Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

The E2 Ubiquitin-conjugating Enzymes Direct Polyubiquitination to Preferred Lysines

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.     

Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction

AtOPT promoter-GUS fusions were constructed for six of the nine known, putative oligopeptide transporters (OPTs) in Arabidopsis thaliana and used to examine AtOPT expression at various stages of plant development. AtOPT1, AtOPT3, AtOPT4, AtOPT6 and AtOPT7 were expressed in the embryonic cotyledons prior to root radicle emergence. Except for AtOPT8, which gave weak expression, all AtOPTs were strongly expressed in post-germinative seedlings with strongest expression in vascular tissues of cotyledons and hypocotyls. Preferential expression of AtOPTs in vascular tissues was also observed in cotyledons, leaves, hypocotyls, roots, flowers, siliques, and seed funiculi of seedlings and adult plants. Differential tissue-specific expression was observed for specific AtOPTs. For example, AtOPT1, AtOPT3 and AtOPT8 were uniquely expressed in pollen. Only AtOPT1 was expressed in growing pollen tubes, while only AtOPT6 was observed in ovules. AtOPT8 was transiently expressed in seeds during early stages of embryogenesis. Iron limitation was found to enhance expression of AtOPT3. These data suggest distinct cellular roles for specific AtOPTs including nitrogen mobilization during germination and senescence, pollen tube growth, pollen and ovule development, seed formation and metal transport.     

Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.     

EGF-ERBB signalling: towards the systems level

Signalling through the ERBB/HER receptors is intricately involved in human cancer and already serves as a target for several cancer drugs. Because of its inherent complexity, it is useful to envision ERBB signalling as a bow-tie-configured, evolvable network, which shares modularity, redundancy and control circuits with robust biological and engineered systems. Because network fragility is an inevitable trade-off of robustness, systems-level understanding is expected to generate therapeutic opportunities to intercept aberrant network activation.     

Inexpensive non-toxic flocculation of microalgae contradicts theories; overcoming a major hurdle to bulk algal production

There are two major energy and cost constraints to bulk production of single cell microalgae for biofuels or feed: expensive culture systems with high capital costs and high energy requirements for mixing and gas exchange; and the cost of harvesting using high-speed continuous centrifugation for dewatering. This report deals with the latter; harvesting by flocculation where theory states that alkaline flocculants neutralize the repelling surface charge of algal cells, allowing them to coalesce into a floc. It had been assumed that with such electrostatic flocculation, the more cells to be flocculated, the more flocculant needed, in a linear stoichiometric fashion, rendering flocculation overly expensive. Counter to theory of electrostatic flocculation, we find that the amount of alkaline flocculant needed is a function of the logarithm of cell density, with dense cultures requiring an order of magnitude less base than dilute suspensions, with flocculation occurring at a lower pH. Various other theories abound that flocculation can be due to multi-valent cross-linking, or co-precipitation with phosphate or with magnesium and calcium, but are clearly not relevant with the flocculants we used. Monovalent bases that cannot cross-link or precipitate phosphate work with the same log-linear stoichiometry as the divalent bases, obviating those theories, leaving electrostatic flocculation as the only tenable theory of flocculation with the materials used. The cost of flocculation of dense cultures with this procedure should be below $1.00/T algae for mixed calcium:magnesium hydroxides.     

Neurotrophin-4 regulates the survival of gustatory neurons earlier in development using a different mechanism than brain-derived neurotrophic factor

The number of neurons in the geniculate ganglion that are available to innervate taste buds is regulated by neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF). Our goal for the current study was to examine the timing and mechanism of NT-4-mediated regulation of geniculate neuron number during development. We discovered that NT-4 mutant mice lose 33% of their geniculate neuronal cells between E10.5 and E11.5. By E11.5, geniculate axons have just reached the tongue and do not yet innervate their gustatory targets; thus, NT-4 does not function as a target-derived growth factor. At E11.5, no difference was observed in proliferating cells or the rate at which cells exit the cell cycle between NT-4 mutant and wild type ganglia. Instead, there was an increase in TUNEL-labeling, indicating an increase in cell death in Ntf4(-/-) mice compared with wild types. However, activated caspase-3, which is up-regulated in the absence of BDNF, was not increased. This finding indicates that cell death initiated by NT-4-removal occurs through a different cell death pathway than BDNF-removal. We observed no additional postnatal loss of taste buds or neurons in Ntf4(-/-) mice. Thus, during early embryonic development, NT-4 produced in the ganglion and along the projection pathway inhibits cell death through an activated caspase-3 independent mechanism. Therefore, compared to BDNF, NT-4 plays distinct roles in gustatory development; differences include timing, source of neurotrophin, and mechanism of action.