The Ubiquitin-like Modifier FAT10 in Dendritic Cell Aggresome-like Induced Structures

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The expression profile of the ubiquitin-like modifier FAT10 in immune cells suggests cell type-specific functions

The TNF and IFN-γ-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) is most prominently expressed in immunological tissues but information regarding basal expression and inducibility of FAT10 in the different types of immune cells is still lacking. Hence, we investigated FAT10 mRNA expression in the major human and murine immune cell subsets, and FAT10 protein expression in human leukocytes. We isolated the different human leukocytes from peripheral blood and the murine immune cell subsets from spleen. The purified leukocytes were left untreated or stimulated with TNF and INF-γ or LPS to induce FAT10 followed by quantitative real-time PCR or western blot analysis. Basal expression of FAT10 mRNA and protein was generally low but strongly up-regulated by IFN-γ and TNF in all immune cell subsets. LPS treatment induced FAT10 expression marginally in human CD8+ T cells and murine granulocytes, but it increased Fat10 expression significantly in murine regulatory T cells. Yet, in human CD8+ T cells, natural killer cells, natural killer T cells, and dendritic cells, the FAT10 mRNA was expressed without induction. Similarly, murine macrophages, monocytes, and regulatory T cells expressed Fat10 in the absence of stimulation. In summary, our findings suggest particular functions of FAT10 in these cell types. Furthermore, we observed not only a cell type-specific but also a species-specific basal FAT10 expression profile. Our data will serve as a guideline for future investigations to further elucidate FAT10’s role in the immune system.publishe

FAT10 localizes in dendritic cell aggresome-like induced structures and contributes to their disassembly

Dendritic cell (DC) aggresome-like induced structures (DALIS) are protein aggregates of polyubiquitylated proteins that form transiently during DC maturation. DALIS scatter randomly throughout the cytosol and serve as antigen storage sites synchronising DC maturation and antigen presentation. Maturation of DCs is accompanied by the induction of the ubiquitin-like modifier FAT10 that localises to aggresomes that are structures similar to DALIS. FAT10 is conjugated to substrate proteins and serves as a signal for their rapid and irreversible degradation by the 26S proteasome similar to, yet independently of ubiquitin, thereby contributing to antigen presentation. Here we have investigated whether FAT10 is involved in the formation and turnover of DALIS and whether proteins accumulating in DALIS can be FAT10ylated. We found that FAT10 localises to DALIS in maturing DCs and that this localisation occurs independently of its conjugation to substrates. Additionally, we investigated the DALIS turnover in FAT10-deficient and -proficient DCs and observed FAT10-mediated disassembly of DALIS. Thus, we report further evidence that FAT10 is involved in antigen processing which may provide a functional rationale why FAT10 is selectively induced upon DC maturation.publishe

SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.publishe