Identification of Intestinal Lamina Propria Plasma Cells by Surface Transmembrane Activator and CAML Interactor Expression.

Plasma cells secrete an abundance of Abs and are a crucial component of our immune system. The intestinal lamina propria harbors the largest population of plasma cells, most of which produce IgA. These Abs can bind to beneficial gut bacteria to reinforce intestinal homeostasis and provide protection against enteric pathogens. Plasma cells downregulate many cell-surface proteins commonly used to identify B cells. In mice, expression of the surface marker CD138 has been widely used to identify plasma cells in lymph nodes, bone marrow, and spleen. Intestinal plasma cells require liberation via extensive tissue processing involving treatment with collagenase. We report that detection of CD138 surface expression is reduced following collagenase treatment. Using a mouse in which yellow fluorescent protein expression is controlled by the plasma cell requisite transcription factor Blimp-1, we show that surface detection of transmembrane activator and CAML interactor captures a significant proportion of Ab-secreting plasma cells in the intestinal lamina propria and gut-draining mesenteric lymph nodes. Additionally, we describe a flow cytometry panel based on the detection of surface markers to identify murine B cell subsets in the intestinal lamina propria and, as a proof of concept, combine it with a cutting-edge fate-tracking system to characterize the fate of germinal center B cells activated in early life. By identifying plasma cells and other key intestinal B subsets in a manner compatible with several downstream applications, including sorting and culturing and in vitro manipulations, this efficient and powerful approach can enhance studies of mucosal immunity.

Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans.

The response to insufficient oxygen (hypoxia) is orchestrated by the conserved hypoxia-inducible factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel with HIF to mediate the physiological hypoxia response. Here, we describe a hypoxia response pathway controlled by Caenorhabditis elegans nuclear hormone receptor NHR-49, an orthologue of mammalian peroxisome proliferator-activated receptor alpha (PPARα). We show that nhr-49 is required for animal survival in hypoxia and is synthetic lethal with hif-1 in this context, demonstrating that these factors act in parallel. RNA-seq analysis shows that in hypoxia nhr-49 regulates a set of genes that are hif-1-independent, including autophagy genes that promote hypoxia survival. We further show that nuclear hormone receptor nhr-67 is a negative regulator and homeodomain-interacting protein kinase hpk-1 is a positive regulator of the NHR-49 pathway. Together, our experiments define a new, essential hypoxia response pathway that acts in parallel with the well-known HIF-mediated hypoxia response.

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development.

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.