Celebrating 100 years of Drosophila research

The seventeenth EMBO Conference on the Molecular and Developmental Biology of Drosophila took place in Kolymbari, Crete, between 20 and 26 June 2010. The conference covered a broad range of topics and much progress was made by combining two or more fields of study. Such combinations included quantitative approaches to cell and developmental biology, dissecting interrelations of physiology and development and integrated genomic analysis.     

A novel three-stage seaweed (Ulva lactuca) biofilter design for integrated mariculture

Seaweed biofilters have proven their usefulness in the treatment of fishpond effluents. However, their performance poses a dilemma: TAN (Total Ammonia N) uptake rate – and with it seaweed yield and protein content – is inversely proportional to TAN uptake efficiency. The ideal for a seaweed biofilter performance would be a high uptake rate together with high uptake efficiency. The novel three-stage seaweed biofilter design described here has solved this dilemma. The design used the finding that the performance of seaweed ponds depended on the flux of TAN through them, and that therefore effluents with reduced TAN concentration could provide the seaweed with a high TAN flux if the water flow increased proportionally. Effluents from a seabream fishpond were passed through a series of three successively smaller (25, 12.5 and 6.25 m², respectively) air-agitated Ulva lactuca ponds. The diminished inflow TAN concentrations to the second and third ponds of the biofilter system were compensated for by the increased water exchange rates, inversely proportional to their sizes. The biofilter performance was evaluated under several TAN loads. TAN was efficiently removed (85–90%), at a high areal rate (up to 2.9 g N m^-2 d^-1) while producing high protein U. lactuca (up to 44% dw) in all three stages, although with mediocre yields (up to 189 g fresh m^-2 d^-1). Performance of each seaweed biofilter pond correlated not with TAN concentration, but with areal TAN loads. The novel three-stage design provides significant functional and economic improvements in seaweed biofiltration of intensive fishpond water.     

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.     

Mutability and importance of a hypermutable cell subpopulation that produces stress-induced mutants in Escherichia coli

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac⁺ mutants, with and without evidence of descent from the HMS, have similar Lac⁺ mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac⁺ mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.     

Identification of trafficking determinants for polytopic rhomboid proteases in Toxoplasma gondii

Rhomboids (ROMs) constitute a family of polytopic serine proteases conserved throughout evolution. The obligate intracellular parasite Toxoplasma gondii possesses six genes coding for ROM-like proteases that are targeted to distinct subcellular compartments: TgROM1 localizes to regulated secretory organelles, micronemes, TgROM2 is present in the Golgi, while TgROM4 and TgROM5 are found in the pellicle of the parasite. The targeting mechanism/s of ROM proteins is an aspect that has not yet been assessed. The existence of TgROM family members localized to different subcellular compartments provides a convenient system to study their sorting mechanisms in a genetically tractable organism that possesses an elaborate secretory pathway and conserved trafficking machineries. In this study, we experimentally established the topology of TgROM1 and TgROM4 at the plasma membrane and applied domain-exchange and site-directed mutagenesis approaches to identify critical sorting determinants on the N-terminal cytosolic domains of TgROM2 and TgROM1 that confer their Golgi and post-Golgi localizations, respectively.     

Resolving the dynamics of EEG generators by multichannel recordings

The voltage recorded over the cortex (ECoG) or over the scalp (EEG) is generated by currents derived from many sources called “generators”. Different patterns and amplitudes are observed in aroused, sleepy, epileptic or other brain states. Differences in amplitude are generally attributed to differences in synchrony among generators. The degree of EEG synchrony is measured by the correlation between electrodes placed over different cortical regions. We present a new way to quantitatively assess the degree of synchronization of these generators via multichannel recordings. We illustrate how situations where there are several groups of generators with different inter-group and intra-group synchronies can be analyzed. Finally, we present a way to identify the organization of groups exhibiting topographic organization. Although the model presented here is highly simplified, several methods are based on averaging activity over increasingly larger areas. These types of measurements may be applied as well to EEG and ECoG recordings.     

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.     

Immunoglobulin light chains dictate vesicular transport-dependent and -independent routes for IgM degradation by the ubiquitin-proteasome pathway

Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.     

Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state

Gap junctions coordinate processes ranging from muscle contraction to ovarian follicle development. Here we show that the gap junction protein Zero population growth (Zpg) is required for germ cell differentiation in the Drosophila ovary. In the absence of Zpg the stem cell daughter destined to differentiate dies. The zpg phenotype is novel, and we used this phenotype to genetically dissect the process of stem cell maintenance and differentiation. Our findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual.