Journal:

Article Title: Cdc42, Rac1, and Rac2 Display Distinct Patterns of Activation during Phagocytosis V⃞

doi: 10.1091/mbc.E03-11-0847

Figure Lengend Snippet: Organization of PBD binding sites relative to actin localization during FcγR-mediated phagocytosis. (A) Time series showing phase-contrast and Ratio images of a macrophage internalizing an IgG-opsonized erythrocyte. The color bar indicates the molar ratio (YFP/CFP). YFP-PBD was recruited to the forming phagosome (1.5–4.5 min) to a much greater extent during closure (5.0–7.5 min) and was cleared from the closed phagosome (8.5 min). (B) Particle-tracking analysis of YFP-actin (open circles) and YFP-PBD (closed circles) indicated that actin was recruited to the phagosome after particle binding (0–1 min) and during extension (1–5 min), and YFP-PBD was recruited throughout phagocytosis, with a pronounced increase in recruitment during closure (5.0–8 min). Data are mean ± SEM for 10 phagocytic events; no more than three events were taken from any one cell, and at least five different cells made up the 10 traces. (C) Simultaneous imaging of YFP-PBD and CFP-actin indicated that actin and the majority of PBD binding sites formed a discrete interface during constriction of the opsonized erythrocyte (constriction is inferred from the deformation of the erythrocyte; see arrow). CFP-actin was recruited to the forming phagosome, moved as a concentrated band over the particle during the extension phase (1.5–6.0 min), and condensed at the point of closure (6–7.5 min). YFP-PBD accumulated significantly on the base of the phagosome as constriction of the particle began (4.5–5.0 min), closely followed the moving band of actin during closure (5.5–7.5 min) and then rapidly dissipated (7.5–8.5 min). Bars, 3 μm. Also see Movie 1.

Article Snippet: To quantify signaling events from multiple phagocytic events, a particle-tracking image analysis algorithm was developed in MetaMorph software (Universal Imaging).

Techniques: Binding Assay, Imaging

Journal:

Article Title: Cdc42, Rac1, and Rac2 Display Distinct Patterns of Activation during Phagocytosis V⃞

doi: 10.1091/mbc.E03-11-0847

Figure Lengend Snippet: Ratiometric imaging and tracking analysis of YFP-Cdc42, YFP-Rac1, YFP-Rac2, and YFP-AtkPH domain relative to CFP during phagocytosis. (A, C, E, and G) Phase-contrast, YFP, and Ratio image time series of RAW macrophages phagocytosing IgG-coated erythrocytes. Color bars indicate the ranges of the Ratio values. (B, D, F, and H) Plots of RP/RC indicating the dynamics of YFP-chimera localization to phagosomes, averaged for 10 phagocytic events each. Error bars are the SE of the mean. (A) YFP-Cdc42 was present at the site of binding (0.5 min), localized to the tips of the advancing pseudopod (1.5–4.5 min) and then remained on the phagosome during and following closure (5.5–8.5 min). (B) Tracking analysis indicated the enhancement of YFP-Cdc42 on multiple phagosomes, but it did not indicate a significant change in localization. (C) YFP-Rac1 was present on plasma membranes before phagocytosis as seen by the Ratio image (0.5 min). The ratio increased as membrane extended around the particle (1.5–4.5 min) and then diminished somewhat during internalization (5.5–8.5 min). (D) Cumulative tracking data indicated that the association of YFP-Rac1 with the phagosome was variable, decreased until closure (>8.0 min) and then remained slightly elevated. (E) YFP-Rac2 localized to the base of the phagosome during extension and closure. (F) Tracking analysis showed YFP-Rac2 slightly increased on the phagosome. (G) The YFP-AktPH domain localized rapidly to the site of particle contact (0.5–1.5 min), continually increased (1.5–4.5 min), and then was cleared from the plasma membrane after closure (5.5–8.5 min). (H) YFP-AktPH was localized to phagosomes throughout formation and closure and then was cleared slowly. Bar, 3 μm.

Article Snippet: To quantify signaling events from multiple phagocytic events, a particle-tracking image analysis algorithm was developed in MetaMorph software (Universal Imaging).

Techniques: Imaging, Binding Assay, Clinical Proteomics, Membrane

Journal:

Article Title: Cdc42, Rac1, and Rac2 Display Distinct Patterns of Activation during Phagocytosis V⃞

doi: 10.1091/mbc.E03-11-0847

Figure Lengend Snippet: FRET stoichiometric imaging of YFP-Cdc42, YFP-Rac1, and YFP-Rac2 activation during phagocytosis of E-IgG. (A) Phase-contrast and EA images for cells expressing YFP-Cdc42 and CFP-PBD. YFP-Cdc42 produced an EA signal as soon as the erythrocyte contacted the macrophage. The high EA was restricted to the advancing tip of the pseudopod as it moved over the particle (1.5–5.5 min) and diminished during the closure phase (5.5–8.5 min). (B) Tracking analysis indicated the rapid association of YFP-Cdc42 with CFP-PBD and persistent FRET throughout pseudopod extension. (C and D) FRET microscopy and stoichiometry of macrophages expressing YFP-Rac1 and CFP-PBD. YFP-Rac1 interacted with CFP-PBD shortly after particle binding and throughout the pseudopod during extension (1.5–5.5 min). The quantity of YFP-Rac1 in complex with CFP-PBD increased transiently on the base of the pseudopod during the closure phase (5.5–7.5 min) and was deactivated on the closed phagosome (8.5 min). (D) This response was consistent when averaged over multiple phagocytic events. (E and F) FRET microscopy of cells expressing YFP-Rac2 and CFP-PBD indicated that Rac2 activation was delayed until closure. (G) Control cell expressing YFP-Cdc42 and CFP showed a uniform value of EA = 0 throughout phagocytosis. (H) Averaged traces from control cells expressing CFP plus YFP-Cdc42 (red), YFP-Rac1 (blue), or YFP-Rac2 (green) never indicated FRET. Bar, 3 μm. Also see Movies 2–4.

Article Snippet: To quantify signaling events from multiple phagocytic events, a particle-tracking image analysis algorithm was developed in MetaMorph software (Universal Imaging).

Techniques: Imaging, Activation Assay, Expressing, Produced, Microscopy, Binding Assay, Control