Vaccinia-related kinase 1 is required for early uterine development in Caenorhabditis elegans

Protein kinases regulate a multitude of processes by reversible phosphorylation of target molecules. Induction of cell proliferation and differentiation are fundamental to development and rely on tightly controlled kinase activities. Vaccinia-Related Kinases (VRKs) have emerged as a multifunctional family of kinases with essential functions conserved, from nematodes and fruit flies, to humans. VRK substrates include chromatin and transcription factors, whereas deregulation of VRKs is implicated in sterility, cancer and neurological defects. In contrast to previous observations, we describe here that Caenorhabditis elegans VRK-1 is expressed in all cell types, including proliferating and post-mitotic cells. Despite the ubiquitous expression pattern, we find that vrk-1 mutants are particularly impaired in uterine development. Our data show that VRK-1 is required for uterine cell proliferation and differentiation. Moreover, the anchor cell, a specialized uterine cell, fails to fuse with neighboring cells to form the utse syncytium in vrk-1 mutants, thus providing further insight on the role of VRKs in organogenesis.We gratefully acknowledge funding from the Spanish Ministry of Economy and Competitiveness (BFU2013-42709P), the Autonomous Government of Andalusia (P08-CVI-3920) and the European Regional Development Fund to P.A. and a postdoctoral contract from the Autonomous Government of Andalusia to A.D.Peer reviewe

Nuclear organization in a Caenorhabditis elegans model for Nestor-Guillermo Progeria Syndrome

Nestor-Guillermo progeria syndrome (NGPS) is a rare disease causing premature aging. This syndrome has features of classic progerias, although NGPS patients have no sign of cardiovascular impairments like in other progeroid syndromes. In contrast, they suffer profound skeletal abnormalities that affect their quality of life. A missense mutation in the BANF1 gene (c.34G > A [p.Ala12Thr]) causes NGPS in humans. BAF-1 is a member of the barriers-to-autointegration factor protein family and when mutated, results in a protein where the alanine 12 is replaced by a threonine residue causing a disruption in the nuclear envelope structure. Since baf-1 is a highly conserved protein present in all animals, we can use C. elegans as organism to model NGPS(In this nematode, it is a glycine mutated to a threonine residue in the same position). Understanding how the mutated BAF-1 protein works and how it interacts with other proteins from the nuclear envelope and with chromatin is essential to learn more about this syndrome and perhaps aging per se.In order to generate a suitable NGPS model, cloning techniques were used to obtain the Crispr/Cas9 plasmids with the protein BAF-1 mutated (BAF-1 G12T), to later on be able to edit the nematode genome having the endogenous baf-1 locus mutated. Also DamID plasmids were generated in order to characterize the interaction profile between BAF-1 G12T and chromatin. Microinjection was performed to obtain the transgenic strains. Setting up crosses between our new strains and others with fluorescent markers, will allow us to obtain the attempted final strain that will carry BAF-1 G12T::Dam in chromosome II and baf-1 mutated in the endogenous locus.Further studies are necessary, if our model is viable, in order to know more about how BAF-1 G12T behaves in C. elegans. To date, the baf-1 G12T::Dam transgenic strain has been generated and we are working on obtaining the other one

NanoBiT based toolkit to study protein-protein interactions in C.elegans

Motivation: The study of interactions between proteins is very relevant in the investigation of molecular biology and biomedicine. In this project we want to generate a new method for the study of protein-protein interactions (PPIs) based on NanoLuc Binary Technology (NanoBiT) and using as model organism C. elegans.Methods: NanoBiT is a two-subunit system, LgBiT (17,6 kDa) and SmBiT (11 amino acids), and based on luminescence. Each subunit will be fused to a specific protein and when the interaction between both proteins occur the subunits will be linked generating a luminescent signal.We are using cloning techniques to generate plasmids with the constructs we are interested in using Sap-Trap protocols and conventional cloning protocols. These plasmids will be inserted as single copies into the genome of C. elegans. Then, different strains of worms will be crossed to obtain the ones we are looking for. We will use the NanoBiT system to be able to check if there is interaction between our proteins of interest.Results: We have obtained several intermediate products in our cloning scheme. These include transsplicing cassettes expressing fluorescent GFP that will facilitate identification of transgenic animals. We have also obtained codon-optimized fragments that encode the two luciferase components SmBiT and LgBiT.Conclusions: If we have sucess with this project we could use the method for a fast analysis of protein interactions

C. elegans epigenetic regulation in development and aging

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License.The precise developmentalmap of the Caenorhabditis elegans cell lineage, as well as a complete genome sequence and feasibility of genetic manipulation make this nematode species highly attractive to study the role of epigenetics during development. Genetic dissection of phenotypical traits, such as formation of egg-laying organs or starvation- resistant dauer larvae, has illustrated how chromatin modifiers may regulate specific cell-fate decisions and behavioral programs. Moreover, the transparent body of C. elegans facilitates non-invasive microscopy to study tissue-specific accumulation of heterochromatin at the nuclear periphery. We also review here recent findings on how small RNA molecules contribute to epigenetic control of gene expression that can be propagated for several generations and eventually determine longevity.This work was supported by the CNRS [to F.P.]; Fondation ARC [SFI20101201659 to F.P.]; Ligue Contre le Cancer [to F.P.]; the Spanish Ministry of Economy and Competitiveness [BFU2010-15478 to P.A.]; the Autonomous Government of Andalusia [P08-CVI-3920 to P.A.]; and the European Regional Development Fund.Peer Reviewe

Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.This work was supported by an ATIP grant from Centre National de la Recherche Scientifique and Human Frontiers Science Program Grant RGP0034/2010 to M.D. W.N. is the recipient of a PhD fellowship from the French Government.Peer Reviewe

HIV-1 Rev oligomerization is not obligatory in the presence of an extra basic domain

BACKGROUND: The HIV-1 Rev regulatory protein binds as an oligomeric complex to viral RNA mediating nuclear export of incompletely spliced and non-spliced viral mRNAs encoding the viral structural proteins. However, the biological significance of the obligatory complex formation of Rev upon the viral RNA is unclear. RESULTS: The activity of various fusion proteins based on the negative oligomerization-defect Rev mutant M4 was tested using Rev dependent reporter constructs. An artificial M4 mutant dimer and an M4 mutant containing an extra basic domain from the HTLV-I Rex protein exhibited nearly full activity when compared to wild type Rev. CONCLUSION: Rev dimerization appears to be required to expose free basic domains whilst the Rev oligomeric complex remains bound to viral RNA via other basic domains

The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License.Regulation of mitosis in time and space is critical for proper cell division. We conducted an RNA interference–based modifier screen to identify novel regulators of mitosis in Caenorhabditis elegans embryos. Of particular interest, this screen revealed that the Nup205 nucleoporin NPP-3 can negatively modulate the timing of mitotic onset. Furthermore, we discovered that NPP-3 and nucleoporins that are associated with it are lost from the nuclear envelope (NE) in the vicinity of centrosomes at the onset of mitosis. We demonstrate that centrosomes are both necessary and sufficient for NPP-3 local loss, which also requires the activity of the Aurora-A kinase AIR-1. Our findings taken together support a model in which centrosomes and AIR-1 promote timely onset of mitosis by locally removing NPP-3 and associated nucleoporins from the NE.V.H. was supported by a Roche postdoctoral fellowship (Mkl/stm 120-2007) and by an MHV postdoctoral fellowship from the Swiss National Science Foundation (PMPD33-118694). Additional support was provided by the Swiss Cancer League (grant KLS 2160-02-2008 to P.G.). Work in the laboratory of P.A. was funded by the Spanish Ministry of Science and Innovation (BFU2010-15478).Peer reviewe

El papel de emerina, proteína que forma parte de la arquitectura nuclear, en la Distrofia Muscular de Emery Dreifuss en el modelo animal Caenorhabditis elegans

AntecedentesEl proyecto parte del hecho de que mutaciones en emerina, una proteína de la envoltura nuclear, son causantes de la distrofia muscular de Emery-Dreifuss [1,2]. Usando el modelo Caenorhabditis elegans, nuestro grupo ha demostrado que animales que no expresan emerina son hipersensibles a l herbicida Aldicarb [3]. Este fenotipo sugiere que emerina tiene un papel importante en la señalización entre las neuronas y los músculos. Además, hemos observado que emerina es importante para el anclaje de cromatina en la envoltura nuclear y la regulación de expresión génica. El objetivo principal de este proyecto es verificar si la hipersensibilidad a Aldicarb está causado por la deleción de emerina y no por otras mutaciones en el fondo genético. El segundo objetivo es determinar si el efecto de Aldicarb sobre emerina tiene lugar en las neuronas y/o en los músculos. Esta información nos permitiría realizar, en el futuro numerosos estudios acerca de la enfermedad. Métodos - Generación de nuevas estirpes mediante cruces entre el mutante de emerina y estirpes que expresan proteínas fluorescentes fusionadas a emerina. Selección positiva de los genotipos mediante PCR y microscopía de fluorescencia de alta resolución.- Generación de plásmidos por clonación para transgénesis por microinyección en las gónadas de C. elegans.- Ensayos de funcionalidad de las proteínas fluorescentes fusionadas a emerina mediante rescate de letalidad embrionaria y rescate de hipersensibilidad a Aldicarb. Silenciamiento del gen lem-2 causa letalidad en ausencia de emerina, y comprobamos si las proteínas fusionadas pueden sustituir a la emerina endógeno. En los ensayos con Aldicarb analizamos la resistencia al parálisis provocado por la droga.ResultadosHoy día como resultados tenemos las estirpes, planteadas al comienzo del mismo. Con ellas hemos realizado los ensayos de hipersensibilidad a Aldicarb, de donde podemos determinar que las estirpes con el alelo de emerina fusionado a GFP y mCherry son capaces de rescatar el fenotipo del mutante de emerina.Hemos realizado además, algunos ensayos de rescate de letalidad embrionaria cuyos resultados de rescate del fenotipo son similares. Además como verificación tenemos en curso la realización de los ensayos con la droga Levamisole, que nos permite distinguir entre efectos en las neuronas y los músculos.En cuanto a la generación de nuevos plásmidos por clonación para la realización de transgénesis, se consigió uno de los planteados, de modo que esperamos conseguir transgénicos en los próximos meses.ConclusionesConcluimos que el defecto en la señalización entre las neuronas y los músculos con alta probabilidad está causado por la mutación en emerina