1γ and FMNL1ΔDAD show membranous and cortical localization and induce bleb formation. A, FMNL1γ and FMNL1ΔDAD induce extensive blebbing in 293T cells. 293T cells were transfected with the pcDNA3.1 vector containing three different isoforms of FMNL1 and analyzed by confocal microscopy after immunofluorescence staining with the rat FMNL1-specific antibody 6F2 followed by Cy3-labeled goat anti-rat antibody (red). Nuclei were stained with DAPI (blue). Scale bars, 2 μm. DIC, differential interference contrast. B, FMNL1γ and FMNL1ΔDAD induce blebbing in K562 cells. FMNL1 isoforms were adenovirally transduced into K562 cells and analyzed by confocal microscopy as described in A. Scale bars, 2 μm. C, FMNL1γ and FMNL1ΔDAD are enhanced in intracellular vesicles of HT1080 and MDA-MB 231 cells. FMNL1 isoforms were adenovirally transduced into HT1080 and MDA-MB 231 cells and analyzed by confocal microscopy as described above. Scale bars, 2 μm. D, F-actin, α-tubulin, and myosin IIb show polarized localization in FMNL1γ-induced blebs in 293T cells. 293T cells were transfected with the pcDNA3.1 vector containing FMNL1γ and stained with rat FMNL1-specific antibody 6F2 followed by Cy3-labeled goat anti-rat antibody (red). Myosin IIb was stained with a rabbit anti-myosin IIb antibody followed by Cy5-labeled goat anti-rabbit antibody (yellow). F-actin was stained with Alexa Fluor 488-conjugated phalloidin (green), and α-tubulin was stained with a mouse anti-α-tubulin antibody followed by Alexa Fluor 488-conjugated goat anti-mouse antibody (green). Nuclei were stained with DAPI (blue). Scale bars, 2 μm. E and F, cell blebbing was quantified by counting 100 FMNL1-transduced cells in 293T cells (E) and K562 cells (F) by independent counting experiments (n ≥ 3), indicating a significant increase of blebbing in cells expressing FMNL1γ and FMNL1ΔDAD (***, p < 0.001). Fluorescent images (A–D) were investigated as described in Fig. 1.

Blebbing induced by FMNL1γ is independent of ROCK and Src but depends on actin, myosin, and microtubules. A and B, 293T cells were transfected with the pcDNA3.1 vector containing FMNL1γ and treated with different bleb inhibitors and solvent controls. Cells were stained after 2 days with the rat FMNL1-specific antibody 6F2 followed by Cy3-labeled goat anti-rat antibody (red). Nuclei were stained with DAPI (blue). Scale bars, 2 μm. Blebbing cells were analyzed by confocal microscopy as described in A. Cell blebbing was quantified by counting 100 FMNL1γ-transduced cells in three independent experiments (B), classifying them as cells exhibiting extended blebs (white bars), limited blebs (black bars), or none (gray bars). DMSO, dimethyl sulfoxide. The S.D. of independent counting experiments is shown (n = 3; ***, p < 0.001).

FMNL1γ contains an N-terminal myristoylation site. A, schematic view of the N terminus of FMNL1. The predicted N-terminal myristoylation site was mutated on positions 2 and 4, glycine and alanine to threonine (gray), respectively. AA, amino acid. B and C, immunoprecipitation and metabolic labeling of FMNL1γ with [³H]myristic acid. 293T cells transfected with FMNL1γ or G2T/A4T were cultured for 16 h with [³H]myristic acid. The indicated proteins were immunoprecipitated with the FMNL1-specific monoclonal antibody 8A8 as well as the isotype control (iso), separated by SDS-PAGE followed by immunoblotting with anti-FMNL1 monoclonal antibody 6F2 (B) or fluorography (C). Molecular mass sizes are indicated in kDa.

Localization and function of FMNL1 are regulated by N-terminal myristoylation. A, localization of FMNLγ at the membrane or intracellular vesicle is abrogated by mutation of the N-terminal myristoylation motif. Different cell lines were transfected or adenovirally transduced with either FMNL1γ or the mutant G2T/A4T followed by staining with the rat FMNL1-specific antibody 6F2 and Cy3-labeled goat anti-rat antibody (red). Nuclei were stained with DAPI (blue). Scale bars, 2 μm. B, localization of FMNL1ΔDAD at the membrane is abrogated by mutation of the N-terminal myristoylation motif. 293T cell lines were transfected with either FMNL1ΔDAD or the mutant G2T/A4TΔDAD followed by staining with the rat FMNL1-specific antibody 6F2 and Cy3-labeled goat anti-rat antibody (red). Nuclei were stained with DAPI (blue). Scale bars, 2 μm. C, 293T cells transfected with the isoform FMNL1γ and FMNL1ΔDAD were treated with the inhibitor 2-hydroxymyristic acid and stained as described above. Scale bars, 2 μm. D, mutation of the N-terminal myristoylation motif reduced membrane blebbing induced by FMNL1γ or FMNL1ΔDAD in 293T cells. For investigation of cell blebbing, 100 transduced cells were analyzed. The bars show the S.D. of independent counting experiments (n = 3; ***, p < 0.001). Fluorescent images (A–C) were investigated as described previously.