The rockefeller university

Vertebrate cells can initiate ciliogenesis from centrioles at the cell center, near the Golgi, forming primary cilia confined or submerged in a deep narrow pit created by membrane invagination. How or why cells maintain submerged cilia is unclear. Here, by characterizing centriole subdistal appendages (sDAP) in cells exclusively growing submerged cilia, we found that a group of sDAP components localize to the centriole proximal end through the cohesion factor C-Napl and that sDAP function redundantly with C-Napl for submerged cilia maintenance. Loss of sDAP and C-Napl has no effect on cilia assembly, but it disrupts stable Golgi-cilia association and allows normally submerged cilia to fully surface, losing the deep membrane invagination. Intriguingly, unlike submerged cilia (stationary), surfaced cilia actively respond to mechanical stimuli with motions and can ectopically recruit Hedgehog signaling components in the absence of agonist. We propose that spatial control of ciliogenesis uncouples or specifies sensory properties of cilia.

Two papers in this issue of Molecular Cell provide insights into how the multisubunit Mediator coactivator complex dynamically links enhancer-bound activators to the RNA polymerase II machinery at the core promoter.

In the search for sequence variants underlying disease, commonly applied filtering steps usually result in a number of candidate variants that cannot further be narrowed down. In autosomal recessive families, disease usually occurs only in one generation so that genetic linkage analysis is unlikely to help. Because homozygous recessive mutations tend to be inherited together with flanking homozygous variants, we developed a statistical method to detect pathogenic variants in autosomal recessive families: We look for differences in patterns of homozygosity around candidate variants between patients and control individuals and expect that such differences are greater for pathogenic variants than random candidate variants. In six autosomal recessive mitochondrial disease families, in which pathogenic homozygous variants have already been identified, our approach succeeded in prioritizing pathogenic mutations. Our method is applicable to single patients from recessive families with at least a few dozen control individuals from the same population; it is easy to use and is highly effective for detecting causative mutations in autosomal recessive families.

Childhood posterior fossa (PF) ependymomas cause substantial morbidity and mortality. These tumors lack recurrent genetic mutations, but a subset of these ependymomas exhibits CpG island (CpGi) hypermethylation [PF group A (PFA)], implicating epigenetic alterations in their pathogenesis. Further, histological grade does not reliably predict prognosis, highlighting the importance of developing more robust prognostic markers. We discovered global H3K27me3 reduction in a subset of these tumors (PF-ve ependymomas) analogous to H3K27M mutant gliomas. PF-ve tumors exhibited many clinical and biological similarities with PFA ependymomas. Genomic H3K27me3 distribution showed an inverse relationship with CpGi methylation, suggesting that CpGi hypermethylation drives low H3K27me3 in PF-ve ependymomas. Despite CpGi hypermethylation and global H3K27me3 reduction, these tumors showed DNA hypomethylation in the rest of the genome and exhibited increased H3K27me3 genomic enrichment at limited genomic loci similar to H3K27M mutant gliomas. Combined integrative analysis of PF-ve ependymomas with H3K27M gliomas uncovered common epigenetic deregulation of select factors that control radial glial biology, and PF radial glia in early human development exhibited reduced H3K27me3. Finally, H3K27me3 immunostaining served as a biomarker of poor prognosis and delineated radiologically invasive tumors, suggesting that reduced H3K27me3 may be a prognostic indicator in PF ependymomas.

Chronic lymphocytic leukemia (CLL) is an incurable leukemia of unknown etiology. Multiple studies suggest that the structure of the variable domains of the surface IGs on these cells, and signaling through them, play key roles in developing the disease. Hence, CLL appears to be driven by antigen-BCR interactions, and identifying the selecting antigens involved in this process is an important goal. We studied the antigen-binding characteristics of 23 CLL-derived, recombinantly-expressed IGs with 5 pathogenic bacteria, determining that CLL IGs differ in bacterial reactivity based on IGHV gene use, mutation status, and association with IGHD and IGHJ genes ("stereotypy"). Although most bacterial-reactive IGs followed the paradigm that IGHV-unmutated IGs were more auto-/poly-reactive, several did not. In addition, some CLL IGs were bacterial mono-reactive, and these displayed IGKV use biases. These findings are consistent with CLL B cells being driven into the leukemogenic process by bacterial as well as auto- antigens. (C) 2016 Published by Elsevier Inc.

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Chemokines and their receptors play a critical role in immune function by directing cell-specific movement. C-C chemokine receptor 7 (CCR7) facilitates entry of T cells into lymph nodes. CCR7-dependent chemotaxis requires either of the cognate ligands C-C chemokine ligand 19 (CCL19) or CCL21. Although CCR7-dependent chemotaxis can be augmented through receptor up-regulation or by increased chemokine concentrations, we found that chemotaxis is also markedly enhanced by serum in vitro. Upon purification, the serum cofactor activity was ascribed to domain 5 of high-molecular-weight kininogen. This peptide was necessary and sufficient for accelerated chemotaxis. The cofactor activity in serum was dependent on coagulation factor XIIa, a serine protease known to induce cleavage of high-molecular-weight kininogen (HK) at sites of inflammation. Within domain 5, we synthesized a 24-amino acid peptide that could recapitulate the activity of intact serum through a mechanism distinct from up-regulating CCR7 expression or promoting chemokine binding to CCR7. This peptide interacts with the extracellular matrix protein thrombospondin 4 (TSP4), and antibodies to TSP4 neutralize its activity. In vivo, an HK domain 5 peptide stimulated homing of both T and B cells to lymph nodes. A circulating cofactor that is activated at inflammatory foci to enhance lymphocyte chemotaxis represents a powerful mechanism coupling inflammation to adaptive immunity.

Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen-targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue-specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.