A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

The CD34⁺ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8⁺ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8⁺ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.     

Structural Plasticity in Human Heterochromatin Protein 1β

As essential components of the molecular machine assembling heterochromatin in eukaryotes, HP1 (Heterochromatin Protein 1) proteins are key regulators of genome function. While several high-resolution structures of the two globular regions of HP1, chromo and chromoshadow domains, in their free form or in complex with recognition-motif peptides are available, less is known about the conformational behavior of the full-length protein. Here, we used NMR spectroscopy in combination with small angle X-ray scattering and dynamic light scattering to characterize the dynamic and structural properties of full-length human HP1β (hHP1β) in solution. We show that the hinge region is highly flexible and enables a largely unrestricted spatial search by the two globular domains for their binding partners. In addition, the binding pockets within the chromo and chromoshadow domains experience internal dynamics that can be useful for the versatile recognition of different binding partners. In particular, we provide evidence for the presence of a distinct structural propensity in free hHP1β that prepares a binding-competent interface for the formation of the intermolecular β-sheet with methylated histone H3. The structural plasticity of hHP1β supports its ability to bind and connect a wide variety of binding partners in epigenetic processes.     

Cell Cycle–Dependent Differentiation Dynamics Balances Growth and Endocrine Differentiation in the Pancreas

Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine, ductal, and acinar cells but become bipotent by embryonic day 13.5, giving rise to endocrine cells and ductal cells. However, the dynamics of individual progenitors balancing self-renewal and lineage-specific differentiation has never been described. Using three-dimensional live imaging and in vivo clonal analysis, we reveal the contribution of individual cells to the global behaviour and demonstrate three modes of progenitor divisions: symmetric renewing, symmetric endocrinogenic, and asymmetric generating a progenitor and an endocrine progenitor. Quantitative analysis shows that the endocrine differentiation process is consistent with a simple model of cell cycle–dependent stochastic priming of progenitors to endocrine fate. The findings provide insights to define control parameters to optimize the generation of β-cells in vitro.     

Loss of Hepatocyte-Nuclear-Factor-1α Impacts on Adult Mouse Intestinal Epithelial Cell Growth and Cell Lineages Differentiation

Background and Aims

Although Hnf1α is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1α on the maintenance of adult small intestinal epithelial functions.

Methodology/Principal Findings

An Hnf1α knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1α displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1α mutant mice. The intestinal epithelium of Hnf1α null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway.

Conclusions/Significance

Together, these results unravel a functional role for Hnf1α in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.     

IL-23 and IL-1β Drive Human Th17 Cell Differentiation and Metabolic Reprogramming in Absence of CD28 Costimulation

1. Download high-res image (119KB)
2. Download full-size image

     

Autophagy during Early Virus–Host Cell Interactions

Autophagy refers to the conserved, multi-step mechanism that delivers cytosolic cargoes to vesicles of the endo-lysosomal system for degradation. It maintains cellular homeostasis by ensuring the continuous degradation of misformed/senescent intracellular components and the associated recycling of nutrients. Autophagy also represents an important cell-intrinsic defense mechanism against invasion by intracellular pathogens, including viruses. Autophagy might oppose viral invasion by targeting viral particles or viral components for degradation. It can also promote the interaction of viral constituents with receptors specialized in the activation of innate immunity pathways or facilitate the activation of anti-viral adaptive immunity. In response to such pressures, viruses have evolved various sophisticated strategies to avoid anti-viral autophagic responses or to manipulate the autophagic machinery to promote their own replication. This review focuses on our current knowledge of autophagy-related events that take place at early stages during interaction of viruses with host cells as well as on their associated consequences in terms of virus replication and cell fate.     

Junctional Music that the Nucleus Hears: Cell–Cell Contact Signaling and the Modulation of Gene Activity

Cell–cell junctions continue to capture the interest of cell and developmental biologists, with an emerging area being the molecular means by which junctional signals relate to gene activity in the nucleus. Although complexities often arise in determining the direct versus indirect nature of such signal transduction, it is clear that such pathways are essential for the function of tissues and that alterations may contribute to many pathological outcomes. This review assesses a variety of cell–cell junction-to-nuclear signaling pathways, and outlines interesting areas for further study.The evolution of multicellular life forms has to a significant extent involved refinements of each cell''s capacity to sense the state of its directly contacting neighbors. This exchange of information often occurs within tissues, with the result that gene activity in the nucleus is altered or maintained accordingly. In this article, we focus on how signals arise at cell–cell junctions and are transduced to the nucleus; we do not include discussion of mechanical/cytoskeletal signals influencing nuclear decisions, and the reader is directed to a recent review of this topic (Ingber 2008).An issue that arises when addressing cell–cell junction(s), referred to as CCJ(s), -to-nuclear signals, is that homotypic or heterotypic junctional proteins responsible for conferring adhesive activity are often in a much larger complex of proteins. These interactions may be either in cis (interacting within the plasma membrane of the cell) or trans orientations (interacting through ectodomain contacts extended between cells). Most of these transmembrane proteins are likely to have the potential to contribute to downstream signaling events, and many may associate with one another only under specific physiological conditions. For example, certain receptor tyrosine kinases (RTKs) associate with particular cadherins, and when associated are relevant to that cadherin''s functions (Wheelock and Johnson 2003; Andl and Rustgi 2005). In this article, we discuss relationships such as these in the context of CCJ-nuclear signaling. A topic not represented here is the CCJ signaling of immune surveillance cells, for example, pathways activated following leukocyte–endothelia contact. This area is of great basic and biomedical interest, but is addressed elsewhere (Dustin 2007).We focus on signaling by a select number of junction types, including adherens, desmosomal, and tight junctions, and to a lesser extent, gap junctions. Details of the structure and function of each of these junctions are presented in other articles (see Meng and Takeichi 2009, Delva et al. 2009, Furuse 2009, and Goodenough and Paul 2009, respectively). These junctions are often represented in textbooks as distinct entities in the context of epithelial tissues, but their structures and how they respond to or generate signaling cues vary according to cellular context. Select components within these junctions may be shared, for example between desmosomal, adherens, and tight junctions, and in some instances, intimate physical proximities are likely to advance these junctions'' functional interrelation. Further, different cell types show less common junctional organizations (Straub et al. 2003; Wuchter et al. 2007), such that the total spectrum of CCJ signals is likely to be impressive, and far beyond what is currently known or understood. Given the interdependence of cell neighbors in forming and maintaining cell groupings, high diversity and sophistication arose in complex organisms, both in CCJ structures themselves and their associated nuclear signaling pathways. Compared with the knowledge accumulated over the past two decades on cell–extracellular matrix signaling via integrins (Abram and Lowell 2009), we know less about signals initiated from forming or mature cell–cell contacts in epithelial, neural, or endothelial tissues. Thus, as the field moves forward, there is the potential to achieve a deepened understanding of how the cell–extracellular matrix and cell–cell adhesion systems are coupled in a signaling context, and how they collectively relate to the adhesion, motility, and differentiation of cells and tissues.     

Human ESC-Derived Chimeric Mouse Models of Huntington’s Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation

1. Download high-res image (223KB)
2. Download full-size image

     

Estimation of Cellobiohydrolase Ⅰ Activity by Numerical Differentiation of Dynamic Ultraviolet Spectroscopy

1,4-β-D-glucan cellobiohydrolase Ⅰ （CBH Ⅰ）, p-nitrophenyl β-D-cellobioside, p-nitrophenol and cellobiose show distinct ultraviolet spectra, allowing the design of an assay to track the dynamic process of p-nitrophenyl β-D-cellobioside hydrolysis by CBH Ⅰ. Based on the linear relationship between p-nitrophenol formation in the hydrolysate and its first derivative absorption curve of AUC340-400 m （area under the curve）, a new sensitive assay for the determination of CBH Ⅰ activity was developed. The dynamic parameters of catalysis reaction, such as Vm and kcat, can all be derived from this result. The influence of β-glucosidase and endoglucanase in crude enzyme sample on the assay was discussed in detail. This approach is useful for accurate determination of the activity of CBHs.