Regulation of protein interaction domains is required for cellular signaling dynamics. Here, we show that the PDZ protein interaction domain from the cell polarity protein Par-6 is regulated by the Rho GTPase Cdc42. Cdc42 binds to a CRIB domain adjacent to the PDZ domain, increasing the affinity of the Par-6 PDZ for its carboxy-terminal ligand by approximately 13-fold. Par-6 PDZ regulation is required for function as mutational disruption of Cdc42-Par-6 PDZ coupling leads to inactivation of Par-6 in polarized MDCK epithelial cells. Structural analysis reveals that the free PDZ domain has several deviations from the canonical PDZ conformation that account for its low ligand affinity. Regulation results from a Cdc42-induced conformational transition in the CRIB-PDZ module that causes the PDZ to assume a canonical, high-affinity PDZ conformation. The coupled CRIB and PDZ architecture of Par-6 reveals how simple binding domains can be combined to yield complex regulation.

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Cdc42 recruits Par-6–aPKC to establish cell polarity from worms to mammals. Although Cdc42 is reported to have no function in Drosophila neuroblasts, a model for cell polarity and asymmetric cell division, we show that Cdc42 colocalizes with Par-6–aPKC at the apical cortex in a Bazooka-dependent manner, and is required for Par-6–aPKC localization. Loss of Cdc42 disrupts neuroblast polarity: cdc42 mutant neuroblasts have cytoplasmic Par-6–aPKC, and this phenotype is mimicked by neuroblast-specific expression of a dominant-negative Cdc42 protein or a Par-6 protein that lacks Cdc42-binding ability. Conversely, expression of constitutively active Cdc42 leads to ectopic Par-6–aPKC localization and corresponding cell polarity defects. Bazooka remains apically enriched in cdc42 mutants. Robust Cdc42 localization requires Par-6, indicating the presence of feedback in this pathway. In addition to regulating Par-6–aPKC localization, Cdc42 increases aPKC activity by relieving Par-6 inhibition. We conclude that Cdc42 regulates aPKC localization and activity downstream of Bazooka, thereby directing neuroblast cell polarity and asymmetric cell division.

Summary Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing—Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds—occurs in single cells (1,2) and in epithelia (3-10). Here we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions as well as around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single cell wound response (2,11), also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions.

SummaryPDZ protein interaction domains are typically selective for C-terminal ligands, however, non-Cterminal, "internal" ligands have also been identified. The PDZ domain from the cell polarity protein Par-6 binds C-terminal ligands and an internal sequence from the protein Pals1/Stardust. The structure of the Pals1-Par-6 PDZ complex reveals that the PDZ ligand-binding site is deformed to allow for internal binding. While binding of the Rho GTPase Cdc42 to a CRIB domain adjacent to the Par-6 PDZ regulates binding of C-terminal ligands, the conformational change that occurs upon binding of Pals1 renders its binding independent of Cdc42. These results suggest a mechanism by which the requirement for a C-terminus can be readily bypassed by PDZ ligands and reveal a complex set of cooperative and competitive interactions in Par-6 that are likely to be important for cell polarity regulation.Protein interaction domains form the backbone of cellular information flow 1 . The PDZ protein interaction domain participates in a wide variety of signaling pathways and is one of the most common in metazoan genomes 2 . Because they often occur in multiple instances in the same polypeptide, PDZ domains are thought to serve an organizational role in signal transduction pathways 3 . Given the large number of PDZ domains, several modes of ligand recognition exist whose mechanisms are still being elucidated.PDZ domains bind to short sequences of five to seven residues in their target proteins 4,5 . Although these recognition sequences have a low information content, specificity is typically enhanced by the requirement that the sequence occurs at the C-terminus. C-terminal recognition is found to fall into several different classes, depending on the identity of critical binding residues 3 . For example, class I PDZ ligands have a consensus sequence of -S-X-V-COOH.The requirement for a C-terminus results from a steric rather than electrostatic mechanism. The peptide-binding pocket is constructed such that residues that extend past the C-terminus clash with a conserved PDZ segment known as the carboxylate-binding loop 3,4 . Although Cterminal ligands possess a negatively charged carboxylate, studies of salt effects on the binding reaction suggest that electrostatic contributions are negligible 6 . Because of the additional specificity provided by the C-terminus, enforcement of C-terminal binding is an important component of PDZ -ligand recognition.While recognition of C-terminal motifs appears to be the dominant mode of PDZ-ligand interaction, non-C-terminal (also known as "internal"), PDZ ligands also exist. Compared to our understanding of C-terminal PDZ ligands, however, the mechanism of internal ligand recognition is much less clear. The best-characterized internal PDZ interactions involve ligands that adopt a specific conformation that adheres to the steric requirements of the PDZ binding Correspondence should be addressed to K.E.P. (prehoda@molbio.uoregon.edu) . pocket. For example, in the hetero-oligomerization of the nNOS and sy...

Maximizing vaccine efficacy is critical, but previous research has failed to provide a one-size-fits-all solution. Although vitamin A and vitamin D supplementation studies have been designed to improve vaccine efficacy, experimental results have been inconclusive. Information is urgently needed to explain study discrepancies and to provide guidance for the future use of vitamin supplements at the time of vaccination. We conducted a randomized, blinded, placebo-controlled study of influenza virus vaccination and vitamin supplementation among 2 to 8 (inclusive) year old children over three seasons, including 2015–2016 (n = 9), 2016–2017 (n = 44), and 2017–2018 (n = 26). Baseline measurements of vitamins A and D were obtained from all participants. Measurements were of serum retinol, retinol-binding protein (RBP, a surrogate for retinol), and 25-hydroxyvitamin D (25(OH)D). Participants were stratified into two groups based on high and low incoming levels of RBP. Children received two doses of the seasonal influenza virus vaccine on days 0 and 28, either with an oral vitamin supplement (termed A&D; 20,000 IU retinyl palmitate and 2000 IU cholecalciferol) or a matched placebo. Hemagglutination inhibition (HAI) antibody responses were evaluated toward all four components of the influenza virus vaccines on days 0, 28, and 56. Our primary data were from season 2016–2017, as enrollment was highest in this season and all children exhibited homogeneous and negative HAI responses toward the Phuket vaccine at study entry. Responses among children who entered the study with insufficient or deficient levels of RBP and 25(OH)D benefited from the A&D supplement (p < 0.001 for the day 28 Phuket response), whereas responses among children with replete levels of RBP and 25(OH)D at baseline were unaffected or weakened (p = 0.02 for the day 28 Phuket response). High baseline RBP levels associated with high HAI titers, particularly for children in the placebo group (baseline RBP correlated positively with Phuket HAI titers on day 28, r = 0.6, p = 0.003). In contrast, high baseline 25(OH)D levels associated with weak HAI titers, particularly for children in the A&D group (baseline 25(OH)D correlated negatively with Phuket HAI titers on day 28, r = −0.5, p = 0.02). Overall, our study demonstrates that vitamin A&D supplementation can improve immune responses to vaccines when children are vitamin A and D-insufficient at baseline. Results provide guidance for the appropriate use of vitamins A and D in future clinical vaccine studies.

Nuclear hormone receptors including the estrogen receptor (ERα) and the retinoic acid receptor regulate a plethora of biological functions including reproduction, circulation and immunity. To understand how estrogen and other nuclear hormones influence antibody production, we characterized total serum antibody isotypes in female and male mice of C57BL/6J, BALB/cJ and C3H/HeJ mouse strains. Antibody levels were higher in females compared to males in all strains and there was a female preference for IgG2b production. Sex-biased patterns were influenced by vitamin levels, and by antigen specificity toward influenza virus or pneumococcus antigens. To help explain sex biases, we examined the direct effects of estrogen on immunoglobulin heavy chain sterile transcript production among purified, lipopolysaccharide-stimulated B cells. Supplemental estrogen in B-cell cultures significantly increased immunoglobulin heavy chain sterile transcripts. Chromatin immunoprecipitation analyses of activated B cells identified significant ERα binding to estrogen response elements (EREs) centered within enhancer elements of the immunoglobulin heavy chain locus, including the Eµ enhancer and hypersensitive site 1,2 (HS1,2) in the 3′ regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus. Taken together, the results highlight: (i) the important targets of ERα in regulatory regions of the immunoglobulin heavy chain locus that influence antibody production, and (ii) the complexity of mechanisms by which estrogen instructs sex-biased antibody production profiles.

Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34+ hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34+ cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34+ cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.