Phosphorylation of the HP1β hinge region sequesters KAP1 in heterochromatin and promotes the exit from naïve pluripotency

Heterochromatin binding protein HP1β plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1β^−/− embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1β is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1β^−/− and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1^−/− cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.     

Characterisation of white and black merino wools: a proteomics study

Wool is an important agricultural commodity with merino wool being rated alongside the finest quality fibres, which include the goat fibres Mohair and Cashmere. Although pigmented wool merinos have become extremely rare, the market for this wool is increasing. In Portugal, there are two merino breeds: white and black, descendants of animals originally bred on the Iberian Peninsula. These breeds have the potential to assist in our understanding of how protein expression relates to wool traits of importance to the textile industry. Herein, we study the characteristics and protein expression profiles of wool from ewes of the Portuguese black and white merino (n=15). Both breeds had very similar results for fibre diameter (25 µm) and curvature (105 to 111°/mm). Significant between-breed differences were found in the two types of keratin-associated proteins (KAPs): high-sulphur proteins (HSPs) and high-glycine–tyrosine proteins (HGTPs). The expression of HSPs, KAP2-3 and KAP2-4, decreased expression in the pigmented animals, whereas KAP13-1 was found in higher amounts. Likewise, the expression of the ultra-high-sulphur proteins, KAP4-3 and KAP4-7-like, was reduced in black sheep to half the levels of the white wools, whereas the HGTPs, KAP6, KAP6-1, KAP6-2 and KAP16-2, were more abundant in black sheep. These results suggest structural differences between the black and white merino wool, because of differences among some KAPs. These differences have important implications for the textile industry.     

Expression of ST3GAL4 Leads to SLex Expression and Induces c-Met Activation and an Invasive Phenotype in Gastric Carcinoma Cells

Sialyl-Lewis X (SLe^x) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLe^x antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLe^x overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLe^x antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLe^x antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation.     

Cutting edge: KAP10, a novel transmembrane adapter protein genetically linked to DAP12 but with unique signaling properties.

Transmembrane adapter proteins are a class of molecules that mediate signals from an extracellular receptor to the cytoplasm of the cell. We have cloned a novel transmembrane adapter protein called KAP10, a approximately 10-kDa protein that is encoded within 100 bp of the DAP12 locus on human chromosome 19. KAP10 is predominantly expressed in immune cells, including NK cells, T cells, and monocytes. We show that KAP10, unlike other transmembrane adapter proteins, binds phosphatidylinositol-3 kinase following phosphorylation of a cytoplasmic YINM motif, which results in activation of Akt. In addition, we identify KAP10 as being able to bind the adapter protein Grb2. Based on our data, we suggest that this molecule is involved in stimulation and costimulation in cells of both myeloid and lymphoid origin.     

The UNI3 Gene Is Required for Assembly of Basal Bodies of Chlamydomonas and Encodes δ-Tubulin, a New Member of the Tubulin Superfamily

We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with α-, β-, and γ-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3–1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3–1 cells is a function of the age of the cell. The uniflagellate uni3–1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3–1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3–1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.     

Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro

The human embryonal carcinoma cell lines $\text{NT2}\text{D1}$ and $\text{NT2}\text{B9}$, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.     

Dynactin polices two-way organelle traffic

How is the bidirectional motion of organelles controlled? In this issue, Deacon et al. (2003) reveal the unexpected finding that dynactin (previously known to control dynein-based motility) binds to kinesin II and regulates anterograde movement of Xenopus melanosomes. This result suggests that dynactin may be a key player in coordinating vesicle traffic in this system.The movement of intracellular cargo is essential for cell survival. In animal cells, membranous organelles are propelled through the cytoplasm by microtubule-based motor proteins. Anterograde movement toward microtubule plus ends at the cell periphery is driven by motor proteins of the kinesin superfamily, whereas retrograde movement toward minus ends at the cell center is largely accomplished by cytoplasmic dynein. In most cells, organelles do not travel smoothly in one direction but frequently switch between plus and minus end–directed travel. The net time spent traveling in the plus versus the minus end direction determines the steady-state distribution of an organelle population within a cell. A long-standing question for those studying organelle transport is how this bidirectional trafficking is coordinated. Is the binding of kinesin and dynein to vesicles mutually exclusive, or are these motors bound at the same time but with their activities coordinately regulated? What molecule(s) might be responsible for linking kinesin and dynein activities? In this issue, Vladimir Gelfand''s group (Deacon et al., 2003) addresses these questions by studying the motor proteins kinesin II and cytoplasmic dynein that move pigment granules in Xenopus melanophore cells. Their results are surprising; the dynactin complex, previously known to bind to cytoplasmic dynein and anchor it to organelles, also interacts with kinesin II and is necessary for plus end–directed motion. The ability of dynactin to physically interact with these two opposite polarity motors suggests that it may be the long sought-after molecular switch that coordinates bidirectional movement in this system.Previous studies hinted that the actions of dynein and kinesin may be controlled via dynactin. Dynactin is a large, multimeric protein complex. Its p150^Glued subunit has binding sites for both microtubules and the intermediate chain of dynein and is thought to be responsible for the association of dynein with many of its cargo organelles (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995; Waterman-Storer et al., 1995). Curiously, the treatment of extruded squid axoplasm with antibodies against p150^Glued inhibited both the anterograde and retrograde movement of organelles along microtubules (Waterman-Storer et al., 1997). These antibodies were known to inhibit the interaction of dynactin with dynein, but their effect on anterograde movement was more difficult to explain. However, genetic studies yielded similar results. Martin et al. (1999) found that mutations in either p150^Glued, the cytoplasmic dynein heavy chain, or kinesin I inhibited both retrograde and anterograde fast axonal transport in Drosophila larvae. This phenotype potentially could be explained by stalled retrograde vesicles sterically blocking the movement of anterograde cargo, but the authors also suggested the possibility of a physical linkage between kinesin, dynein, and dynactin. This theory was further tested by tracking the movement of lipid droplets in Drosophila embryos (Gross et al., 2002b). A mild defect in the dynein heavy chain impaired several aspects of minus end–directed transport of lipid droplets: run lengths, velocities, and the opposing optical trap force required to halt droplet movement were all decreased. Surprisingly, this mutation produced similar effects on droplets moving toward the microtubule plus ends. Embryos expressing a mutant p150^Glued protein that partially impaired dynactin function also exhibited impaired movement in both the plus and minus end directions. Collectively, these results suggested that dynactin might be involved in coordinating the bidirectional movement of organelles. However, these studies did not provide a molecular explanation of how this mechanism might work.To study the mechanism of coordination of bidirectional vesicle movement, Deacon et al. (2003) used Xenopus melanophores due to the unique ability to experimentally control the directional movement of their pigmented melanosomes (Daniolos et al., 1990). Upon treatment of melanophores with melatonin, the cAMP concentration in the cytoplasm drops and the melanosomes move with a net minus end–directed bias and aggregate toward the cell center. Treatment with melanocyte-stimulating hormone (MSH)^* restores cAMP levels, and the melanosomes exhibit a plus end–directed bias and disperse throughout the cell. Aggregation is accomplished by cytoplasmic dynein (Nilsson and Wallin, 1997), whereas dispersion requires the combined actions of kinesin II and the actin-based motor myosin V (Rogers and Gelfand, 1998; Tuma et al., 1998; Gross et al., 2002a). Kinesin II is a heterotrimeric protein consisting of two motor subunits and a third nonmotor subunit known as kinesin-associated protein (KAP) (Cole et al., 1992). KAP is thought to be involved in binding kinesin II to its cargo, although the mechanism for this interaction is not known.The role of dynactin in melanosome transport was investigated by disrupting dynactin function via the overexpression of dynamitin (Echeverri et al., 1996), a crucial subunit that holds the dynactin complex together. To ensure that all observed melanosome movement occurred on the microtubule cytoskeleton, actin filaments were depolymerized with latrunculin B. Here, the authors report that melanosome movement to both the plus and minus ends of microtubules was inhibited by dynamitin overexpression, suggesting a role for dynactin in coordinating bidirectional movement. They considered whether this result might be explained if both kinesin II and dynein bound to dynactin and thereby docked onto membranes. To test this idea, kinesin II was immunoprecipitated with a series of antibodies, and the authors found that dynactin was pulled down along with this kinesin motor in all cases. The reverse experiment of immunoprecipitating with p150^Glued antibodies also brought down kinesin II. Blot overlays of purified melanosomes with p150^Glued detected an interaction with a 115-kD protein, the expected size of Xenopus KAP. Subsequent overlay and affinity pull-down experiments with purified proteins confirmed the direct binding of p150^Glued to KAP. By constructing a series of GST fusion proteins, Deacon et al. (2003) were able to map the site of this interaction to residues 600–811 of p150^Glued and the COOH-terminal domain of KAP. Interestingly, this region of p150^Glued also interacts with the dynein intermediate chain, which raised the question of whether kinesin II and dynein might compete for binding to dynactin. Using a blot overlay competition assay, the authors found that the COOH-terminal KAP domain blocked the binding of p150^Glued to the dynein intermediate chain, whereas the NH₂-terminal KAP domain, used as a control, did not. This result confirms that the two motors cannot bind dynactin simultaneously.If these biochemical results are relevant to melanosome movement, then overexpression of KAP should inhibit both anterograde and retrograde traffic. Indeed, overexpession of Xenopus KAP or just its COOH-terminal fragment inhibited bidirectional melanosome movement. As a control, NH₂-terminal KAP had no effect on retrograde movement and only a small effect on anterograde movement, perhaps due to interactions with the kinesin II motor subunits. Together, the results of Deacon et al. (2003) demonstrate that kinesin II, via its KAP subunit, binds to the p150^Glued subunit of dynactin and that this interaction is important for kinesin II–mediated movement of melanosomes.Although the authors identify the p150^Glued subunit of dynactin as a key player in coordinating the bidirectional movement of melanosomes, the mechanism is still unclear. Their biochemical results showing competitive binding to dynactin suggest that binding of kinesin II and dynein to melanosomes may be mutually exclusive events; however, previous work has shown that this is not the case. In a recent paper from the same authors (Gross et al., 2002a), as well as an earlier study from Reese and Haimo (Reese and Haimo, 2000), the relative amounts of kinesin II and dynein bound to purified melanosomes did not change when cells were treated with melatonin to stimulate aggregation or with MSH to stimulate dispersion. Thus, it is possible that proteins other than dynactin might bind kinesin II and dynein to melanosomes. This question also could be addressed by determining if motor binding to melanophores is diminished in cells overexpressing KAP or dynamitin. Unfortunately, Deacon et al. (2003) were not able to answer this question by biochemical isolation of melanosomes and motor quantitation because transfected cells were only a small percentage of the total population. Another possible model is that dynactin is not needed for recruiting kinesin II and dynein to melanosomes but is somehow involved in regulating the activation or organization of motors already bound to the membrane.Future studies will no doubt explore whether dynactin is involved in bidirectional transport in systems other than melanophores. In intraflagellar transport, kinesin II and cytoplasmic dynein 2 are involved in moving nonmembranous particles between the cell body and the tip of the flagella or cilia (Rosenbaum and Witman, 2002). It will be interesting to determine whether dynactin plays a role in this type of cargo transport. In neurons, kinesin I is responsible for moving organelles from the cell body to the axon terminal. As discussed above, Martin et al. (1999) found that mutations in either kinesin I heavy chain, dynein, or p150^Glued all produced the same phenotype in Drosophila larvae neurons, suggesting that dynactin may play a role in coordinating bidirectional movement in this system as well. Immunoprecipitation of neuronal p150^Glued, however, brought down only dynein but not kinesin I. This finding may result from the fact that kinesin I, which possesses a light chain unrelated to the KAP subunit, could be linked indirectly to dynactin by another protein. Thus, this study by Deacon et al. (2003) has opened up a new area of exploration and dynactin will undoubtedly receive closer scrutiny from kinesin researchers in the future.     

Dis3L2 regulates cell proliferation and tissue growth through a conserved mechanism

Dis3L2 is a highly conserved 3’-5’ exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms’ tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.     

The Peptide-binding Cavity Is Essential for Als3-mediated Adhesion of Candida albicans to Human Cells

The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion.     

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

Background

The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.

Results

We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.

Conclusion

We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.     

EGFR Inhibition Abrogates Leiomyosarcoma Cell Chemoresistance through Inactivation of Survival Pathways and Impairment of CSC Potential

Background

Tumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome.

Methodology/Principal Findings

We expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as “tumor spheres” and as “monolayers” in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells.

Conclusions/Significance

EGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients.     

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.