Fig 2: Gonococci enter macrophages despite a block in phagocytosis.(A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L. pneumophila ?dotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5µM) and microtubules polymerization (nocodazole, 3µM); (B) the small GTPases Cdc42 (ML-141, 5µM), Rac1/2/3 (EHT 1864, 5µM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1µM) and ROCK1/2 kinases (GSK269962, 1µM); (C) the phagosome regulators Dynamin (dynasore, 30µM) and Phosphoinositide-3 kinase (LY294002, 10µM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25µM), Arp2/3 complex (CK869, 10µM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20µM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the ACTR2 KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N.g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project [112]: MAP1LC3A (https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line); MAP1LC3B (https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line); MAP1LC3C (https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N.g colonies or L.p ?dotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for ForminINH and MyosinIIINH, E), not significant (ns.).

Fig 3: VEGF increases growth cone motility and the growth rate by signaling through VEGFR-2. Cdc42 phosphorylates N-WASP, resulting in the activation of ARP2/3. Activated Arp2/3 can bind actin, initiating polymerization and branching. Inhibition of N-WASP via wiskostatin prevents the activation of Arp2/3, and thus also G-actin polymerization to F-actin. VEGF signaling via Cdc42 also activates LIMK, which inactivates cofilin, preventing the fragmentation of F-actin into G-actin. BMS-5 inhibits LIMK, shifting the equilibrium away from Ssh-1, resulting in more active cofilin. Arrows in blue indicate a positive impact on axonal growth, whereas red arrows indicate a repulsive effect.

Fig 4: Decreased ADF/cofilin phosphorylation and Drebrin levels in synaptosomes of APP/PS1 mice (see Fig. 6-1). A, Phosphorylation of cofilin (Ser3) is reduced in synaptosomes isolated from ADL APP/PS1 mice compared with age-matched WT mice. Statistical analysis: p-cofilin/cofilin, p = 0.0181, t = 2.631, df = 16; p-cofilin/tubulin, p = 0.0238, t = 2.498, df = 16; cofilin/tubulin, p = 0.7692, t = 0.3051, df = 16; n = 8–9 per group. B, C, ADF/cofilin-actin rods observed in primary neurons derived from APP/PS1 mice. Representative confocal images of primary cortical neurons from WT and APP/PS1 mice immunostained for actin (red) and cofilin (green). Arrows indicates ADF/cofilin-actin rods (B'). Insets show magnified images. Scale bar, 5 µm. D, Drebrin levels are reduced in synaptosomes isolated from ADL APP/PS1 mice compared with age-matched WT mice. Statistical analysis: Drebrin/tubulin, p = 0.0458, t = 2.146, df = 18; n = 10 per group. E, F, Arp2 and Arp3 protein levels are unaffected in synaptosomes isolated from ADL APP/PS1 mice compared with age-matched WT mice. Statistical analysis: Arp2/tubulin, p = 0.4899, t = 0.7090, df = 14; Arp3/tubulin, p = 0.9445, t = 0.07088, df = 14; n = 8 per group. Unpaired two-tailed t test was used. Asterisks indicate statistical significance for WT versus APP/PS1.

Fig 5: Distributional changes of anti-Arp2 and phalloidin rhodamine, subsequent to the application of NGF, VEGF, or wiskostatin. (a) Controls (n = 52) show a congruent distribution pattern of Arp2 (green) and actin (red). Arp2 showed a stronger fluorescent level in lamellipodia under every condition examined. Stimulation with NGF (b) (n = 50) and additional VEGF (c) (n = 50) lead to a highly significant increase in the mean circumference and area in both Arp2 and actin compared to standard conditions (*** = p < 0.001). After inhibition of Arp2/3-signaling by application of the N-WASP-inhibitor (d) wiskostatin under control conditions (n = 55) and after stimulation with VEGF plus NGF (e) (n = 57), the growth cones did not differ significantly from controls. The table (f) shows the statistical data for growth cones, stained with anti-Arp2. The graphs (g) show the mean circumference and the mean area (h) of the analyzed growth cones. Scale bars = 10 µm. Error bars = SEM.