A motif in LILRB2 critical for Angptl2 binding and activation.

A better understanding of the interaction between extrinsic factors and surface receptors on stem cells will greatly benefit stem cell research and applications. Recently, we showed that several angiopoietin-like proteins (Angptls) bind and activate the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support ex vivo expansion of hematopoietic stem cells (HSCs) and leukemia development. However, the molecular basis for the interaction between Angptls and LILRB2 was unclear. Here, we demonstrate that Angptl2 expressed in mammalian cells forms high-molecular-weight species and that ligand multimerization is required for activation of LILRB2 for downstream signaling. A novel motif in the first and fourth Ig domains of LILRB2 was identified that is necessary for the receptor to be bound and activated by Angptl2. The binding of Angptl2 to LILRB2 is more potent than and not completely overlapped with the binding of another ligand, HLA-G. Immobilized anti-LILRB2 antibodies induce a more potent activation of LILRB2 than Angptl2, and we developed a serum-free culture containing defined cytokines and immobilized anti-LILRB2 that supports a net expansion of repopulating human cord blood HSCs. Our elucidation of the mode of Angptl binding to LILRB2 enabled the development of a new approach for ex vivo expansion of human HSCs.

Sodium and potassium competition in potassium-selective and non-selective channels.

Potassium channels selectively conduct K(+), primarily to the exclusion of Na(+), despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na(+) and K(+) binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K(+) occupancy within the filter upon Na(+) competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K(+)-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K(+) sites. A double-barrier mechanism is proposed to explain K(+) channel selectivity at low K(+) concentrations.

Distinct gating mechanisms revealed by the structures of a multi-ligand gated K(+) channel.

The gating ring-forming RCK domain regulates channel gating in response to various cellular chemical stimuli in eukaryotic Slo channel families and the majority of ligand-gated prokaryotic K(+) channels and transporters. Here we present structural and functional studies of a dual RCK-containing, multi-ligand gated K(+) channel from Geobacter sulfurreducens, named GsuK. We demonstrate that ADP and NAD(+) activate the GsuK channel, whereas Ca(2+) serves as an allosteric inhibitor. Multiple crystal structures elucidate the structural basis of multi-ligand gating in GsuK, and also reveal a unique ion conduction pore with segmented inner helices. Structural comparison leads us to propose a novel pore opening mechanics that is distinct from other K(+) channels.DOI:http://dx.doi.org/10.7554/eLife.00184.001.

Actin-binding protein drebrin E is involved in junction dynamics during spermatogenesis.

The actin-based cytoskeleton plays a critical role in the seminiferous epithelium during spermatogenesis by conferring cell shape, adhesion, structural support and cell polarity to both Sertoli and developing germ cells, which are essential for spermatogonial stem cell renewal, maintenance of the stem cell niche, cell cycle progression, mitosis, meiosis, spermiogenesis and spermiation. However, few functional studies are found in the literature, which explore the functional significance of actin dynamics in these events. This by and large is due to a lack of information on the proteins that regulate actin dynamics. Herein, we report drebrin E is an integrated component of the apical ectoplasmic specialization (apical ES) and the basal ES at the blood-testis barrier (BTB) in the seminiferous epithelium of the adult rat testis. Using immunohistochemistry and dual-labeled immunofluorescence analysis, drebrin E was found to display a stage-specific localization at the apical ES, as well as at the basal ES at the BTB during the seminiferous epithelial cycle of spermatogenesis. Drebrin E was first detected in stage V tubules at the basal ES with the highest expression at the BTB at stages V and VI, but it diminished considerably by stages VII and VIII and was almost non-detectable until stage IV. At the apical ES, drebrin E was also first detected at stage V, surrounding the entire head of the elongating spermatid, but by stage VI its localization had "shifted" to localize most intensely and almost exclusively to the concave side of the spermatid head. In stage VII tubules, drebrin E co-localized with actin, as well as with two other actin regulatory proteins Eps8 (epidermal growth factor receptor pathway substrate 8, an actin capping and bundling protein) and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to regulate actin nucleation and branching). The localization of drebrin E at the apical ES was compromised following treatment of rats with adjudin, which is known to exert its destructive effects primarily at the apical ES by inducing premature loss of elongating/elongated spermatids from the epithelium, mimicking "spermiation." Instead of being restricted to the concave side of spermatid heads, drebrin E was found to be mis-localized in the seminiferous epithelium of adjudin-treated rats; it was also present on the convex side of elongating spermatids, but these cells were mis-oriented so that their heads no longer pointed toward the basement membrane. The expression of drebrin E by Sertoli cells was also found to be modulated by TGFß3 and TNFα. Since Arp3, but not Eps8, was found to bind drebrin E; and cytokines were also shown to affect the cellular distribution of drebrin E and enhance the interaction between drebrin E and Arp3, these findings illustrate that cytokines may regulate BTB dynamics during the epithelial cycle by recruiting drebrin E and Arp3 to the BTB microenvironment to induce changes in the configuration of actin filament bundles at the basal ES. In summary, these findings illustrate drebrin E is working in concert with Arp3 to regulate actin filament bundles at both the apical and the basal ES in the testis, conferring adhesion and cell polarity at both sites during spermatogenesis.