Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

Mutant p53 amplifies a dynamin-1/APPL1 endosome feedback loop that regulates recycling and migration.

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors downstream of gain-of-function (GOF) mutant p53. We report that dynamin-1 (Dyn1) is up-regulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Dyn1 is required for the recruitment and accumulation of the signaling scaffold, APPL1, to a spatially localized subpopulation of endosomes at the cell perimeter. We developed new tools to quantify peripherally localized early endosomes and measure the rapid recycling of integrins. We report that these perimeter APPL1 endosomes modulate Akt signaling and activate Dyn1 to create a positive feedback loop required for rapid recycling of EGFR and ß1 integrins, increased focal adhesion turnover, and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus that integrates signaling and receptor trafficking, which can be co-opted and amplified in mutant p53-driven cancer cells to increase migration and invasion.

Severe Hemorrhagic Shock Induces Acute Activation and Expansion of IL-8+/IL-10+ Neutrophils with Enhanced Oxidative Reactivity in Non-Human Primates.

BACKGROUND: Neutrophilic inflammation is a mediator of morbidity and mortality in response to hemorrhagic shock. Although injury-induced neutrophil margination has long been observed, the nature of neutrophils' role in the "second hit" paradigm remains to be fully elucidated. We sought to extensively characterize neutrophil phenotype and functionality in response to severe hemorrhage in non-human primates (NHPs). METHODS: NHPs (n = 8) were subjected to severe hemorrhagic shock and resuscitation. Blood was obtained at baseline (T = 0 min), end of shock (T = 60 min), end of resuscitation (T = 180 min), T = 360 min, and 24 h (T = 1440 min). Neutrophils were quantified by complete blood count and flow cytometry. IL-8 and IL-10 production was determined by intracellular flow cytometry. Oxidation of dihydrorhodamine-123 (DHR-123) was used to determine neutrophil oxidative bursts (untreated), priming (+fMLP), and burst capacity (+PMA/ionomycin) via microplate reader ex vivo. Data are reported as mean ±â€ŠSEM; statistical significance was measured using repeated measures ANOVA with Bonferroni adjustment. P < 0.05 is considered significant. RESULTS: CD45CD11bCD16 neutrophils doubled postinjury (P < 0.0001); this was due to activated IL-8/IL-10 neutrophils that increased in frequency in relation to resting IL-8IL-10 cells. At 24 h, the proportions of activated to resting neutrophils returned to baseline levels. Resuscitative measures initially decreased neutrophil oxidative output; however, oxidative bursts, priming, and burst capacity were significantly increased at 24 h (P < 0.0025, 0.0124, and 0.0118, respectively). CONCLUSION: These results demonstrate an acute expansion and phenotypic activation of circulating neutrophils postinjury followed by a return to homeostatic proportions within 24 h; paradoxically, phenotypically "resting" neutrophils at 24 h have significantly higher oxidative potential, predisposing for exaggerated inflammatory responses. These data are consistent with clinical literature and provide important functional insight into neutrophil-mediated shock pathology.