Involving society in science: Reflections on meaningful and impactful stakeholder engagement in fundamental research.

Open Science calls for transparent science and involvement of various stakeholders. Here are examples of and advice for meaningful stakeholder engagement.

Multiple and surprising new functions for emerin, a nuclear membrane protein.

Emerin is an integral protein of the nuclear inner membrane. Emerin is not essential, but its loss of function causes Emery-Dreifuss muscular dystrophy. We summarize significant recent progress in understanding emerin, which was previously known to interact with barrier-to-autointegration factor and lamins. New partners include transcription repressors, an mRNA splicing regulator, a nuclear membrane protein named nesprin, nuclear myosin I and F-actin. These interactors imply multiple roles for emerin in the nucleus, some of which overlap with related LEM-domain proteins.

Barrier-to-autointegration factor phosphorylation on Ser-4 regulates emerin binding to lamin A in vitro and emerin localization in vivo.

Barrier-to-autointegration factor (BAF) is a conserved 10-kDa chromatin protein essential in proliferating cells. BAF dimers bind double-stranded DNA, histone H3, histone H1.1, lamin A, and transcription regulators, plus emerin and other LEM-domain nuclear proteins. Two-dimensional gel analysis showed that endogenous human and Xenopus BAF are posttranslationally modified by phosphorylation and potentially other modifications and that they are hyperphosphorylated during mitosis. The invariant Ser-4 residue on BAF is a major site of phosphorylation during both interphase and mitosis. In HeLa cells that overexpressed the phosphomimetic BAF missense mutant S4E, but not S4A, emerin mislocalized from the nuclear envelope, suggesting Ser-4-nonphosphorylated BAF normally promotes emerin localization at the nuclear envelope. Supporting this model, wild-type BAF but not mutant S4E enhanced emerin binding to lamin A in vitro. Thus, Ser-4-unphosphorylated BAF has a positive role in localizing emerin; this role may be disease relevant because loss or mislocalization of emerin causes Emery-Dreifuss muscular dystrophy. Our findings further suggest Ser-4 phosphorylation inhibits BAF binding to emerin and lamin A, and thereby weakens emerin-lamin interactions during both mitosis and interphase.

What MAN1 does to the Smads. TGFbeta/BMP signaling and the nuclear envelope.

The inner nuclear membrane protein MAN1 has been identified as an important factor in transforming growth factor beta/bone morphogenic protein (TGFbeta/BMP) signaling. Loss of MAN1 results in three autosomal dominant diseases in humans; all three characterized by increased bone density. Xenopus embryos lacking MAN1 develop severe morphological defects. Both in humans and in Xenopus embryos the defects originate from deregulation of TGFbeta/BMP signaling. Several independent studies have shown that MAN1 is antagonizing TGFbeta/BMP signaling through binding to regulatory Smads. Here, recent progress in understanding MAN1 functions is summarized and a model for MAN1-dependent regulation of TGFbeta/BMP signaling is proposed.

Nuclear membrane protein emerin: roles in gene regulation, actin dynamics and human disease.

Loss of emerin, a nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy (EDMD), characterized by muscle weakening, contractures of major tendons and potentially lethal cardiac conduction system defects. Emerin has a LEM-domain and therefore binds barrier-to-autointegration factor (BAF), a conserved chromatin protein essential for cell division. BAF recruits emerin to chromatin and regulates higher-order chromatin structure during nuclear assembly. Emerin also binds filaments formed by A-type lamins, mutations in which also cause EDMD. Other partners for emerin include nesprin-1alpha and transcriptional regulators such as germ cell-less (GCL). The binding affinities of these partners range from 4nM (nesprin-1alpha) to 200 nM (BAF), and are physiologically significant. Biochemical studies therefore provide a valid means to predict the properties of emerin-lamin complexes in vivo. Emerin and lamin A together form stable complexes with either BAF or GCL in vitro. BAF, however, competes with GCL for binding to emerin in vitro. These and additional partners, notably actin and nuclear myosin II, suggest disease-relevant roles for emerin in gene regulation and the mechanical interity of the nucleus.

KSHV ORF67 encoded lytic protein localizes on the nuclear membrane and alters emerin distribution.

p29, a newly identified Kaposi's sarcoma-associated herpesvirus (KSHV) protein, is the product of ORF67, the positional homolog of the conserved herpesvirus protein UL34. Like its homologues in other herpesviruses, p29 is expressed early during viral lytic cycle, and is localized on the nuclear rim. Upon chemical induction of viral replication in primary effusion lymphoma cells, p29 interacts with p33, encoded by ORF69, the positional homolog of the conserved herpesvirus protein UL31, and both proteins colocalize on the nuclear membrane. IFA and biochemical analysis of infected or transfected cells showed that p29 expression resulted in delocalization and hyperphosphorylation of emerin, whereas other nuclear lamin associated proteins, such as LUMA, LB1 and LBR were not affected. Mislocalization of emerin was robustly increased upon combined expression of p29 and p33, suggesting that emerin destabilization might represent the first step in nuclear lamina disassembling, a process necessary for nucleocapsid maturation.

GlialCAM, a protein defective in a leukodystrophy, serves as a ClC-2 Cl(-) channel auxiliary subunit.

Ion fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl(-) channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.

PP2A regulates BMP signalling by interacting with BMP receptor complexes and by dephosphorylating both the C-terminus and the linker region of Smad1.

Phosphorylation of Smads is a crucial regulatory step in the signal transduction pathway initiated by bone morphogenetic proteins (BMPs). Although the dephosphorylation events terminating the pathway in the nucleus have been characterized, little is known about the dephosphorylation of Smads in the cytoplasm. In a proteomic screen for proteins interacting with the BMP type-II receptor, we found the regulatory Bbeta subunit of PP2A. PP2A is one of the major serine/threonine phosphatases involved in cell-cycle regulation and signal transduction. Here, we present data showing that the Bbeta subunit of PP2A interacts with both BMP type-I and type-II receptors. Furthermore, we demonstrate that several B subunits can associate with the BMP type-II receptor, independently of the kinase activity of the receptor and the catalytic subunit of PP2A. By contrast, the PP2A catalytic subunit is required for PP2A function at the receptor complex. This function of PP2A is the dephosphorylation of Smad1, mainly in the linker region. PP2A-mediated dephosphorylation of the BMP-Smad linker region leads to increased nuclear translocation of Smads and overall amplification of the BMP signal. Although other phosphatases identified within the BMP pathway are all shown to inhibit signalling, PP2A is the first example for a signalling stimulatory phosphatase within this pathway.

LUMA interacts with emerin and influences its distribution at the inner nuclear membrane.

We present here a first characterization of LUMA, an unique integral inner nuclear membrane (INM) protein. LUMA is a highly conserved protein even in some bacteria and shares a PFAM domain of unknown function with orthologs from many species. Assessing LUMA topology by using protease protection of membrane-inserted LUMA and antibody epitope accessibility assays reveals that LUMA contains four transmembrane domains and a large hydrophilic domain located between membrane spans 1 and 2. The large hydrophilic domain is exposed to the perinuclear space whereas both LUMA termini reside cyto- or nucleoplasmically. Nuclear envelope targeting of LUMA mainly depends on the membrane spans. LUMA's transmembrane domains also promote homooligomerization. LUMA binds A- and B-type lamins and depends on A-type lamins for its INM localization. Furthermore, it interacts with emerin. Both downregulation of LUMA and overexpression of dominant-negative acting LUMA fragments causes redistribution of emerin. We propose that LUMA functions as a tetraspanin-like membrane organizer and has the potential to contribute to the pathomechanism of dystrophic diseases, such as Emery-Dreifuss muscular dystrophy.

Sun2 is a novel mammalian inner nuclear membrane protein.

Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning. In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells. In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein. The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals. The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes. In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization. Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents. Furthermore, Sun2 is enriched in purified HeLa cell nuclei. Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a sub-population present in small clusters. Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes. From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein. Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases.