Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca²⁺-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca²⁺-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.     

Metabolic Regulation by the Mitochondrial Phosphatase PTPMT1 Is Required for Hematopoietic Stem Cell Differentiation

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Highlights? PTPMT1 depletion causes cell cycle delay and differentiation block in HSCs ? The HSC pool in PTPMT1 knockout mice is drastically (～40-fold) expanded ? Mitochondrial metabolism is altered and AMPK is highly activated in knockout HSCs ? PTPMT1 PIP substrates directly enhance fatty-acid-induced activation of UCP2     

c-Jun NH2-Terminal Kinase Is Required for Lineage-Specific Differentiation but Not Stem Cell Self-Renewal

The c-Jun NH₂-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1^−/− Jnk2^−/− ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.The c-Jun NH₂-terminal kinase (JNK) is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. JNK is encoded by two ubiquitously expressed genes (Jnk1 and Jnk2) and by a third gene (Jnk3) that is selectively expressed in neurons (14). Gene disruption studies demonstrate that mice without Jnk1 or Jnk2 are viable, but compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality (14). Murine embryonic fibroblasts (MEFs) isolated from Jnk1^−/− Jnk2^−/− mice exhibit a severe growth retardation phenotype (54). The markedly reduced growth of Jnk1^−/− Jnk2^−/− MEFs is consistent with the finding that JNK is critically required for the regulation of AP1-dependent gene expression (56) that is implicated in cellular proliferation (26). Thus, Jnk1^−/− Jnk2^−/− MEFs express low levels of AP1 proteins (e.g., c-Jun and JunD) and exhibit marked defects in AP1 target gene expression (34, 56). This loss of AP1 function is mediated, in part, by reduced phosphorylation of the activation domain of Jun family proteins and ATF2 (56).More recent studies using a conditional gene ablation strategy have demonstrated that compound JNK deficiency causes rapid senescence (12). This conclusion was confirmed by using chemical genetic analysis with MEFs isolated from mice with a germ line mutation that sensitizes JNK to inhibition by a predesigned small-molecule drug (12, 25). This form of senescence was found to be p53 dependent (12) and resembles the p53-dependent senescence of c-Jun^−/− MEFs (49). These data indicate that JNK plays a critical role in cellular proliferation. Indeed, it is possible that the p53-dependent senescence observed in JNK-deficient cells may contribute to aging. This is because altered p53 function is established to be an important determinant of early aging (36, 55). Importantly, this role of p53 in aging appears to be distinct from p53-mediated tumor suppression and DNA damage responses (21, 39, 43).One aspect of the aging process is a reduction in the regenerative capacity of stem cells (50). Indeed, it has been established that altered p53 activity associated with aging causes decreased stem cell function (8, 18, 42) and that disruption of the p53 pathway can increase stem cell function (1). Since JNK can influence p53-dependent senescence (12), these data indicate that JNK may be important for stem cell proliferation and self-renewal potential.Embryonic stem (ES) cells proliferate and are capable of both self-renewal and differentiation to multiple cell types. Indeed, murine ES cells can differentiate to create all tissues within a mouse. The profound growth retardation and rapid p53-dependent senescence of Jnk1^−/− Jnk2^−/− MEFs (12) suggests that JNK may play a critical role in the normal function of ES cells, including self-renewal and differentiation potential. The purpose of the present study was to test this hypothesis. Our approach was to isolate ES cells from wild-type and JNK-deficient mice. We demonstrate that JNK is not required for self-renewal or the proliferation of ES cells. However, JNK is required for ES cell differentiation.     

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background

Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells.

Methodology/Principal Findings

Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G₀/G₁ phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice.

Conclusions/Significance

These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.     

ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary

The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.     

SLOW MOTION Is Required for Within-Plant Auxin Homeostasis and Normal Timing of Lateral Organ Initiation at the Shoot Meristem in Arabidopsis

The regular arrangement of leaves and flowers around a plant''s stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation.     

Alternative Cultures for Human Pluripotent Stem Cell Production,Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.     

CLCA2 Interactor EVA1 Is Required for Mammary Epithelial Cell Differentiation

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized human mammary epithelial cells (HMEC) caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The primary sequence of the CLCA2 TMS was found to be conserved in CLCA2 orthologs throughout mammals, suggesting that its interaction with EVA1 co-evolved with the mammary gland. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.