Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization.

Autophagy is an essential catabolic intracellular pathway that maintains homeostasis by degrading long-lived proteins, damaged organelles, and provides an energy source during nutrient starvation. It is now understood that autophagy has discrete functions as a selective lysosomal degradation pathway targeting large cytosolic structural and signaling complexes to influence cell motility and adhesion. We provide evidence suggesting the primary autophagy regulators Atg5 and FIP200 both play a role in cell motility and extracellular matrix adhesion. However, their loss of function has a differential impact on focal adhesion composition and organization, as well as signaling in response to fibronectin induced cell spreading. This differential impact on focal adhesions is illustrated by smaller focal adhesion complexes and a decrease in FAK, paxillin, and vinculin expression associated with FIP200 loss of function. In contrast, Atg5 loss of function results in production of large and stable focal adhesions, characterized by their retention of phosphorylated FAK and Src, which correlates with increased vinculin and FAK protein expression. Importantly, autophagy is upregulated during processes associated with focal adhesion reorganization and their exhibits colocalization of autophagosomes with focal adhesion cargo. Interestingly, FIP200 localizes to vinculin-rich focal adhesions and its loss negatively regulates FAK phosphorylation. These data collectively suggest FIP200 and Atg5 may have both autophagy-dependent and -independent functions that provide distinct mechanisms and impacts on focal adhesion dynamics associated with cell motility.

Tollip coordinates Parkin-dependent trafficking of mitochondrial-derived vesicles.

Multiple mitochondrial quality control pathways exist to maintain the health of mitochondria and ensure cell homeostasis. Here, we investigate the role of the endosomal adaptor Tollip during the mitochondrial stress response and identify its interaction and colocalisation with the Parkinson's disease-associated E3 ubiquitin ligase Parkin. The interaction between Tollip and Parkin is dependent on the ubiquitin-binding CUE domain of Tollip, but independent of Tom1 and mitophagy. Interestingly, this interaction is independent of Parkin mitochondrial recruitment and ligase activity but requires an intact ubiquitin-like (UBL) domain. Importantly, Tollip regulates Parkin-dependent endosomal trafficking of a discrete subset of mitochondrial-derived vesicles (MDVs) to facilitate delivery to lysosomes. Retromer function and an interaction with Tom1 allow Tollip to facilitate late endosome/lysosome trafficking in response to mitochondrial stress. We find that upregulation of TOM20-positive MDVs upon mitochondrial stress requires Tollip interaction with ubiquitin, endosomal membranes and Tom1 to ensure their trafficking to the lysosomes. Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial-derived cargo to the lysosomes.

Lycopene acts through inhibition of IκB kinase to suppress NF-κB signaling in human prostate and breast cancer cells.

We studied the effect of the potent dietary antioxidant lycopene on multiple points along the nuclear factor kappa B (NF-κB) signaling pathway in prostate and breast cancer cells. Lycopene significantly inhibited prostate and breast cancer cell growth at physiologically relevant concentrations of ≥1.25 µM. Similar concentrations also caused a 30-40 % reduction in inhibitor of kappa B (IκB) phosphorylation in the cells, as determined by western blotting. Furthermore, the same degree of inhibition by lycopene was observed for NF-κB transcriptional activity, as determined by reporter gene assay. Concomitant with this, immunofluorescence staining of lycopene-treated cells showed a significant suppression (≥25 %) of TNF-induced NF-κB p65 subunit nuclear translocation. Further probing of lycopene's effects on upstream elements of the NF-κB pathway showed a 25 % inhibition of both activity of recombinant IκB kinase ß (IKKß) kinase in a cell-free in vitro assay, as well as activity of IKKß immunoprecipitated from MDA-MB-231 cells treated with lycopene. In conclusion, the anticancer properties of lycopene may occur through inhibition of the NF-κB signaling pathway, beginning at the early stage of cytoplasmic IKK kinase activity, which then leads to reduced NF-κB-responsive gene regulation. Furthermore, these effects in cancer cells were observed at concentrations of lycopene that are relevant and achievable in vivo.