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1.  DAZL Limits Pluripotency, Differentiation, and Apoptosis in Developing Primordial Germ Cells 
Stem Cell Reports  2014;3(5):892-904.
The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.
Graphical Abstract
•DAZL colocalizes with a network of translational inhibitors in granular structures•DAZL interacts with mRNA transcripts of key pluripotency genes and Caspases•Loss of DAZL function leads to prolonged expression of pluripotency genes•Loss of DAZL leads to expression of Caspases, resulting in apoptosis in PGCs
In this article, Geijsen and colleagues demonstrate that DAZL acts as a translational suppressor of pluripotency, differentiation, and apoptosis in developing primordial germ cells. As such, DAZL limits pluripotency while simultaneously preventing somatic differentiation and provides an elegant fail-safe mechanism into the PGC system, in which the loss of pluripotency regulation simultaneously triggers germ cell death and prevents germ cell tumor formation.
PMCID: PMC4235140  PMID: 25418731
2.  CUL4A contributes to the biology of basal-like breast tumors through modulation of cell growth and antitumor immune response 
Oncotarget  2014;5(8):2330-2343.
The CUL4A E3 ubiquitin ligase is involved in the regulation of many cellular processes and its amplification and/or overexpression has been observed in breast cancer. The 13q34 amplification, which is associated with the basal-like breast cancer subtype, has been proposed as one of the mechanism behind CUL4A up-regulation. However, the specific contribution of CUL4A to the biology of basal-like breast tumors has not yet been elucidated. In this work, by using cellular models of basal phenotype, we show the inhibitory effect of CUL4A silencing in the proliferation and growth of breast cancer cells both, in vitro and in vivo. We also demonstrate the transforming capacity of CUL4A exogenous overexpression in the 184B5 human mammary epithelial cells in vitro. Our results suggest a synergistic effect between CUL4A high levels and the activation of the RAS pathway in the tumorigenesis of basal-like breast cancer tumors. In addition, by using a proteomics approach we have defined novel candidate proteins and pathways that might mediate the oncogenic effect of CUL4A. In particular, we report a putative role of CUL4A in bypassing the immune system in breast cancer through the down-regulation of several molecules involved in the immune surveillance. These findings provide insight into the oncogenic properties of CUL4A in basal-like breast cancer and highlight the therapeutic opportunities to target CUL4A.
PMCID: PMC4039166  PMID: 24870930
CUL4A; 13q34 amplification; breast cancer; basal-like; proteomics; immune system
3.  Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction 
The Journal of Cell Biology  2013;203(2):265-282.
β(1,3)glucan is critical for contractile ring positioning and for coupling septum synthesis to constriction of the contractile ring and plasma membrane extension during cytokinesis.
Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.
PMCID: PMC3812973  PMID: 24165938
4.  The Combined Expression Patterns of Ikaros Isoforms Characterize Different Hematological Tumor Subtypes 
PLoS ONE  2013;8(12):e82411.
A variety of genetic alterations are considered hallmarks of cancer development and progression. The Ikaros gene family, encoding for key transcription factors in hematopoietic development, provides several examples as genetic defects in these genes are associated with the development of different types of leukemia. However, the complex patterns of expression of isoforms in Ikaros family genes has prevented their use as clinical markers.
In this study, we propose the use of the expression profiles of the Ikaros isoforms to classify various hematological tumor diseases. We have standardized a quantitative PCR protocol to estimate the expression levels of the Ikaros gene exons. Our analysis reveals that these levels are associated with specific types of leukemia and we have found differences in the levels of expression relative to five interexonic Ikaros regions for all diseases studied. In conclusion, our method has allowed us to precisely discriminate between B-ALL, CLL and MM cases. Differences between the groups of lymphoid and myeloid pathologies were also identified in the same way.
PMCID: PMC3855751  PMID: 24324784
5.  F-actin asymmetry and the endoplasmic reticulum–associated TCC-1 protein contribute to stereotypic spindle movements in the Caenorhabditis elegans embryo 
Molecular Biology of the Cell  2013;24(14):2201-2215.
The position of the spindle apparatus determines the plane of cell cleavage and, therefore, the size and position of daughter cells, as well as the decision between symmetric and asymmetric cell division. We show that asymmetry in cortical actin and, remarkably, an endoplasmic reticulum–localized protein contribute to proper spindle positioning in the Caenorhabditis elegans embryo.
The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα–GPR–LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα–GPR–LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.
PMCID: PMC3708726  PMID: 23699393
6.  A Proteomic Characterization of Factors Enriched at Nascent DNA Molecules 
Cell reports  2013;3(4):1105-1116.
DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is wide-spread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.
PMCID: PMC3714744  PMID: 23545495
7.  Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission 
The Journal of Cell Biology  2012;198(4):637-656.
The α(1-3)glucan synthase Ags1 is essential for both secondary septum formation and the primary septum structural strength needed to counter cell turgor pressure during cell separation.
Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of α(1-3)glucan in septation and cell separation. We show that α(1-3)glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore α(1-3)glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure.
PMCID: PMC3514033  PMID: 22891259
8.  Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis 
Molecular Biology of the Cell  2012;23(23):4526-4542.
The phosphoinositide 3-kinase (PI3K) pathway is mutated in approximately half of tumors; it is therefore important to define its functions. This study shows that PI3Kα activity regulates mitotic entry and spindle orientation; in contrast, PI3Kβ controls dynein/dynactin and Aurora B activation at kinetochores and, in turn, chromosome segregation.
Class IA phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP3). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110β. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110β activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110β function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP3 generation, prometaphase progression, and spindle orientation. In contrast, p110β was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110β function in preserving genomic stability during mitosis.
PMCID: PMC3510015  PMID: 23051731
9.  Phosphoinositide 3-kinase beta controls replication factor C assembly and function 
Nucleic Acids Research  2012;41(2):855-868.
Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1– and RFC–RAD17 complexes, but also RFC–CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes.
PMCID: PMC3553946  PMID: 23175608
10.  Incidence and prognosis of intra-abdominal hypertension in critically ill medical patients: a prospective epidemiological study 
Annals of Intensive Care  2012;2(Suppl 1):S3.
The aim of this study was to determine the incidence of intra-abdominal hypertension (IAH) in patients with two or more categorized risk factors (CRF) for IAH, and their morbidity and mortality during their intensive care unit (ICU) stay.
Prospective cohort study carried out at a medical ICU. A total of 151 medical patients were enrolled during a period of 3 months. After ICU whole staff training, we conducted daily screening of the four CRF for IAH based on the World Society of Abdominal Compartment Syndrome (WSACS) guidelines (namely, diminished abdominal wall compliance, increased intraluminal content, increased abdominal content, and capillary leak syndrome or fluid resuscitation). In those patients with risk factors of at least two different categories (≥2 CRF), intra-abdominal pressure (IAP) was measured every 8 h during ICU stay. Data included demographics, main diagnosis on admission, severity scores, cumulative fluid balance, daily mean IAP, resolution of IAH, days of ICU and hospital stay, and mortality.
Eighty-seven patients (57.6%) had ≥2 CRF for IAH, 59 (67.8%) out of whom developed IAH. Patients with ≥2 CRF had a significantly higher mortality rate (41.4 vs. 14.3%, p < 0.001). Patients with IAH had higher body mass index, severity scores, organ dysfunctions/failures, number of CRF for IAH, days of ICU/hospital stay and hospital mortality rate (45.8 vs. 32.1%, p = 0.22). Non-resolution of IAH was associated with a higher mortality rate (64.7 vs. 35.3%, p = 0.001). None of the cohort patients developed abdominal compartment syndrome. The multivariate analysis showed that IAH development (odds ratio (OR) 4.09; 95% confidence interval (CI) 0.83-20.12) was a non-independent risk factor for mortality, and its non-resolution (OR 13.15; 95% CI 22.13-81.92) was an independent risk factor for mortality.
Critically ill medical patients admitted to ICU with ≥2 CRF have high morbidity, mortality rate, and incidence of IAH, so IAP should be measured and monitored as recommended by the WSACS. Our study highlights the importance of implementing screening and assessment protocols for an early diagnosis of IAH.
PMCID: PMC3390290  PMID: 22873419
intra-abdominal hypertension; abdominal compartment syndrome; intra-abdominal pressure; multiple organ failure; critically ill patients; intensive care.
11.  Nuclear but Not Cytosolic Phosphoinositide 3-Kinase Beta Has an Essential Function in Cell Survival ▿  
Molecular and Cellular Biology  2011;31(10):2122-2133.
Class IA phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival.
PMCID: PMC3133359  PMID: 21383062
12.  Perspectives in stem cell proteomics 
Genome Medicine  2009;1(4):45.
A brief report on the Perspectives in Stem Cell Proteomics Conference, Hinxton, UK, 22-23 March, 2009.
PMCID: PMC2684666  PMID: 19439029

Results 1-12 (12)