Genetic control of kinetochore-driven microtubule growth in Drosophila mitosis

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but allows KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast/orbit/chb (CLASP1), mei-38 (TPX2), mars (HURP), dgt6 (HAUS6), Eb1 (MAPRE1/EB1), Patronin (CAMSAP2), asp (ASPM) and Klp10A (KIF2A). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6 and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1 and Patronin positively regulate polymerization, bundling and stabilization of regrowing MTs until a bipolar spindle is reformed.

Glycosylation reduces the glycan-independent immunomodulatory effect of recombinant Orysata lectin in Drosophila S2 cells

Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins.

First person – Chujun Zhang

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Chujun Zhang is first author on ‘ Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells’, published in JCS. Chujun is a PhD student in the lab of Prof. Catherine Rabouille at Hubrecht Institute of the KNAW and UMC Utrecht, Utrecht, The Netherlands, investigating pathways leading to stress assemblies formation.

Activation of salt Inducible Kinases, IRE1 and PERK leads to Sec bodies formation in Drosophila S2 cells

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the ER exit sites under the stress of amino-acid starvation. Here we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt inducible kinases (SIK) by Na+ stress, that when it is strong is sufficient. The second is activation of IRE1 and PERK downstream of ER stress induced by absence of amino- acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK and IRE1/PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work pioneers the role of SIK in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino-acids and salt.

Effects of Kathon, a Chemical Used Widely as a Microbicide, on the Survival of Two Species of Mosquitoes

In recent decades, demands for novel insecticides against mosquitoes are soaring, yet candidate chemicals with desirable properties are limited. Kathon is a broad-spectrum isothiazolinone microbicide, but other applications remain uncharacterized. First, we treated larvae of Culex quinquefasciatus and Aedes albopictus, two major mosquito vectors of human viral diseases, with Kathon at 15 mg/L (a concentration considered safe in cosmetic and body care products), and at lower concentrations, and found that Kathon treatment resulted in high mortality of larvae. Second, sublethal concentration of Kathon can cause significantly prolonged larval development of C. quinquefasciatus. Third, we explored the effects of two constituents of Kathon, chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT), on the survival of larvae, and found that CMIT was the major toxic component. Further, we explored the mechanisms of action of Kathon against insect cells and found that Kathon reduces cell viability and adenosine triphosphate production but promotes the release of lactate dehydrogenase in Drosophila melanogaster S2 cells. Our results indicate that Kathon is highly toxic to mosquito larvae, and we highlight its potential in the development of new larvicides for mosquito control.

RiboTRIBE: Monitoring Translation with ADAR-meditated RNA Editing

RNA translation is tightly regulated to ensure proper protein expression in cells and tissues. Translation is often assayed with biochemical assays such as ribosome profiling and TRAP, which are effective in many contexts. These assays are however not ideal with limiting amounts of biological material when it can be difficult or even impossible to make an extract with sufficient signal or sufficient signal:noise. Because of our interest in translational regulation within the few Drosophila adult circadian neurons, we fused the ADAR catalytic domain (ADARcd) to several small subunit ribosomal proteins and assayed mRNA editing in Drosophila S2 cells. The strategy is named RiboTRIBE and is analogous to a recently published APOBEC-based method. The list of RiboTRIBE-edited transcripts overlaps well with ribosome profiling targets, especially with more highly ranked targets. There is also an enriched number of editing sites in ribosome-associated mRNA comparing to total mRNA, indicating that editing occurs preferentially on polyribosome-associated transcripts. The use of cycloheximide to freeze translating ribosomes causes a substantial increase in the number of RiboTRIBE targets, which is decreased by pretreating cells with the chain terminating drug puromycin. The data taken together indicate that RiboTRIBE successfully identifies transcripts undergoing active translation.

Trim9 and Klp61F promote polymerization of new dendritic microtubules along parallel microtubules

Axons and dendrites are distinguished by microtubule polarity. In Drosophila, dendrites are dominated by minus-end-out microtubules while axons contain plus-end-out microtubules. Local nucleation in dendrites generates microtubules in both orientations. To understand why dendritic nucleation does not disrupt polarity, we used live imaging to analyze the fate of microtubules generated at branch points. We found that they had different rates of success exiting the branch based on orientation: correctly oriented minus-end-out microtubules succeeded in leaving about twice as often as incorrectly oriented microtubules. Increased success relied on other microtubules in a parallel orientation. From a candidate screen, we identified Trim9 and kinesin-5 (Klp61F) as machinery that promoted growth of new microtubules. In S2 cells, EB1 recruited Trim9 to microtubules. Klp61F promoted microtubule growth in vitro and in vivo, and could recruit Trim9 in S2 cells. In summary, the data argue that Trim9 and kinesin-5 act together at microtubule plus ends to help polymerizing microtubules parallel to pre-existing ones resist catastrophe.

Stonewall prevents expression of testis-enriched genes and binds to insulator elements in D. melanogaster

Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. In Drosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell-daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq on stwl mutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered that testis-enriched genes, including the differentiation factor bgcn and a large testis-specific cluster on chromosome 2, are upregulated or ectopically expressed in stwl mutant ovaries. Surprisingly, we also found that RNAi knockdown of stwl in somatic S2 cells results in ectopic expression of these genes. Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl binds upstream of transcription start sites and localizes to heterochromatic sequences. We also find that Stwl is enriched at repetitive sequences associated with telomeres. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation by binding insulators and establishing chromatin boundaries.