CD14 controls the LPS-induced endocytosis of Toll-like receptor 4.

The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.

Phosphoinositide binding by the Toll adaptor dMyD88 controls antibacterial responses in Drosophila.

The cell biological principles that govern innate immune responses in Drosophila are unknown. Here, we report that Toll signaling in flies was dictated by the subcellular localization of the adaptor protein dMyD88. dMyD88 was located at the plasma membrane by a process dependent on a C-terminal phosphoinositide-binding domain. In vivo analysis revealed that lipid binding by dMyD88 was necessary for its antimicrobial and developmental functions as well as for the recruitment of the downstream cytosolic adaptor Tube to the cell surface. These data are reminiscent of the interactions between the mammalian Toll adaptors MyD88 and TIRAP with one major exception. In the mammalian system, MyD88 is the cytosolic adaptor that depends on the phosphoinositide-binding protein TIRAP for its recruitment to the cell surface. We therefore propose that dMyD88 is the functional homolog of TIRAP and that both proteins function as sorting adaptors to recruit downstream signaling adaptors to activated receptors.

MEN1 and FANCD2 mediate distinct mechanisms of DNA crosslink repair.

Cells mutant for multiple endocrine neoplasia type I (MEN1) or any of the Fanconi anemia (FA) genes are hypersensitive to the killing effects of crosslinking agents, but the precise roles of these genes in the response to interstrand crosslinks (ICLs) are unknown. To determine if MEN1 and the FA genes function cooperatively in the same repair process or in distinct repair processes, we exploited Drosophila genetics to compare the mutation frequency and spectra of MEN1 and FANCD2 mutants and to perform genetic interaction studies. We created a novel in vivo reporter system in Drosophila based on the supF gene and showed that MEN1 mutant flies were extremely prone to single base deletions within a homopolymeric tract. FANCD2 mutants, on the other hand, had a mutation frequency and spectrum similar to wild type using this assay. In contrast to the supF results, both MEN1 and FANCD2 mutants were hypermutable using a different assay based on the lats tumor suppressor gene. The lats assay showed that FANCD2 mutants had a high frequency of large deletions, which the supF assay was not able to detect, while large deletions were rare in MEN1 mutants. Genetic interaction studies showed that neither overexpression nor loss of MEN1 modified the ICL sensitivity of FANCD2 mutants. The strikingly different mutation spectra of MEN1 and FANCD2 mutants together with lack of evidence for genetic interaction between these genes indicate MEN1 plays an essential role in ICL repair distinct from the Fanconi anemia genes.

Drosophila homologs of FANCD2 and FANCL function in DNA repair.

Fanconi anemia (FA) is a genetically heterogeneous disease characterized by developmental defects, progressive bone marrow failure and cancer susceptibility. Cells derived from patients with FA show spontaneous chromosomal aberrations and hypersensitivity to cross-linking agents, indicating a cellular defect in DNA repair. Among the 12 FA genes, only FANCD2, FANCL and FANCM have Drosophila homologs. Given this difference between the human and Drosophila FA pathways, it is unknown whether the fly homologs function in DNA repair. Here, we report that knockdown of Drosophila FANCD2 or FANCL leads to specific hypersensitivity to cross-linking agents. Further analysis revealed that FANCD2 and FANCL function in a linear pathway with FANCL being necessary for the monoubiquitination of FANCD2. FANCD2 mutants also exhibited the same defect in the ionizing radiation-inducible S-phase checkpoint that is seen in mammalian cells deficient for this gene. Finally, in an assay for inactivating mutations, FANCD2 mutants have an elevated mutation rate in response to nitrogen mustard, indicating that these flies are hypermutable. Taken together, these data demonstrate that Drosophila FANCD2 and FANCL play a critical role in DNA repair. Because of the lack of other FA genes, further studies will determine whether the conserved FA genes function as the minimal machinery or whether additional genes are involved in the Drosophila FA pathway.

Deciphering the function of nucleic acid sensing TLRs one regulatory step at a time.

While initial studies of Toll-like Receptor (TLR) signaling mainly focused on genetic analysis of signal transduction, recent work has highlighted the importance of understanding the basic cell biology underlying receptor function. Nowhere is this issue more important than in the study of the nucleic acid-sensing TLRs. These receptors face the unique challenge of distinguishing microbial nucleic acids from similar host-derived molecules. The physiological cost of not making this distinction can be readily observed in studies of autoimmunity, a cause of which is often the inappropriate detection of self nucleic acids. In this review, we highlight recent research that has revealed myriad ways in which mammalian cells control the function of nucleic acid-sensing TLRs. A theme is now emerging whereby these receptors are subject to sequential regulatory mechanisms that control protein transport to their sites of signal transduction, as well as their access microbial nucleic acids.

Hypermutability in a Drosophila model for multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type I (MEN1) is an autosomal dominant cancer predisposition syndrome, the gene for which encodes a nuclear protein, menin. The biochemical function of this protein has not been completely elucidated, but several studies have shown a role in transcriptional modulation through recruitment of histone deacetylase. The mechanism by which MEN1 mutations cause tumorigenesis is unknown. The Drosophila homolog of MEN1, Mnn1, encodes a protein 50% identical to human menin. In order to further elucidate the function of MEN1, we generated a null allele of this gene in Drosophila and showed that homozygous inactivation results in morphologically normal flies that are hypersensitive to ionizing radiation and two DNA cross-linking agents, nitrogen mustard and cisplatinum. The spectrum of agents to which mutant flies are sensitive and analysis of the molecular mechanisms of this sensitivity suggest a defect in nucleotide excision repair. Drosophila Mnn1 mutants have an elevated rate of both sporadic and DNA damage-induced mutations. In a genetic background heterozygous for lats, a Drosophila and vertebrate tumor suppressor gene, homozygous inactivation of Mnn1 enhanced somatic mutation of the second allele of lats and formation of multiple primary tumors. Our data indicate that Mnn1 is a novel member of the class of autosomal dominant cancer genes that function in maintenance of genomic integrity, similar to the BRCA and HNPCC genes.