The annelid worm Platynereis dumerilii (Lophotrochozoa) exhibits ancestral developmental, body plan and genomic characteristics and possesses two types of eyes: adult pigment cup eyes and larval two-celled eyes. Platynereis therefore represents a useful model organism for the study of eye evolution in annelids. My research goal has been to characterize the differentiating Platynereis adult and larval eyes on the molecular level in order to explore the evolutionary history of these two types of eyes and their cell types: rhabdomeric photoreceptor cells (rPRCs) and pigment cells (PCs). This aim has been addressed by using the ‘molecular fingerprint’ (MFP) approach for the comparative study of cell types. I first identified specific molecular markers for each of the cell types in both types of eyes. These were then used to establish a comprehensive MFP of these cell types that included both effectors genes (differentiation genes expressed in eye and neuronal cell types) and transcription factors which play a role in eye and neuronal specification. This was achieved by means of gene expression studies, using wholemount double in-situ hybridization and 3D in-silico alignments. The data obtained reveal that Platynereis adult and larval eyes are composed of six cell types, based on MFP comparisons: adult eyes ventral and dorsal rPRCs, adult eyes ventral and dorsal PCs, larval eye rPRCs and larval eye pigment cells. The distribution of the adult rPRCs and PCs into two (ventral and dorsal) cell types relates to the fact that Platynereis develops two pairs of adult eyes that appear to differ in terms of their molecular regulation. It also revealed that many transcription factors regulating eye development in Drosophila and/or vertebrates are also expressed in the differentiating Platynereis eyes. Surprisingly, some of these are adult eye-specific and some are larval eyespecific, meaning that the adult and larval eyes of Platynereis show a distinct MFP, corroborating that they represent different types of eyes. On the other hand, some shared effector genes were identified between the rPRCs and PCs of the adult eyes, as well as of the larval eyes. This finding implicates that the rPRCs and PCs of Platynereis are sister cell types that can be traced back to a single ancestral multifunctional cell type precursor. Hierarchical clustering analysis based on the MFP results mirrors the ‘phylogeny’ of the different eyes cell types, in which the larval eyes cell types cluster together as do the two types of adult eyes rPRCs and PCs. In order to gain more insight into the developmental regulation of both eyes in Platynereis, I chose to assess the role of the conserved hedgehog (Hh) signaling pathway in Platynereis eye development. By using the antagonist cyclopamine to inhibit the hedgehog pathway in Platynereis embryos, I found out that Platynereis Hh pathway plays a role in adult but not in larval eye development. This adds another key distinction between the adult and larval eyes of Platynereis. These results support the view that annelid eyes originated from one multifunctional single cell prototype eye that bore characteristics of both PRCs and PCs. It was first duplicated to give rise to adult and larval eye precursors to then diversify into the PRCs and PCs present in today’s annelid eyes.
Determination of cell shape is an essential mechanism for cells like neurons, epithelial and fungal. In general, these cells are able to control their shape by positioning mechanisms that regulate cell growth and cell polarity. One key element for such a process is the microtubule cytoskeleton, which is organized into higher order assemblies in polarized cells. Therefore, an important question is to understand how these assemblies are established and maintained during interphase. We addressed this question by making use of mutants of the fission yeast Schizosaccharomyces pombe to study the organization of Interphase Microtubule Assemblies in high ultra-structural detail, using a new emerging technology – electron tomography. Mto1p, Ase1p and Klp2p are key Microtubule Associated Proteins required for the organization of microtubules into interphase microtubule arrays (IMAs). We reconstructed high resolution 3D volumes of mto1, ase1 and klp2 deletion strains (and all double mutants) in order to study the formation and organization of IMAs. We show that all mutants lacking ase1 maintain microtubule overlap regions but with altered inter-microtubule spacing. In addition, in ase1Δ klp2Δ cells the microtubules appear to have lost their connection to the spindle pole body. Interphase microtubule arrays in klp2Δ cells are mostly composed of only two microtubules instead of 2 to 9 in wild type. A similar phenotype is found in cells lacking Mto1p protein and cells lacking Mto1p and Klp2p proteins. Cells lacking Ase1p and Mto1p proteins form an interphase microtubule arrays composed of three microtubules, which extend the whole cell length. Both mto1Δ and mto1Δ ase1Δ cells present intra-nuclear microtubules but with a different organization. Finally, we show that impaired interphase microtubule arrays affect the mitochondria network structure in these deletion strains. The electron tomography analysis of the Ase1p deletion strains suggest the existence of other putative proteins involved in the microtubule bundling process. We show that most microtubules ends in mto1Δ cells have open end structures allowing microtubules to show treadmilling behavior. Furthermore, cells lacking Mto1p and/or Klp2 show IMAs with only two MTs, suggesting a role of Klp2p in microtubule nucleation. We propose a mechanistic model to tentatively explain the formation of stable IMAs. Finally we discussed the effects of defective IMAs at the cellular level by showing how the mitochondria network is affected in the deletion mutants compared with wild type cells.
Cells in a multicellular organism need to monitor their environment for nutritional cues, growth and stress signals in order to adapt their metabolism and growth to the changing conditions. The Target of Rapamycin (TOR) signaling pathway is an evolutionary conserved cellular protein network that controls responses to these signals. TOR signaling is a major research topic because of its role in several prevalent human disorders, including cancer and diabetes, but our understanding of TOR pathway is still far from complete. This work is aimed at improving our understanding of TOR signaling in two ways: by developing new research tools to dissect the dynamics of TOR signaling in cells and by characterizing the function of a novel component in the TOR pathway. The first chapter presents the project to develop a probe, based on the Fluorescence Resonance Energy Transfer (FRET) method, for measuring TOR activity in cell culture. I used several approaches to design a FRET probe, based on a substrate of TOR kinase called 4EBP1. These probes did not prove to be useful because fusions to fluorophore domains abolished biological regulation of 4EBP1. An alternative strategy whereby a small tag was introduced into 4EBP1 and then used for in vivo labeling, could solve this problem. Unfortunately, when this probe was expressed in the cells, its interaction with a corresponding partner did not produce a reliable FRET signal, as determined by three different methods. Nevertheless, the obtained results can be used as a basis for future attempts to develop probes for TOR kinase. The second chapter describes the genetic analysis of the MAP4K3 function in Drosophila. MAP4K3 is a new component of the TOR pathway proposed to mediate nutrient sensing by TOR. I identified a strong hypomorphic MAP4K3 mutant and investigated the phenotypes caused by absence of this protein. MAP4K3 mutant flies were viable but weak. Mutant animals demonstrated delayed growth, reduced cell and organ size. Furthermore, they were lean, displaying reduced fat, which could be rescued by genetically increasing TOR activity. This suggests that the observed metabolic defect is due to low TOR activity. The mechanism of MAP4K3 action may involve the MAP kinase pathway and/or modifying activity of Rag GTPases, which can interact with MAP4K3 in cell culture. These results establish MAP4K3 as a regulator of metabolism and growth and open up new possibilities for manipulating TOR signaling.
Post-transcriptional regulation of gene expression relies on multiple mechanisms to elicit translational control and/or mRNA decay. RNA silencing pathways operate at both stages, targeting a significant fraction of the transcriptome, namely those mRNAs complementary to short interfering RNAs (siRNAs) and microRNAs (miRNAs). Using Drosophila S2 cells, I have carried out a genome-wide search for transcripts regulated by these pathways. mRNA expression profiles were obtained for cells depleted of AGO1, AGO2, PIWI or Aubergine, members of the Argonaute family of proteins essential for RNA silencing, and analyzed alongside profiles from cells depleted of the miRNA-processing enzyme Drosha. Changes in transcript levels in Drosha-depleted cells correlated closely with those in the AGO1 knockdown, demonstrating that miRNA targets change level following inhibition of the miRNA pathway and supporting the idea that miRNAs can cause degradation of the targeted transcripts, and do not just repress translation as previously thought. Furthermore, it was found that a subset of miRNA targets is also regulated by AGO2; together with evidence that AGO1 and AGO2 silence the expression of a common set of mobile genetic elements, this suggests a degree of functional overlap for AGO1 and AGO2 in the Drosophila RNA silencing pathway. I next focused on the Drosophila protein Belle, a member of the conserved family of DEAD-box RNA helicases. Most members of this protein family exhibit NTPase activity stimulated by or dependent on RNA binding and use the energy derived from NTP hydrolysis to unwind double-stranded RNA or disrupt RNA/protein interactions. Many DEAD-box proteins localize to RNA granules such as maternal and neuronal transport mRNPs, polar granules, and P bodies. Belle is a component of nuage within nurse cells and polar granules within the oocyte. It is an essential protein required for larval growth, as well as male and female fertility, and has a putative role in RNA silencing. I show that Belle is required for cell viability. In cells depleted of Belle, general protein synthesis is inhibited. However, a luciferase mRNA reporter with Belle tethered to the 3′ UTR is translationally repressed without any reduction in mRNA levels. Tethering of Belle to the reporter mRNA induces the formation of heavy mRNP complexes. Translational repression is abolished when Belle contains mutations disrupting the putative helicase activity. Using a biochemical and computational approaches, I found that in Drosophila S2 cells, Belle is part of an interaction network consisting of proteins implicated in general protein synthesis as well as selective translational control mechanisms. Belle interacts with translation initiation factors and ribosomal proteins, supporting the idea that Belle is required for general translation efficiency. However it also associates with translationally regulated mRNAs and proteins involved in mRNA translational control and localization, suggesting that Belle may be implicated in mRNP assembly and transport as well as localized mRNA translation. Finally, I show that Belle interacts with AGO2 and other components of the RISC, suggesting that Belle may function as an auxiliary factor in the RNA silencing pathway.
Neurofibromatosis type I (NF1) is an inherited neurocutaneous disorder with a high incidence of 1 in 3500 newborns. Clinical manifestations include pigment anomalies, Lisch nodules and the formation of different tumors like neurofibroma. NF1 is caused by alterations of the NF1 gene, encoding the Ras specific GTPase activating protein Neurofibromin, which participates in several major signaling pathways. A structural proteomics approach recently led to the discovery of an unpredicted pleckstrin homology (PH)- and a Sec14-like domain. In this thesis I have investigated the biochemical properties of the NF1-SecPH module. NF1-SecPH can bind glycerophospholipids with a preference for phosphatidylethanolamine and -glycerol (PtdEtn, -Gro), of which PtdEtn is abundant in Neurofibromin containing cells and thus a likely physiological ligand. It was furthermore possible to crystallize NF1-SecPH in complex with glycerophospholipids which is the first structure of a CRAL Trio domain bound to such ligands and shows that PtdEtn binds to the interior of the Nf1-Sec portion. Lipid exchange experiments revealed that PtdEtn and PtdGro are readily exchanged, but phosphatidylcholine, -serine and -inositol (PtdCho, -Ser, -Ins) are only incorporated to a minor degree. The lipid exchange activity can be modulated by soluble headgroups of phosphorylated PtdIns derivatives (PIPs), which is consistent with a regulatory interaction between Nf1-Sec and NF1-PH. While some patient derived mutants show significant structural alterations compared to the cellular NF1-SecPH module, their properties with respect to lipid content and PIP binding are only affected slightly. Localization studies in the presence and absence of stimuli did not reveal a specific compartment association compared to other PH domain containing proteins. Taken together, PtdEtn is probably a physiological ligand of NF1- SecPH, which seems able to incorporate membrane derived lipids in a regulated fashion.
Peptide-based fluorescent reporters of enzyme activity in living cells have been proposed to complement, or provide alternatives to, genetically-encoded probes, such as those based on the Green Fluorescent Protein and its colourful variants. The use of well-established chemical methodologies – especially solid phase synthesis – and the vast array of fluorescent dyes available allow the preparation and the evaluation of compounds with different specificities, making a wide range of investigations possible. However, cell uptake of these compounds is generally poor, and normally requires invasive methods such as microinjection or electroporation. In these conditions, the usefulness of these probes can be severely limited. The discovery of the so-called cell-penetrating peptides (CPPs), more than ten years ago, has actually revealed that some of these compounds can cross efficiently the cell membranes and are able to deliver hydrophilic cargoes, such as DNA, proteins or even nanoparticles inside the cell. Unfortunately, much of the initial enthusiasm dissipated when it was found that endocytosis plays a major role in the uptake process. The entrapment in endosomal vesicles may in fact impose strict limits in terms of modification and degradation of the cargo of interest. This thesis describes the synthesis of fluorescent peptides and focuses on the problem of their delivery into the cytoplasm of living cells. A negatively charged peptide corresponding to one of the autophosphorylation sequences of the epithelial grow factor receptor (EGFR), namely DADEY992L, was taken as an example to investigate the possibility of creating a cell-permeable fluorescent sensor for the EGFR kinase activity in living cells. The use of a conjugate of the cell-penetrating peptide Penetratin and the probe revealed an endosomal localization in one cell type, and surprisingly no uptake in a second cell line tested. Different fluorescent derivatives of Penetratin demonstrated variations in cell uptake under different experimental conditions. During this study, red fluorescent derivatives of the cell-penetrating TAT peptide were also assessed. Although they also showed predominantly accumulation in membrane-bound compartments, these appeared to be different from the endosomal vesicles. One TAT variant in particular, namely TAT(K-LRh), may be potentially exploited as a novel vector, provided that further studies will clarify unambiguously the compartment of its accumulation. To overcome the limits inherent in the use of Penetratin conjugates, a different strategy was implemented, which relied on the chemical modification of the probe with bioactivatable protecting groups. The rationale behind these modifications was to increase the hydrophobic character of the peptide and allow its diffusion into the cells. In particular, acetoxymethyl (AM) esters were used to temporarily mask the negative charges of the aspartate and glutamate residues present in the peptide sequence. These biodegradable esters are promptly removed by ubiquitous non-specific esterases present in cells. The modified fluorescent peptide was able to penetrate easily and distribute homogeneously into the cytoplasm and the nucleus of several cell lines, with a mechanism resembling passive diffusion. Unfortunately, FRET-based assays did not measure any interaction with the EGFR. In vitro analyses showed unmasking of the carboxylate groups, albeit with modifications to the peptide backbone, which may have resulted from intramolecular condensations and formation of aspartimide residues. In particular, mass spectrometry data support this model. A loss of substrate specificity may be expected to result from such modifications, which may explain why the fluorescent peptide did not appear to interact with its enzyme target despite intracellular delivery having been achieved. This work highlights the feasibility of a tailored modification of peptides, either by masking negatively charged groups as, for example, AM esters, or by conjugating to variant of the TAT peptide, for the intracellular delivery of peptidic cargoes. Further refinements may solve the problems of poor endosomal escape or backbone modifications, and may yet offer small molecule alternatives to genetically encoded probes.