Inventing Engineered Organoids for end-stage liver failure patients

Journal of Molecular Histology - End-stage liver disease (ESLD) is a term used clinically in reference to a group of liver diseases with liver transplantation as the choice of treatment. Due to the...     

The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth

Cilia are specialized organelles that play an important role in several biological processes, including mechanosensation, photoperception, and osmosignaling. Mutations in proteins localized to cilia have been implicated in a growing number of human diseases. In this study, we demonstrate that the von Hippel-Lindau (VHL) protein (pVHL) is a ciliary protein that controls ciliogenesis in kidney cells. Knockdown of pVHL impeded the formation of cilia in mouse inner medullary collecting duct 3 kidney cells, whereas the expression of pVHL in VHL-negative renal cancer cells rescued the ciliogenesis defect. Using green fluorescent protein-tagged end-binding protein 1 to label microtubule plus ends, we found that pVHL does not affect the microtubule growth rate but is needed to orient the growth of microtubules toward the cell periphery, a prerequisite for the formation of cilia. Furthermore, pVHL interacts with the Par3-Par6-atypical PKC complex, suggesting a mechanism for linking polarity pathways to microtubule capture and ciliogenesis.     

Identifying the neural circuitry of alcohol craving and relapse vulnerability

With no further intervention, relapse rates in detoxified alcoholics are high and usually exceed 80% of all detoxified patients. It has been suggested that stress and exposure to priming doses of alcohol and to alcohol-associated stimuli (cues) contribute to the relapse risk after detoxification. This article focuses on neuronal correlates of cue responses in detoxified alcoholics. Current brain imaging studies indicate that dysfunction of dopaminergic, glutamatergic and opioidergic neurotransmission in the brain reward system (ventral striatum including the nucleus accumbens) can be associated with alcohol craving and functional brain activation in neuronal systems that process attentional relevant stimuli, reward expectancy and experience. Increased functional brain activation elicited by such alcohol-associated cues predicted an increased relapse risk, whereas high brain activity elicited by affectively positive stimuli may represent a protective factor and was correlated with a decreased prospective relapse risk. These findings are discussed with respect to psychotherapeutic and pharmacological treatment options.     

NPHP proteins: gatekeepers of the ciliary compartment

The cilia and the cytoplasm are separated by a region called the transition zone, where wedge-shaped structures link the microtubule doublets of the axoneme to the ciliary membrane, thereby forming a ciliary “gate.” In this issue, Craige et al. (J. Cell Biol. doi:10.1083/jcb.201006105) demonstrate in Chlamydomonas reinhardtii that Nphp6/cep290, which is mutated in nephronophthisis (NPHP), is an integral component of these connectors and maintains the structural integrity of this gate.Cilia, tiny hairlike organelles that protrude from the cell surface, are located on almost all polarized cell types of the human body. Although the basic structures of different types of cilia are similar, they exert various tissue-specific functions during development, tissue morphogenesis, and homeostasis. Their prevalence and involvement in various cellular functions could explain why cilia-related disorders (ciliopathies) can affect many organ systems. Ciliopathies can either involve single organs, such as cystic kidney disease, or can occur as multisystemic disorders, such as Bardet Biedl syndrome and nephronophthisis (NPHP)-related disorders with phenotypically variable and overlapping disease manifestations (Badano et al., 2006; Fliegauf et al., 2007). Among syndromic forms of cystic kidney diseases, NPHP is the most common and complex disorder in childhood. NPHP comprises a genetically heterogenous group of renal cystic disorders with an autosomal recessive inheritance pattern. NPHP can cause end-stage renal disease in early infancy, childhood, and adolescence, as well as in adulthood, and can be associated with extra-renal disease manifestations such as ocular motor apraxia (Cogan syndrome), retinitis pigmentosa, Leber congenital amaurosis, coloboma of the optic nerve, cerebellar vermis aplasia (Joubert syndrome), liver fibrosis, cranioectodermal dysplasia, cone-shaped epiphyses, asphyxiating thoracic dysplasia (Jeune’s syndrome), Ellis-van Creveld syndrome, and, rarely, situs inversus (Omran and Ermisch-Omran, 2008). In addition, it has been shown that NPHP mutations can cause Meckel syndrome, a perinatal lethal disease characterized by congenital cystic kidney disease and encephalocele.Several genes responsible for NPHP have been identified (summarized in Omran and Ermisch-Omran, 2008), and many of the encoded proteins, such as NPHP1, NPHP2 (inversin), NPHP3, NPHP4 (nephroretinin), NPHP6, and NPHP8, have been found to interact with each other (Olbrich et al., 2003; Mollet et al., 2005; Delous et al., 2007; Bergmann et al., 2008). Although important mechanistic insights in the pathogenesis of NPHP have been established, such as perturbed Wnt signaling, the exact functional role of NPHP proteins still remained enigmatic (Simons et al., 2005; Bergmann et al., 2008). In this issue, Craige et al. shed new light in the function of NPHP6. They demonstrate that NPHP6 is a structural component of the champagne glass–shaped structures that link the microtubular doublets of the axoneme to the ciliary necklace, a distinct portion of the ciliary membrane first described almost 40 yr ago (Gilula and Satir, 1972) . Up to now, nothing was known about the protein composition of this unique structure at the ciliary base.The ciliary compartment including the ciliary membrane is equipped with a distinct composition of proteins, and the compartment border is located at the transition zone, where intraflagellar transport (IFT) particles are involved in active transport of cargoes from and to the ciliary compartment across the compartment border driven by two kinesin-2 family members: the heterotrimeric KIF3A–KIF3B–KAP complex and the homodimeric KIF17 motor (Fig. 1). Interestingly, several studies demonstrated that NPHP proteins sublocalize to the ciliary base of primary cilia (NPHP1, NPHP4, NPHP6, NPHP8, NPHP9, and NPHP11) as well as to the connecting cilium of the photoreceptor (NPHP1, NPHP5, and NPHP6), which is considered to be the orthologous structure of the transition zone (Olbrich et al., 2003; Mollet et al., 2005; Otto et al., 2005; Sayer et al., 2006; Delous et al., 2007; Bergmann et al., 2008; Otto et al., 2008; Valente et al., 2010). Detailed analyses of proteins such as NPHP1 revealed specific and exclusive localization at the transition zone (Fig. 1 A), which suggests a possible gatekeeper-like functional role of NPHP proteins at the ciliary compartment border to control delivery and exit of proteins to and from the cilium, respectively (Fliegauf et al., 2006). During ciliogenesis, NPHP1 becomes immediately recruited to the transition zone, which indicates that NPHP proteins may also be important for formation of this organelle. Interestingly, localization of these proteins to the transition zone has been evolutionary conserved and is also observed in Caenorhabditis elegans (Jauregui et al., 2008).Open in a separate windowFigure 1.NPHP proteins function at the ciliary gate (transition zone). (A) Localization of nephrocystin (red, NPHP1) at the transition zone is shown in murine (mIMCD3) immotile renal cilia (top), immotile canine renal MDCK cilia (middle), and motile human respiratory cilia (bottom). The ciliary axoneme is stained with antibodies targeting acetylated α-tubulin (green). Bars, 5 µm. (B) The triplet microtubule structure of the basal body is converted into the axonemal doublet structure at the transition zone of primary cilia. Proximal transition y-shaped fibers (red) connect each outer microtubule doublet to the membrane and mark the border at which IFT proteins start to shuffle cargoes to and from the ciliary compartment. The ciliary compartment, including the ciliary membrane, is therefore equipped with a distinct composition of proteins such as polycystin-2 and BBS proteins (i.e., BBS4), which differs from the cytoplasm and the apical plasma membrane. NPHP6/CEP290 as well as other NPHP proteins (e.g., NPHP1) localize at the transition zone and probably function as gatekeepers that control access and exit of proteins to and from the ciliary compartment, respectively.In this issue Craige et al. (2010) exploit the excellent genetic and biochemical tools available in Chlamydomonas reinhardtii to investigate the role of cep290/Nphp6 in the regulation of ciliary protein trafficking. Using immunoelectron microscopy, they show that cep290 localizes to the wedge-shaped structures that bridge and connect the flagellar membrane to the axonemal outer doublets within the transition zone. Further ultrastructural studies revealed defects of those structures in cep290 mutants, which indicates that cep290 is essential for integrity of the ciliary “gate” and an integral component of this poorly characterized structure. Detailed analyses of anterograde and retrograde IFT transport kinetics did not reveal gross alterations, which indicated that cep290 does not regulate IFT motor activity. Mass spectrometry analyses of flagella identified a complex pattern of abnormal protein composition. Biochemistry analyses of the flagella found increased amounts of IFT complex B proteins and BBS4, and decreased levels of the IFT complex A protein IFT139 as well as polycystin-2, which confirms that cep290 functions as a gatekeeper to control protein content of the flagella compartment. Alteration of polycystin-2 and BBS4 levels might even explain the complex clinical phenotype of cystic kidney disease and BBS-like findings present in children affected by CEP290/NPHP6 mutations (den Hollander et al., 2006; Sayer et al., 2006; Valente et al., 2006; Baala et al., 2007).Craige et al. (2010) also make some interesting observations that could be relevant to somatic gene therapy. Using dikaryon rescue studies, they show that cep290 is a dynamic protein that shuttles between the cytoplasm and the transition zone and that can incorporate into preassembled mutant transition zones and restore function. These results could be applied toward targeted gene therapy in NPHP-related diseases, such as Leber congenital amaurosis, a retinal degeneration disease in which cep290 is frequently mutated. Expression of CEP290 by gene therapy vectors in photoreceptors of patients could restore ciliary function.The cellular biological findings presented by Craige et al. (2010) are of major scientific interest because they open a new NPHP research field focusing on the ciliary compartment border. Future studies will address the roles of other interacting NPHP proteins for the integrity and/or function of the ciliary gate. Cell type–specific differences of the composition of the ciliary gate might account for the phenotypic differences observed in NPHP patients. Recent findings indicate similarities between the mechanisms regulating nuclear and ciliary import. Consistently, ciliary targeting of the IFT motor protein KIF17 has been shown to be regulated by a ciliary-cytoplasmic gradient of the small GTPase Ran, with high levels of GTP-bound Ran (RanGTP) in the cilium (Dishinger et al., 2010). Furthermore, KIF17 interacts with the nuclear import protein importin-β2 in a manner dependent on the ciliary localization signals and inhibited by RanGTP. Thus, the wedge-shaped fibers may function as the ciliary equivalent of the nuclear pore. Further work will shed light on the relationship between the different components of this interesting structure.     

In vivo fluxes in the ammonium-assimilatory pathways in corynebacterium glutamicum studied by 15N nuclear magnetic resonance

Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)-glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.     

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.     

Purification and some catalytic properties of phosphoglucomutase from maize leaves

Phosphoglucomutase (EC 2.7.5.1, PGM) was purified to homogeneity from maize (Zea mays L.) leaves. The enzyme had specific activity 11. 7 U/mg protein and molecular mass (determined by gel-chromatography) of 133 +/- 4 kD. The molecular mass of PGM subunits determined by SDS-electrophoresis was 66 +/- 3 kD. The enzyme had Km for glucose-1-phosphate and glucose-1,6-diphosphate of 20.0 +/- 0.9 and 16.0 +/- 0.8 &mgr;M, respectively. Concentrations of glucose-1-phosphate and glucose-1,6-diphosphate above 3 and 0.4 mM, respectively, cause substrate inhibition. The enzyme activity was maximal at pH 8.0 and temperature 35 degreesC. Magnesium ions activate the enzyme and manganese ions inhibit it. 3-Phosphoglycerate is an uncompetitive inhibitor of the enzyme (Ki = 1.22 +/- 0.05 mM). Fructose-6-phosphate, 6-phosphogluconate, and ADP activate PGM, whereas ATP, UTP, and AMP inhibit the enzyme. Citrate was also a potent inhibitor, inhibitory effects of isocitrate and cis-aconitate being less pronounced.