Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: The adapter protein Abi1 promotes cell migration. ( A ) Protein expression of human airway smooth muscle (HASM) cells expressing control (Ctrl) shRNA or Abi1 shRNA, or rescue cells was evaluated by immunoblot analysis. Data are mean values of experiments from 5 batches of cell culture. Error bars indicate SD. ( B ) Migration of HASM cells was tracked using time-lapse microscopy. Images were taken every 10 min for 16 h. Migration plots (35–46 cells from each group) were generated using the NIH ImageJ software. Red tracks indicate downward migration whereas black tracks indicate upward migration. ( C )–( E ) Abi1 knockdown (KD) reduced accumulated distance, Euclidean distance and speed of cell migration. Rescue of Abi1 recovered these parameters. ( F ), ( G ) Migration of HASM cells was examined by using the wound healing assay. Scale bar: 100 µm. n = 10 experiments. Error bars indicate SD. * p < 0.05. One ANOVA was used for statistical analysis.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Expressing, Control, shRNA, Western Blot, Cell Culture, Time-lapse Microscopy, Generated, Software, Knockdown, Wound Healing Assay

Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: Abi1 complexes with Pfn-1 and they colocalize at the leading cell edge. ( A ) Schematic illustration of Abi1 full-length (FL) and the truncated fragment. Abi1 contains proline-rich (PP) and SH3 domains in its C terminus, and the WAVE binding (WAB) and homeodomain homology region (HHR) in its N terminus. ( B ) FL Abi1, but not Abi1 mutant lacking proline-rich domain (∆PP), complexes with Pfn-1. EYFP-tagged FL Abi1 or ∆PP were cotransfected into cells with Flag-Pfn-1. Cell extracts were immunoprecipitated with GFP antibody and blotted with GFP antibody and Flag antibody. Blots are representative of three identical experiments. ( C ) Extracts of HASM cells were immunoprecipitated with Pfn-1 antibody and blotted with antibodies against Abi1 and Pfn-1. Blots are representative of three identical experiments. ( D ) Abi1 and Pfn-1 were colocalized at the leading edge. Cells were immunostained for Abi1 and Pfn-1, and examined using a confocal microscope. Scale bar: 10 µm. ( E ) Abi1 and F-actin coordinately spread at the cell edge of live HASM cells. Space and time of labeled Abi1 and F-actin in cells were monitored live using a TIRF microscope. The red line indicates the position where kymographs are assessed. Scale bar: 5 µm.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Mutagenesis, Immunoprecipitation, Microscopy, Labeling

Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: Proline-rich domain of Abi1 is important for leading edge localization of Pfn-1 and cell migration. ( A ), ( B ) Abi1 KD attenuated the cellular localization of Pfn-1, but not pVASP (Ser-157). Immunofluorescent (IF) and black white (BW) images of each cell are shown. BW images of each cell clearly show location of the leading edge. Arrows point to the leading cell edge. Data are mean values from at least 30 cells. Error bars represent SD. Scale bar: 10 µm. ( C ), ( D ) WT Abi1, but not Abi1∆PP mutant, localized at the cell periphery. Also, the expression of the Abi1 mutant reduced the positioning of Pfn-1 at the cell edge. Note: WT and mutant Abi1 also localize in the nuclear/perinuclear region. Data are mean values from at least 32 cells for each group. Error bars represent SD. Scale bar: 20 µm. Arrows point to the leading edge. ( E ) – ( G ) WT or Abi1∆PP mutant were re-expressed in Abi1 KD cells followed by assessment of migration. Uninfected (UI) cells were also used as a control. Abi1∆PP mutant inhibited accumulated distance, Euclidean distance and speed of cells (at least 38 cells for each group). Error bars indicate SD. ** p < 0.01; NS, not significant. The t test was used for statistical analysis.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Mutagenesis, Expressing, Control

Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: Differential role of Abi1 in spatial localization of migration-associated proteins. ( A ) Abi1 KD attenuated localization of c-Abl, pN-WASP and F-actin at the leading edge without affecting cortactin positioning. In addition, Abi1 KD did not affect vinculin relative intensity and area. Scale bar, 20 µm. White arrows point to the leading edges. Red arrows point to focal adhesions. ( B ) Data are mean values of experiments from at least 32 cells for each group. Error bars indicate SD. ** p < 0.01. NS, not significant. The t test was used for statistical analysis.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Migration

Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: c-Abl regulates the recruitment of Abi1 and Pfn-1 to the leading edge. ( A ) c-Abl is localized at the tip of lamellipodia. Scale bar, 10 µm. ( B ) Immunoblot analysis of stable c-Abl knockdown cells and control cells. Data are mean values of experiments from five batches of cell culture. Error bars indicate SD. ( C ) KD of c-Abl reduced the localization of Abi1 and Pfn-1 at the leading edge. Scale bar, 10 µm. Data are mean values of experiments from at least 30 cells for each group. Error bars indicate SD. ** p < 0.01. Arrows point to the leading edge. The t test was used for statistical analysis.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Knockdown, Control, Cell Culture

Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: Integrin β1 controls the positioning of Abi1 and Pfn-1 at tip of protrusion. ( A ) Integrin β1 and Abi1 are colocalized in the leading cell edge. Scale bar, 10 µm. ( B ) Coimmunoprecipitation analysis show that Abi1 complexes with integrin β1 in smooth muscle cells. Blots are representative of three identical experiments. ( C ) Immunoblot analysis of HASM cells treated with β 1 sense (S) or antisense (AS) oligodeoxynucleotides. Data are mean values of experiments from 5 batches of cell culture. Error bars indicate SD. ( D ) KD of integrin β1 reduced the localization of Abi1 and Pfn-1 in the leading edge. Scale bar, 10 µm. Data are mean values of experiments from at least 32 cells for each group. Error bars indicate SD. ** p < 0.01. Arrows point to the leading edge. The t test was used for statistical analysis.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Cell Culture

Journal: Scientific Reports

Article Title: Distinctive roles of Abi1 in regulating actin-associated proteins during human smooth muscle cell migration

doi: 10.1038/s41598-020-67781-1

Figure Lengend Snippet: Proposed mechanisms for the regulation of smooth muscle cell migration. In response to extracellular cues (e.g. the ECM), integrin β1 locates to the tip of lamellipodia, which recruits Abi1 to the leading cell edge. Abi1 then recruits Pfn-1, which promotes actin polymerization at barbed end, lamellipodial formation and migration. Abi1 also activates N-WASP, which facilitates actin branching and mesh assembly. Abi1 does not affect the localization and activation of VASP and cortactin (Cort) at the leading edge.

Article Snippet: Briefly, lentiviruses encoding Abi1 shRNA (sc-40306-V), control shRNA (sc-108080) and c-Abl shRNA (sc-29843-V) were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Activation Assay