Development of a Rainbow Trout (Oncorhynchus mykiss) Intestinal In Vitro Platform for Profiling Amino Acid Digestion and Absorption of a Complete Diet.

The ever-increasing number and variation of raw materials utilized to provide alternative feed formulations continues to allow for a more sustainable and flexible approach. Testing all these options in vivo is still the most robust and reliable manner to pick the best raw material candidates, but it requires the use of large numbers of animals and is time-consuming and expensive. Therefore, we are developing an in vitro platform that can provide a reliable evaluation of new ingredients. The main aim of this work was to combine an in vitro digestion protocol of extruded, commercially relevant aquafeeds with the exposure of intestinal epithelial cells to the extracted bio-available fraction (BAF). The results show that 250,000 cells/cm2 represents the optimal seeding density and that up to 50% BAF concentration for up to 24 h had no negative effects on the epithelial barrier morphology and function. It is possible to determine amino acid digestibility and bioavailability in all the experimental conditions (with and without BSA, at 25% and 50% dilution) and at all time points (0, 6, and 24 h). However, BAF concentration, the medium used for its dilution, and the length of exposure to the different epithelial cell lines can all influence the results and, therefore, must be selected according to the final aim of the experiment.

Stable and dynamic microtubules coordinately determine and maintain Drosophila bristle shape.

Within interphase cells, microtubules (MTs) are organized in a cell-specific manner to support cell shape and function. Here, we report that coordination between stable and dynamic MTs determines and maintains the highly elongated bristle cell shape. By following MT-decorating hooks and by tracking EB1 we identified two MT populations within bristles: a stable MT population polarized with their minus ends distal to the cell body, and a dynamic MT population that exhibits mixed polarity. Manipulating MT dynamics by Klp10A downregulation demonstrates that MTs can initiate new shaft extensions and thus possess the ability to determine growth direction. Actin filament bundling subsequently supports the newly formed shaft extensions. Analysis of ik2 mutant bristles, established by elongation defects in the Drosophila ikkε homolog, led to the observation that stable and dynamic MT orientation and polarized organization are important for proper bristle elongation. Thus, we demonstrate for the first time that coordination between stable and dynamic MT sets that are axially organized yet differently polarized drives cell elongation.

Drosophila javelin-like encodes a novel microtubule-associated protein and is required for mRNA localization during oogenesis.

Asymmetrical localization of mRNA transcripts during Drosophila oogenesis determines the anteroposterior and dorsoventral axes of the Drosophila embryo. Correct localization of these mRNAs requires both microtubule (MT) and actin networks. In this study, we have identified a novel gene, CG43162, that regulates mRNA localization during oogenesis and also affects bristle development. We also showed that the Drosophila gene javelin-like, which was identified based on its bristle phenotype, is an allele of the CG43162 gene. We demonstrated that female mutants for jvl produce ventralized eggs owing to the defects in the localization and translation of gurken mRNA during mid-oogenesis. Mutations in jvl also affect oskar and bicoid mRNA localization. Analysis of cytoskeleton organization in the mutants reveal defects in both MT and actin networks. We showed that Jvl protein colocalizes with MT network in Schneider cells, in mammalian cells and in the Drosophila oocyte. Both in the oocyte and in the bristle cells, the protein localizes to a region where MT minus-ends are enriched. Jvl physically interacts with SpnF and is required for its localization. We found that overexpression of Jvl in the germline affects MT-dependent processes: oocyte growth and oocyte nucleus anchoring. Thus, our results show that we have identified a novel MT-associated protein that affects mRNA localization in the oocyte by regulating MT organization.

The Drosophila javelin gene encodes a novel actin-associated protein required for actin assembly in the bristle.

The Drosophila melanogaster bristle is a highly polarized cell that builds specialized cytoskeletal structures. Whereas actin is required for increasing bristle length, microtubules are essential for bristle axial growth. To identify new proteins involved in cytoskeleton organization during bristle development, we focused on identifying and characterizing the javelin (jv) locus. We found that in a jv mutant, the bristle tip is swollen and abnormal organization of bristle grooves is seen over the entire bristle. Using confocal and electron microscopy, we found that in jv mutant bristles, actin bundles do not form properly due to a loss of actin filaments within the bundle. We show that jv is an allele of the predicted CG32397 gene that encodes a protein with no homologs outside insects. Expression of the Jv protein fused to a green fluorescent protein (GFP) shows that the protein is colocalized with actin bundles in the bristle. Moreover, expression of Jv-GFP within the germ line led to the formation of ectopic actin bundles that surround the nucleus of nurse cells. Thus, we report that Jv is a novel actin-associated protein required for actin assembly during Drosophila bristle development.

The highly elongated Drosophila mechanosensory bristle--a new model for studying polarized microtubule function.

Microtubules (MTs) are polar polymers that can facilitate asymmetric distribution of cell components, a process important for polarized cell growth. The highly elongated and polarized Drosophila mechnosensory bristle cytoplasm is filled with short MTs that constitute a significant component of the shaft cytoplasm. Inhibition of MT assembly affects biased axial growth in the bristle and highlights the importance of MTs for this process. We demonstrate that the vast majority of bristle MTs are organized in a polarized manner, minus-ends out. We also show that genetic disruption of the MT polarity affects the polar distribution of cell components and leads to an alteration in the biased axial shape of the bristle shaft. Thus, we suggest that the asymmetric organization of the MT population within the bristle cell shaft is necessary for the proper axial elongation of this cellular extension. We would also like to emphasize the benefits of using the bristle cell as a model for studying MTs and MT-binding proteins because changes to this cytoskeletal component result in easily recognized at the phenotypes.

Asymmetric microtubule function is an essential requirement for polarized organization of the Drosophila bristle.

While previous studies have shown that microtubules (MTs) are essential for maintaining the highly biased axial growth of the Drosophila bristle, the mechanism for this process has remained vague. We report that the MT minus-end marker, Nod-KHC, accumulates at the bristle tip, suggesting that the MT network in the bristle is organized minus end out. Potential markers for studying the importance of properly polarized MTs to bristle axial growth are Ik2 and Spindle-F (Spn-F), since mutations in spn-F and ik2 affect bristle development. We demonstrate that Spn-F and Ik2 are localized to the bristle tip and that mutations in ik2 and spn-F affect bristle MT and actin organization. Specifically, mutation in ik2 affects polarized bristle MT function. It was previously found that the hook mutant exhibited defects in bristle polarity and that hook is involved in endocytic trafficking. We found that Hook is localized at the bristle tip and that this localization is affected in ik2 mutants, suggesting that the contribution of MTs within the bristle shaft is important for correct endocytic trafficking. Thus, our results show that MTs are organized in a polarized manner within the highly elongated bristle and that this organization is essential for biased bristle axial growth.

The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis.

BACKGROUND: IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2. RESULTS: We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization. CONCLUSION: We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.