PICOT binding to the polycomb group protein, EED, alters H3K27 methylation at the MYT1 PRC2 target gene.

PICOT is a ubiquitous protein that has no functional redundant ortholog and is critical for mouse embryonic development. It is involved in the regulation of signal transduction in T lymphocytes and cardiac muscle, and in cellular iron metabolism and biogenesis of Fe/S proteins. However, very little is known about the physiological role of PICOT and its mechanism of action, and on its upstream regulators or downstream target molecules. In attempt to identify new PICOT interaction partners, we adopted the yeast two-hybrid system and screened a Jurkat T cell cDNA library using the full-length human PICOT cDNA as a bait. We found that PICOT interacts with embryonic ectoderm development (EED), a Polycomb Group (PcG) protein that serves as a core component of the Polycomb repressive complex 2 (PRC2) and contributes to the regulation of chromatin remodeling and cell differentiation. Using bead immobilized GST-PICOT and GST-EED fusion proteins in a pull-down assay and reciprocal coimmunoprecipitation studies we demonstrated that the interaction between PICOT and EED also occurs in human Jurkat T cells. In addition, immunofluorescence staining of Jurkat T cells revealed partial colocalization of PICOT and EED, predominantly in the cell nuclei. A pull-down assay using the GST-EED fusion protein and lysates of cells expressing different Myc-tagged truncation products of PICOT revealed that binding of EED is mediated by each of the two C-terminal PICOT homology domains and suggests that simultaneous interaction via both domains increases the binding affinity. Furthermore, PICOT knock-down in Jurkat T cells resulted in a reduced histone H3 lysine 27 trimethylation (H3K27me3) at the PRC2 target gene, myelin transcription factor 1 (MYT1), suggesting that PICOT binding to EED alters PRC2-regulated transcriptional repression, and potentially contributes to the epigenetic regulation of chromatin silencing and remodeling.

External pH is a cue for the behavioral switch that determines surface motility and biofilm formation of Alicyclobacillus acidoterrestris.

Bacteria use different strategies to survive unfavorable environmental conditions. Alicyclobacillus acidoterrestris is a bacterium capable of surviving extremely harsh conditions, for instance, during industrial food processing. A. acidoterrestris is a spore-forming, thermoacidophilic, nonpathogenic bacterium that commonly contaminates commercial pasteurized fruit juices and is, therefore, considered a major microbiological contaminant in the juice industry. The purpose of this study was to elucidate whether A. acidoterrestris is capable of multicellular behavior by testing its ability of biofilm formation and surface motility. A. acidoterrestris was found to be proficient in migration over a surface that is apparently powered by flagella. It was further shown that lowering the external pH leads to inhibition in surface motility of these bacteria. Concomitantly, the reduction in the external pH triggered biofilm formation of A. acidoterrestris cells. Thus, although no significant biofilm was formed at pH 4.5, robust cell adhesion and confluent biofilm formation was seen below the pH 3.6. These findings indicate that the reduction of external pH is an environmental cue for the behavioral switch that inhibits surface motility and triggers biofilm formation of A. acidoterrestris. Gaining insight into the multicellular behavior that facilitates A. acidoterrestris survival in food contact surfaces may contribute to the development of novel antimicrobial means to prevent cross-contamination caused by this bacterium.

Butyric acid released during milk lipolysis triggers biofilm formation of Bacillus species.

Bacillus species form biofilms within milking pipelines and on surfaces of equipment in the dairy industry which represent a continuous hygiene problem and can lead to serious economic losses due to food spoilage and equipment impairment. Although much is known about the mechanism by which the model organism Bacillus subtilis forms biofilms in laboratory mediums in vitro, little is known of how these biofilms are formed in natural environments such as milk. Besides, little is known of the signaling pathways leading to biofilm formation in other Bacillus species, such as Bacillus cereus and Bacillus licheniformis, both of which are known to contaminate milk. In this study, we report that milk triggers the formation of biofilm-related structures, termed bundles. We show this to be a conserved phenomenon among all Bacillus members tested. Moreover, we demonstrate that the tasA gene, which encodes a major portion of the matrix which holds the biofilm together, is vital for this process. Furthermore, we show that the free fatty acid (FFA) - butyric acid (BA), which is released during lipolysis of milk fat and demonstrates antimicrobial activity, is the potent trigger for biofilm bundle formation. We finally show that BA-triggered biofilm bundle formation is mediated by the histidine kinase, KinD. Taken together, these observations indicate that BA, which is a major FFA within milk triggers biofilm formation in a conserved mechanism among members of the Bacillus genus.

miR-142 orchestrates a network of actin cytoskeleton regulators during megakaryopoiesis.

Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142, we utilized a novel reporter and a loss-of-function mouse allele that we have recently generated. In this study, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Genetic ablation of miR-142 caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterized a network of miR-142-3p targets which collectively control actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal hemostasis.DOI: http://dx.doi.org/10.7554/eLife.01964.001.

Could microRNAs contribute to the maintenance of ß cell identity?

Normal physiology depends on defined functional output of differentiated cells. However, differentiated cells are often surprisingly fragile. As an example, phenotypic collapse and dedifferentiation of ß cells were recently discovered in the pathogenesis of type 2 diabetes (T2D). These discoveries necessitate the investigation of mechanisms that function to maintain robust cell type identity. microRNAs (miRNAs), which are small non-coding RNAs, are known to impart robustness to development. miRNAs are interlaced within networks, that include also transcriptional and epigenetic regulators, for continuous control of lineage-specific gene expression. In this Opinion article, we provide a framework for conceptualizing how miRNAs might participate in adult ß cell identity and suggest that miRNAs may function as important genetic components in metabolic disorders, including diabetes.

Draft Genome Sequence of Alicyclobacillus acidoterrestris Strain ATCC 49025.

Alicyclobacillus acidoterrestris is a spore-forming Gram-positive, thermo-acidophilic, nonpathogenic bacterium which contaminates commercial pasteurized fruit juices. The draft genome sequence for A. acidoterrestris strain ATCC 49025 is reported here, providing genetic data relevant to the successful adaptation and survival of this strain in its ecological niche.

Kindlin-3 is required for the stabilization of TCR-stimulated LFA-1:ICAM-1 bonds critical for lymphocyte arrest and spreading on dendritic cells.

Kindlin-3 is a key lymphocyte function-associated antigen-1 (LFA-1) coactivator deleted in leukocyte adhesion deficiency-III (LAD-III). In the present study, we investigated the involvement of this adaptor in lymphocyte motility and TCR-triggered arrest on ICAM-1 or on dendritic cells (DCs). Kindlin-3-null primary T cells from a LAD-III patient migrated normally on the major lymph node chemokine CCL21 and engaged in normal TCR signaling. However, TCR activation of Kindlin-3-null T lymphocytes failed to trigger the robust LFA-1-mediated T-cell spreading on ICAM-1 and ICAM-1-expressing DCs that is observed in normal lymphocytes. Kindlin-3 was also essential for cytoskeletal anchorage of the LFA-1 heterodimer and for microclustering of LFA-1 within ventral focal dots of TCR-stimulated lymphocytes spread on ICAM-1. Surprisingly, LFA-1 on Kindlin-3-null lymphocytes migrating over CCL21 acquired normal expression of an epitope associated with the conformational activation of the key headpiece domain, ß I. This activated LFA-1 was highly responsive to TCR-triggered ICAM-1-driven stop signals in normal T cells locomoting on CCL21, but not in their Kindlin-3-null T-cell counterparts. We suggest that Kindlin-3 selectively contributes to a final TCR-triggered outside-in stabilization of bonds generated between chemokine-primed LFA-1 molecules and cell-surface ICAM-1.

Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state.

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.

Hodgkin's lymphoma cells exhibit high expression levels of the PICOT protein.

PICOT was originally discovered as a protein kinase C (PKC) binding protein in human Jurkat T-lymphocytes in which it was found to modulate PKCtheta-dependent functions. In addition, RT-PCR analysis suggested the expression of PICOT in a wide range of organs and cell types, including cells that are devoid of PKCtheta. We aimed at analyzing the expression of the PICOT protein in mouse lymphoid organs, and to compare them with those of Jurkat T-lymphocytes and other cell lines. We also analyzed whether PICOT expression in T-lymphocytes is dependent on the presence of PKCtheta, and whether it correlates with cell growth rate. Western blot analyses demonstrated PICOT expression in all lymphoid organs and cell lines tested. In addition, similar expression levels were observed in lymphoid organs of wild-type and PKCtheta-null mice, suggesting that PICOT expression in T-lymphocytes is independent of PKCtheta. However, PICOT expression levels were higher in Jurkat T-lymphocytes and other lymphoma cell lines compared to freshly isolated lymphocytes, while T-lymphocyte mitogens, such as concanavalin A, increased PICOT expression concomitantly with the induction of a faster T-lymphocyte growth rate. Finally, immunohistochemistry of freshly-isolated lymph nodes from Hodgkin's lymphoma patients revealed significantly higher levels of PICOT in Hodgkin's cells, compared to the normal surrounding lymphocytes. The present results show a direct correlation between PICOT expression levels and increased cell growth, both in vitro and in vivo, and suggest that immunostaining of PICOT might be useful for in situ identification of transformed cells, such as those of Hodgkin's lymphoma.

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions.

Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3-null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative beta(1) Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4-adhesive functions in human lymphocytes.

A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-triggered integrin activation but not in chemokine-triggered motility of human T lymphocytes and CD34+ cells.

The chemokine CXCL12 promotes migration of human leukocytes, hematopoietic progenitors, and tumor cells. The binding of CXCL12 to its receptor CXCR4 triggers Gi protein signals for motility and integrin activation in many cell types. CXCR7 is a second, recently identified receptor for CXCL12, but its role as an intrinsic G-protein-coupled receptor (GPCR) has been debated. We report that CXCR7 fails to support on its own any CXCL12-triggered integrin activation or motility in human T lymphocytes or CD34(+) progenitors. CXCR7 is also scarcely expressed on the surface of both cell types and concentrates right underneath the plasma membrane with partial colocalization in early endosomes. Nevertheless, various specific CXCR7 blockers get access to this pool and attenuate the ability of CXCR4 to properly rearrange by surface-bound CXCL12, a critical step in the ability of the GPCR to trigger optimal CXCL12-mediated stimulation of integrin activation in T lymphocytes as well as in CD34(+) cells. In contrast, CXCL12-triggered CXCR4 signaling to early targets, such as Akt as well as CXCR4-mediated chemotaxis, is insensitive to identical CXCR7 blocking. Our findings suggest that although CXCR7 is not an intrinsic signaling receptor for CXCL12 on lymphocytes or CD34(+) cells, its blocking can be useful for therapeutic interference with CXCR4-mediated activation of integrins.

RhoA is involved in LFA-1 extension triggered by CXCL12 but not in a novel outside-in LFA-1 activation facilitated by CXCL9.

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer's patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1-3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.

Membranal cholesterol is not required for L-selectin adhesiveness in primary lymphocytes but controls a chemokine-induced destabilization of L-selectin rolling adhesions.

Cholesterol-enriched lipid microdomains regulate L-selectin signaling, but the role of membrane cholesterol in L-selectin adhesion is unclear. Arrest chemokines are a subset of endothelial chemokines that rapidly activate leukocyte integrin adhesiveness under shear flow. In the absence of integrin ligands, these chemokines destabilize L-selectin-mediated leukocyte rolling. In the present study, we investigated how cholesterol extraction from the plasma membrane of peripheral blood T or B cells affects L-selectin adhesions and their destabilization by arrest chemokines. Unlike the Jurkat T cell line, whose L-selectin-mediated adhesion is cholesterol dependent, in primary human PBLs and in murine B cells and B cell lines, cholesterol depletion did not impair any intrinsic adhesiveness of L-selectin, consistent with low selectin partitioning into lipid rafts in these cells. However, cholesterol raft disruption impaired the ability of two arrest chemokines, CXCL12 and CXCL13, but not of a third arrest chemokine, CCL21, to destabilize L-selectin-mediated rolling of T lymphocytes. Actin capping by brief incubation with cytochalasin D impaired the ability of all three chemokines to destabilize L-selectin rolling. Blocking of the actin regulatory phosphatidylinositol lipid, phosphatidylinositol 4,5-bisphosphate, did not affect chemokine-mediated destabilization of L-selectin adhesions. Collectively, our results suggest that L-selectin adhesions are inhibited by actin-associated, cholesterol-stabilized assemblies of CXCL12- and CXCL13-binding receptors on both T and B lymphocytes. Thus, the regulation of L-selectin by cholesterol-enriched microdomains varies with the cell type as well as with the identity of the destabilizing chemokine.

A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets.

Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in beta(1), beta(2), and beta(3) integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired alpha(IIb)beta(3) activation by key agonists. beta(2) integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFalpha-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil beta(2) integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.

DOCK2 regulates chemokine-triggered lateral lymphocyte motility but not transendothelial migration.

Rac GTPases are key regulators of leukocyte motility. In lymphocytes, chemokine-mediated Rac activation depends on the CDM adaptor DOCK2. The present studies addressed the role of DOCK2 in chemokine-triggered lymphocyte adhesion and motility. Rapid chemokine-triggered activation of both LFA-1 and VLA-4 integrins took place normally in DOCK2-/- T lymphocytes under various shear flow conditions. Consequently, DOCK2-/- T cells arrested normally on TNFalpha-activated endothelial cells in response to integrin stimulatory chemokine signals, and their resistance to detachment was similar to that of wild-type (wt) T lymphocytes. Nevertheless, DOCK2-/- T lymphocytes exhibited reduced microvillar collapse and lamellipodium extension in response to chemokine signals, ruling out a role for these events in integrin-mediated adhesion strengthening. Strikingly, arrested DOCK2-/- lymphocytes transmigrated through a CCL21-presenting endothelial barrier with similar efficiency and rate as wt lymphocytes but, unlike wt lymphocytes, could not locomote away from the transmigration site of the basal endothelial side. DOCK2-/- lymphocytes also failed to laterally migrate over multiple integrin ligands coimmobilized with chemokines. This is a first indication that T lymphocytes use 2 different chemokine-triggered actin remodeling programs: the first, DOCK2 dependent, to locomote laterally along apical and basal endothelial surfaces; the second, DOCK2 independent, to cross through a chemokine-bearing endothelial barrier.