SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin‐2

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.     

Induction of Circadian Rhythm in Cultured Human Mesenchymal Stem Cells by Serum Shock and cAMP Analogs in Vitro

Circadian clocks have been shown to operate developmentally in mouse and human hematopoietic stem and progenitor cells in vivo, but little is known about their possible oscillations in vitro. Here, we show that repeated circadian oscillations could be induced in both cultured bone marrow‐derived mesenchymal‐ and adipose‐derived stem cells (MSCs and ASCs, respectively) by serum shock. In particular, the novel finding of rhythmic clock gene expression induced by cAMP analogs showed similarities as well as differences to serum‐induced oscillations. Rhythmic PER1 expression was found in serum‐shocked MSCs, suggesting the phosphorylation status of PER1 is important for its activity in circadian rhythms. Furthermore, immunofluoresent staining showed that the localization of PER1 was dependent on the level of PER1 expression. These inducible self‐sustained circadian clocks in primary cultures of human MSCs in vitro with rhythmic changes in expression levels, phosphorylation, and localization of clock protein, PER1, may be of importance for maintaining the induced oscillations in stem cells. Therefore, the established cell models described here appear to be valuable for studying the molecular mechanism driving and coordinating the circadian network between stem and stromal cells.     

The Specificity of Targeted Vaccines for APC Surface Molecules Influences the Immune Response Phenotype

Different diseases require different immune responses for efficient protection. Thus, prophylactic vaccines should prime the immune system for the particular type of response needed for protection against a given infectious agent. We have here tested fusion DNA vaccines which encode proteins that bivalently target influenza hemagglutinins (HA) to different surface molecules on antigen presenting cells (APC). We demonstrate that targeting to MHC class II molecules predominantly induced an antibody/Th2 response, whereas targeting to CCR1/3/5 predominantly induced a CD8⁺/Th1 T cell response. With respect to antibodies, the polarizing effect was even more pronounced upon intramuscular (i.m) delivery as compared to intradermal (i.d.) vaccination. Despite these differences in induced immune responses, both vaccines protected against a viral challenge with influenza H1N1. Substitution of HA with ovalbumin (OVA) demonstrated that polarization of immune responses, as a consequence of APC targeting specificity, could be extended to other antigens. Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of adaptive immune responses by specifically targeting different surface molecules on APCs.