CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.

Autophagy has emerged as an important antimicrobial host defense mechanism that not only orchestrates the systemic immune response, but also functions in a cell autonomous manner to directly eliminate invading pathogens. Pathogenic bacteria such as Salmonella have evolved adaptations to protect themselves from autophagic elimination. Here we show that signaling through the non-receptor tyrosine kinase focal adhesion kinase (FAK) is actively manipulated by the Salmonella SPI-2 system in macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface of the Salmonella-containing vacuole (SCV), leading to amplified signaling through the Akt-mTOR axis and inhibition of the autophagic response. In FAK-deficient macrophages, Akt/mTOR signaling is attenuated and autophagic capture of intracellular bacteria is enhanced, resulting in reduced bacterial survival. We further demonstrate that enhanced autophagy in FAK−/− macrophages requires the activity of Atg5 and ULK1 in a process that is distinct from LC3-assisted phagocytosis (LAP). In vivo, selective knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral infection with S. typhimurium. Clearance was correlated with reduced infiltration of inflammatory cell types into infected tissues and reduced tissue damage. Together, these data demonstrate that FAK is specifically targeted by S. typhimurium as a novel means of suppressing autophagy in macrophages, thereby enhancing their intracellular survival.

Summary Background Early, sorting endosomes are a major crossroad of membrane traffic, at the intersection of the endocytic and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations is mediated by a number of distinct coat protein complexes, including AP-1, AP-3 and GGAs. Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal membrane, but the mechanisms of coat recruitment and assembly at this site remain poorly understood. Results Here we report that the endosomal Rab protein Rab4 orchestrates a GTPase cascade which results in the sequential recruitment of the Arf-like protein Arl1, the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2, and the class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1/2 activity with Brefeldin A results in the loss of AP-1, AP-3 and GGA-3, but not Arl1, from endosomal membranes and the formation of elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal membranes, inhibits the formation of tubular subdomains and blocks recruitment of BIG1/2, Arfs and adaptor complexes to the endosome. Conclusion Together these findings indicate that Arl1 links Rab4-dependent formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that constitute the endosomal sorting machinery.