Figure 1.
Directional lamellipodium formation initiates migration. (A) 3D reconstruction of a polarized migrating human platelet stained for the fibrinogen receptor GPIIBIIIA and its activated form (stained with antibody clone PAC-1). (B) p-MLC, Arp2/3, and F-actin immunofluorescence staining of migrating human platelets. Asterisks (∗) indicate the formation of pseudonuclei (PN) at the trailing edge. (C) Histogram of colocalization of Myh9 (upper panel, green), Arp2/3 (lower panel, green), and F-actin (pink) expression, along the green lines in Figure 1B. (D) Time-resolved morphology tracking of the PN (green) and outer shape of a human platelet (pink) in the initiation phase of migration. (E) Representative phase contrast (PH) micrographs of live imaging of a human platelet starting to spread and eventually migrating. Green: PN, pink: lamellipodial leading edge. Scale bar, 5 μm. (F) Longitudinal depiction of PN and leading-edge tracking analysis. (G) Representative micrographs and kymograph analysis (upper panel) of a spread platelet initiating migratory behavior. Kymograph analyses graphically display the spatial position of pixels (and therefore platelets) on the indicated white line (y-axis) across time (x-axis); consequently, the retracted, black PN of the platelet moves down the white line (y-axis) once migration is initiated (white arrowhead). Scale bar, 5 μm. (H) Micrographs of live imaging of a murine LifeAct-eGFP platelet spreading and initiating migration reveals dynamic actin waves at the leading edge (pink arrows); the green circle marks the PN of platelet. (I) Spatiotemporal tracking of dynamic actin waves (black arrowheads) along the leading edge (green spotted line) of a murine LifeAct-eGFP. Bottom panel: histogram of LifeAct eGFP mean fluorescence intensity (MFI) (normalized to maximum intensity) measured along the green spotted line marking the leading edge (upper panel) at the indicated time points. The black arrowheads represent the peak of the actin wave moving along the leading edge for the indicated time points. Panels A-I: assays were performed on fibrinogen/albumin matrices. Scale bars, 5 μm.

Directional lamellipodium formation initiates migration. (A) 3D reconstruction of a polarized migrating human platelet stained for the fibrinogen receptor GPIIBIIIA and its activated form (stained with antibody clone PAC-1). (B) p-MLC, Arp2/3, and F-actin immunofluorescence staining of migrating human platelets. Asterisks (∗) indicate the formation of pseudonuclei (PN) at the trailing edge. (C) Histogram of colocalization of Myh9 (upper panel, green), Arp2/3 (lower panel, green), and F-actin (pink) expression, along the green lines in Figure 1B. (D) Time-resolved morphology tracking of the PN (green) and outer shape of a human platelet (pink) in the initiation phase of migration. (E) Representative phase contrast (PH) micrographs of live imaging of a human platelet starting to spread and eventually migrating. Green: PN, pink: lamellipodial leading edge. Scale bar, 5 μm. (F) Longitudinal depiction of PN and leading-edge tracking analysis. (G) Representative micrographs and kymograph analysis (upper panel) of a spread platelet initiating migratory behavior. Kymograph analyses graphically display the spatial position of pixels (and therefore platelets) on the indicated white line (y-axis) across time (x-axis); consequently, the retracted, black PN of the platelet moves down the white line (y-axis) once migration is initiated (white arrowhead). Scale bar, 5 μm. (H) Micrographs of live imaging of a murine LifeAct-eGFP platelet spreading and initiating migration reveals dynamic actin waves at the leading edge (pink arrows); the green circle marks the PN of platelet. (I) Spatiotemporal tracking of dynamic actin waves (black arrowheads) along the leading edge (green spotted line) of a murine LifeAct-eGFP. Bottom panel: histogram of LifeAct eGFP mean fluorescence intensity (MFI) (normalized to maximum intensity) measured along the green spotted line marking the leading edge (upper panel) at the indicated time points. The black arrowheads represent the peak of the actin wave moving along the leading edge for the indicated time points. Panels A-I: assays were performed on fibrinogen/albumin matrices. Scale bars, 5 μm.

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