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

Journal: Annals of Clinical and Translational Neurology

Article Title: Elevated levels of extracellular vesicles in progranulin‐deficient mice and FTD‐ GRN Patients

doi: 10.1002/acn3.51242

Figure Lengend Snippet: Elevated Levels of Brain EVs in 12‐ to 13‐month‐old Grn –/– Mice. Brain EVs were isolated from wild‐type, Grn +/– , and Grn –/– littermates, and the levels of EVs in fraction 2 were compared using several methods. A, Nanoparticle tracking analysis revealed more vesicles of exosomal size in Grn –/– mice than wild‐type (RM ANOVA genotype x particle size interaction, P < 0.0001, * P < 0.05 by Dunnett’s post hoc test). B, This increase in exosome‐sized vesicles persisted when corrected for hemibrain weight in Grn –/– mice (ANOVA effect of genotype, P = 0.0133, ** P = 0.0070 by Dunnett’s post hoc test). C, Fraction 2 from Grn –/– mice also contained more total protein than wild‐type mice (ANOVA effect of genotype, P = 0.0040, ** P = 0.0021 by Dunnett’s post hoc test). Finally, fraction 2 from Grn –/– mice contained significantly more HSP‐70 (D, ANOVA effect of genotype, P = 0.0206, * P = 0.0138 by Dunnett’s post hoc test) and trended toward having higher levels of CD81 (E, ANOVA effect of genotype, P = 0.0562) and flotillin‐1 (F, ANOVA effect of genotype, P = 0.0857) than wild‐type. G, The other fractions contained undetectable levels of these proteins. All data are corrected for hemibrain weight except for the nanoparticle tracking profiles in A. n = 10–13 mice per genotype. H = brain homogenate.

Article Snippet: Nanoparticle tracking profiles (concentration by particle size) were analyzed by repeated measures ANOVA with factors of particle size and genotype (or patient group) using GraphPad Prism 8.

Techniques: Isolation

Journal: Annals of Clinical and Translational Neurology

Article Title: Elevated levels of extracellular vesicles in progranulin‐deficient mice and FTD‐ GRN Patients

doi: 10.1002/acn3.51242

Figure Lengend Snippet: Elevated Levels of EVs in Frontal Cortex of Patients with FTD‐ GRN . EVs were isolated from frozen post mortem samples of inferior frontal gyrus from controls (n = 5) and patients with FTD‐ GRN (n = 13) as shown in Figure . A, B, As with mouse brain samples, fraction 2 was enriched for EV marker proteins and total protein content. C, D, Fraction 2 from post mortem samples contained vesicles of typical EV morphology under transmission electron microscopy. E, Nanoparticle tracking analysis revealed vesicles of the size for exosomes and microvesicles, although there was not an overall difference in vesicle concentration between FTD‐ GRN patients and controls (E, RM ANOVA effect of group, P = 0.51). However, levels of the EV marker proteins HSP‐70 (F, Mann‐Whitney test, P = 0.0396) and CD81 (G, Mann‐Whitney test, P = 0.046) and were elevated in fraction 2 from FTD‐ GRN patients. Representative blots are shown in I. All data are corrected for slice weight except for the nanoparticle tracking profiles in E. Independent images of representative EVs at different magnifications are shown in C and D with 100 nm scale bars.

Article Snippet: Nanoparticle tracking profiles (concentration by particle size) were analyzed by repeated measures ANOVA with factors of particle size and genotype (or patient group) using GraphPad Prism 8.

Techniques: Isolation, Marker, Transmission Assay, Electron Microscopy, Concentration Assay, MANN-WHITNEY

Journal: Annals of Clinical and Translational Neurology

Article Title: Elevated levels of extracellular vesicles in progranulin‐deficient mice and FTD‐ GRN Patients

doi: 10.1002/acn3.51242

Figure Lengend Snippet: Elevated Levels of Plasma EVs in Patients with FTD‐ GRN . EVs were isolated by differential centrifugation of frozen plasma samples from controls (n = 8), presymptomatic GRN carriers (n = 7), and symptomatic GRN patients (n = 12). A, EV pellets contained EV marker proteins, but lacked markers for other organelles (Grp94 – ER/Golgi, cytochrome C – mitochondria, histone H3 – nucleus). Lysate from HEK‐293 cells (HEK) was used as a positive control for organelle markers. B and C Plasma EVs exhibited typical morphology under transmission electron microscopy (scale bars = 100 nm). D, Nanoparticle tracking analysis revealed that plasma from symptomatic GRN patients contained significantly more particles of exosomal size than controls and presymptomatic GRN carriers (RM ANOVA effect of group, P = 0.0447, group x size interaction, P < 0.0001, * P = 0.0415 by Dunnett’s post hoc test, E, ANOVA effect of group, P = 0.0162, * P < 0.05 by Dunnett’s post hoc test). F–I, Similarly, western blot revealed elevated levels of the EV marker proteins CD9 (F, Kruskal‐Wallis test effect of group, P = 0.0021, ** P < 0.01 by Dunn’s post hoc test) and flotillin‐1 (G, Kruskal‐Wallis test effect of group, P = 0.0182, * P < 0.05 by Dunn’s post hoc test) in symptomatic GRN patients compared to both controls and presymptomatic GRN carriers. C is an enlarged portion of B, as shown by dashed lines.

Article Snippet: Nanoparticle tracking profiles (concentration by particle size) were analyzed by repeated measures ANOVA with factors of particle size and genotype (or patient group) using GraphPad Prism 8.

Techniques: Clinical Proteomics, Isolation, Centrifugation, Marker, Positive Control, Transmission Assay, Electron Microscopy, Western Blot