Intracellular Galectin-9 Controls Dendritic Cell Function by Maintaining Plasma Membrane Rigidity

Biological Sciences; Molecular Biology; Cell BiologyEndogenous extracellular Galectins constitute a novel mechanism of membrane protein organization at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organization and function. By combining functional, super-resolution and atomic force microscopy experiments to analyze membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure by modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organization. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type-lectin receptor-mediated pathogen uptake by DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionarily conserved

Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer

Prostate tumor growth initially depends on androgens, which act via the androgen receptor (AR). Despite androgen ablation therapy, tumors eventually progress to a castrate-resistant stage in which the AR remains active. The mechanisms are poorly understood but it may be that changes in levels or activity of AR coregulators affect trafficking and activation of the receptor. A key stage in AR signaling occurs in the cytoplasm, where unliganded receptor is associated with the heat shock protein (HSP)90 foldosome complex. p23, a key component of this complex, is best characterized as a cochaperone for HSP90 but also has HSP90-independent activity and has been re-ported as having differential effects on the activity of different steroid receptors. Here we report that p23 increases activity of the AR, and this appears to involve steps both in the cytoplasm (increasing ligand-binding capacity, possibly via direct interaction with AR) and the nucleus (en-hancing AR occupancy at target promoters). We show, for the first time, that AR and p23 can interact, perhaps directly, when HSP90 is not present in the same complex. The effects of p23 on AR activity are at least partly HSP90 independent because a mutant form of p23, unable to bind HSP90, nevertheless increases AR activity. In human prostate tumors, nuclear p23 was higher in malignant prostate cells compared with benign/normal cells, supporting the utility of p23 as a therapeutic target in prostate cancer. © 2012 by The Endocrine Society

The polycomb group protein ring1b/rnf2 is specifically required for craniofacial development

Polycomb group (PcG) genes are chromatin modifiers that mediate epigenetic silencing of target genes. PcG-mediated epigenetic silencing is implicated in embryonic development, stem cell plasticity, cell fate maintenance, cellular differentiation and cancer. However, analysis of the roles of PcG proteins in maintaining differentiation programs during vertebrate embryogenesis has been hampered due to the early embryonic lethality of several PcG knock-outs in the mouse. Here, we show that zebrafish Ring1b/Rnf2, the single E3 ubiquitin ligase in the Polycomb Repressive Complex 1, critically regulates the developmental program of craniofacial cell lineages. Zebrafish ring1b mutants display a severe craniofacial phenotype, which includes an almost complete absence of all cranial cartilage, bone and musculature. We show that Cranial Neural Crest (CNC)-derived cartilage precursors migrate correctly into the pharyngeal arches, but fail to differentiate into chondrocytes. This phenotype is specific for cartilage precursors, since other neural crest-derived cell lineages, including glia, neurons and chromatophores, are formed normally in ring1b mutants. Our results therefore reveal a critical and specific role for Ring1b in promoting the differentiation of cranial neural crest cells into chondrocytes. The molecular mechanisms underlying the pathogenesis of craniofacial abnormalities, which are among the most common genetic birth defects in humans, remain poorly understood. The zebrafish ring1b mutant provides a molecular model for investigating these mechanisms and may lead to the discovery of new treatments or preventions of craniofacial abnormalitie

Apoptosis is slightly increased in the pharyngeal arch of <i>ring1b</i> mutants.

<p>Lateral views of WT and <i>ring1b</i> embryos at 36 hpf stained for TUNEL. In WT embryos two small clusters of TUNEL-positive apoptotic cells were detected in the pharyngeal arch region just posterior to the eye (A, arrows). These clusters appear to contain more apoptotic cells in the <i>ring1b</i> mutants (B, arrows). The arrowhead indicates the otic vesicle (ov). WT embryos at 36 hpf contain few apoptotic cells in the trunk (C), whereas there is an increase in overall apoptosis particularly in the trunk and the tail in <i>ring1b</i> mutants (D, arrows).</p

<i>Ring1b</i> mutants lack almost all head cartilage elements.

<p>Lateral view of WT and <i>ring1b</i> live embryos at 72 hpf (A, B). Alcian-Blue stained head cartilages of WT (C, E, G and H) and <i>ring1b</i> (D, F, H and J) mutants at the indicated developmental points, ventral views. The paired trabeculae have elongated and fused posteriorly in WT embryos at 56 hpf (E) and by 72 hpf the elaborate cartilagenous skeleton of the head has been established (I). In contrast, no cartilage is visible in <i>ring1b</i> mutants except for two minute cartilage deposits at 72 hpf <i>ring1b</i> mutants (J: arrowheads). ch: ceratohyal; ep: ethmoid plate; hys: hyosymplectic; m: Meckel’s cartilage; pc: parachordal, pq: palatoquadrate; tc: trabeculae.</p

Cranial musculature development is severely impaired in <i>ring1b</i> mutants.

<p>Ventral views of WT and <i>ring1b</i> embryos stained with the MF20 antibody (A-F). The anterior mandibularis (am) has not formed in <i>ring1b</i> mutants at 56 hpf (B), the sternohyoideus (sh) is reduced at 65 hpf (D) and at 72 hpf, cranial muscles are almost completely absent (F). hh: hyohyoideus; ih: interhyoideus; ima: intermandibularis anterioris; imp: intermandibularis posterioris.</p

Loss of endochondral and dermal ossification in <i>ring1b</i> mutants.

<p>Ventral (A–D, E, F) and lateral (A’–D’, E’,) views of <i>in situ</i> hybridizations with riboprobes against the indicated genes in WT and <i>ring1b</i> mutants at 68–72 hpf. In WT embryos, <i>runx2a</i> and <i>runx2b</i> are expressed in hypertrophic pharyngeal arch-derived chondrocytes, as well as in the dermal ossification centers of the operculum, parasphenoid and cleithrum. Weak expression is detected in pharyngeal arches and the parasphenoid of <i>ring1b</i> mutants (B, B’, D, D’). At 72 hpf, <i>col10a1</i> is expressed in developing dermal bones in WT embryos, but not in <i>ring1b</i> mutants (E–F). cl: cleithrum; de: dentary; h: hyoid; m: mandibular; mx: maxilla; pa: pharyngeal arches; pq: palatoquadrate; ps: parasphenoid, op: operculum cl: the cleithrum. Numbers indicate the respective pharyngeal arches.</p

Cranial neural crest cells migrate into the pharyngeal arches of <i>ring1b</i> mutants.

<p>Dorsal (A–H) and lateral (A’–H’) views of WT and <i>ring1b</i> mutants at 32 hpf stained with the indicated genes. Pre-chondrogenic gene expression in CNCs is largely unaffected in <i>ring1b</i> mutants. <i>Dlx2a</i> is expressed at normal levels in the pharyngeal arches of <i>ring1b</i> mutants (A–B’). Separation of <i>dlx2a</i>-positive CNCs into distinct groups of cells that populate the posterior arches is slightly affected in <i>ring1b</i> mutants A–B’). <i>Hand2</i> expression in the <i>ring1b</i> pharyngeal arches is identical to that of WT siblings (C–D’). <i>Hoxa3a</i> expression is reduced in the <i>ring1b</i> posterior pharyngeal arches (E–F’). Expression of the pharyngeal pouch marker <i>cyp26a1</i> is severely reduced in <i>ring1b</i> mutants (G–H’).</p

Intracellular Galectin-9 Controls Dendritic Cell Function by Maintaining Plasma Membrane Rigidity

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Tetraspanin CD53 controls T cell immunity through regulation of CD45RO stability, mobility, and function

International audienceT cells depend on the phosphatase CD45 to initiate T cell receptor signaling. Although the critical role of CD45 in T cells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in T cell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and T cell activation. CD53-negative T cells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing T cells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for T cell immunity