The endoplasmic reticulum (ER) depends on extensive association with the microtubule (MT) cytoskeleton for its structure and mitotic inheritance. However, mechanisms that underlie coupling of ER ...membranes to MTs are poorly understood. We have identified thousand and one amino acid kinase 2 (TAOK2) as a pleiotropic protein kinase that mediates tethering of ER to MTs. In human cells, TAOK2 localizes in distinct ER subdomains via transmembrane helices and an adjacent amphipathic region. Through its C-terminal tail, TAOK2 directly binds MTs, coupling ER membranes to the MT cytoskeleton. In TAOK2 knockout cells, although ER-membrane dynamics are increased, movement of ER along growing MT plus ends is disrupted. ER-MT tethering is tightly regulated by catalytic activity of TAOK2, perturbation of which leads to defects in ER morphology, association with MTs, and cell division. Our study identifies TAOK2 as an ER-MT tether and reveals a kinase-regulated mechanism for control of ER dynamics.
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•TAOK2 is an ER kinase that directly binds microtubules, coupling ER to microtubules•Loss of TAOK2 increases ER and microtubule dynamics•TAOK2 associates with EB1 and STIM1 to regulate ER movement on microtubule plus ends•Catalytic activity of TAOK2 drives untethering of ER from the mitotic spindle
Nourbakhsh, Ferreccio, et al. reveal that the protein kinase TAOK2 is a molecular tether that couples the endoplasmic reticulum to the microtubule cytoskeleton. TAOK2-mediated tethering is autoregulated by its catalytic activity, which is essential for ER motility and ER-membrane dissociation from the mitotic spindle during cell division.
For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that ...embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs). Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.
Pluripotent stem cells have distinct metabolic requirements, and reprogramming cells to pluripotency requires a shift from oxidative to glycolytic metabolism. Here, we show that this shift occurs ...early during reprogramming of human cells and requires hypoxia-inducible factors (HIFs) in a stage-specific manner. HIF1α and HIF2α are both necessary to initiate this metabolic switch and for the acquisition of pluripotency, and the stabilization of either protein during early phases of reprogramming is sufficient to induce the switch to glycolytic metabolism. In contrast, stabilization of HIF2α during later stages represses reprogramming, partly because of the upregulation of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL inhibits induced pluripotent stem cell (iPSC) generation by repressing apoptotic caspase 3 activity specifically in cells undergoing reprogramming but not human embryonic stem cells (hESCs), and inhibiting TRAIL activity enhances human iPSC generation. These results shed light on the mechanisms underlying the metabolic shifts associated with the acquisition of a pluripotent identity during reprogramming.
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•HIF1α and HIF2α are required during early stages of reprogramming•HIF2α inhibits reprogramming when expressed during later stages of the process•HIF2α regulates expression of TRAIL, which inhibits reprogramming•TRAIL represses caspase 3 activity specifically in cells undergoing reprogramming
Both HIF1α and HIF2α are required during early stages of reprogramming human cells to iPSCs, whereas HIF2α inhibits later stages of reprogramming by regulating the expression of TRAIL.
The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific ...developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein–protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.
To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained ...karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology-directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.
Pluripotent stem cells hold great promise for the future of regenerative medicine. Though many studies have examined the genetic determinants of pluripotency, the role of metabolism in stemness ...acquisition and maintenance remains an active area of investigation. Due to its role in the acquisition of different metabolic states, hypoxia, via action of the Hypoxia Inducible Factors (HIFs), is also suspected to regulate pluripotency. Indeed this low oxygen level is a key feature of a variety of stem cell niches. We thus decided to study the roles of HIFs in the acquisition and maintenance of pluripotency. First, we tested the role of HIFs in stemness acquisition by reprogramming human fibroblasts into induced pluripotent stem cells (iPSCs). We found that even though both HIF1α and HIF2α are needed for iPSC formation, HIF2α overexpression towards the later stages of reprogramming inhibits colony formation through TRAIL activation. Then, in the human embryonic stem cell paradigm we showed that HIF1α is required for the transition from the naïve to the primed hESC, two separate pluripotent states characterized by distinct metabolic profiles. Together these results underline the crucial role of hypoxia and HIFs in the regulation of stemness and metabolism. Another area in which metabolism can act to regulate pluripotency is through the regulation of epigenetics. Previous studies showed that energy metabolism and the availability of certain metabolites can influence stem cells and their stemness capacities. The Polycomb repressive complex 2 (PRC2) has methyltransferase activity on histones, primarily adding the repressive trimethylation mark on histone 3 lysine 27 (H3K27me3). We investigated the role of JARID2, a key protein of this complex, in the maintenance of pluripotency of hESCs. Interestingly, the loss of JARID2 in naïve hESC (2iL-I-F) induces a concomitant loss of H3K27me3 and pluripotency markers. A variety of solid tumors contain a subpopulation of cancer stem cells (CSCs), cancer cells characterized by their ability to self-renew, initiate tumors and lead to metastasis. CSCs share several features with normal adult stem cells in terms of metabolic profile. Clarifying the role of metabolism in stemness acquisition could have interesting applications for cancer stem cells. Interestingly, both primed stem cells and cancer stem cells share a similar glycolytic metabolism. Adult stem cells in the model organism Drosophila melanogaster have been recently used as a model to study cancer stem cells. In vivo studies allow for considerations of the interactions between stem cells and the stem cell niche. These interactions have been shown to be critical in the resistance of stem cells to apoptosis following exposure to ionizing radiation. Germline stem cells are able to survive IR damage through Pvf-1 mediated Tie-2 receptor activation. The human homologs Angiopoietin1 (Ang1) and Tie2 are therefore interesting targets for inhibitors and activators in human stem cells. A combination of in silico designed scaffolds with the binding domain of Ang1, F-domain was used to activate the Tie2 receptor.
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