Sequential action of Rab proteins along the endocytic-recycling pathway

The endocytic pathway in eukaryotic cells is organized into distinct compartments. Early endosomes are heterogeneous and dynamic organelles, which are found both spread through the cortical cytoplasm (sorting-early endosomes) and in the perinuclear region (recycling endosomes). Endocytosed molecules that enter sorting endosomes can be directed to the degradative pathway or recycled back to the plasma membrane, either directly or by passing through perinuclear-recycling endosomes. Very little is known about the molecular mechanisms that regulate transport between these different compartments and the functional properties of endocytic organelles. In this thesis, I have investigated the molecular regulation of the endocytic-recycling pathway and, in particular, how endocytosis and recycling are co-ordinated at the level of early endosomes. To this end, I have addressed the function of Rab5, Rab4, and Rabll, three small GTPases that control trafficking from the plasma membrane to the early endosomes, sorting of cargo within early endosomes and recycling to the plasma membrane, respectively. Rab proteins function in their GTP-bound active state through the recruitment of effector proteins to the membrane compartment where they are localized. The identification of a large number of Rab5 effectors, together with the realization that these proteins act co-operatively, led to the proposal that this GTPase organizes the endosomal membrane into a biochemically and functionally distinct membrane domain. If generalized to other family members, this model would predict that other Rab proteins present on endosomes such as Rab4 and Rabll should be localized to discrete, non-overlapping membrane domains distinct from the one occupied by Rab5. Moreover, the finding that one Rab5 effector, Rahaptin-5, interacts also with Rah4 raises the possibility that the activity of these two GTPases is molecularly co-ordinated. Given the complementary role of Rab5 and Rab4 in regulating entry in, and exit out, of early endosomes the coupling of their activity through a common effector may ensure co-ordination of the endocytic and recycling functions of early endosomes. I have experimentally tested the working hypothesis predicting that divalent Rab effectors may regulate the association between contiguous Rab domains to allow the sequential transport of cargo along the endocytic-recycling pathway. To provide evidence for this model, two complementary approaches have been undertaken. First, I have developed an in vitro recycling assay to identify and characterize molecules involved in this process. Second, I have conducted a high-resolution morphological analysis of the endocytic recycling pathway in stable cell lines expressing GFP-tagged versions of Rab proteins. The results obtained support the idea that endosomes are indeed organized into distinct domains, harbouring Rab5, Rab4 and Rabll. The association between these domains follows a non-random distribution giving rise to three major populations of endosomes: one containing Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rabll. Upon endocytosis, recycling cargo first enters Rab5 domains, and then progresses through Rab5/Rab4 and Rab4/Rabll endosomes before returning to the plasma membrane. Based on these observations, I then addressed the question of how the communication between neighbouring domains is regulated by specifically searching for Rab5 and Rab4 common effectors using an affinity chromatography approach. The results of these experiments led me to identify Rahenosyn-5 as a novel Rab5 and Rab4 common effector. Subsequently, I demonstrated that Rabenosyn-5, as well as Rabaptin-5 over-expression specifically increased the association between Rab5 and Rab4 endosomal domains and stimulated transferrin recycling. Concomitantly, the fraction of Rah4+Rabll positive structures was reduced and transport to peri-nuclear recycling endosomes decreased. Thus, divalent Rab5 and Rab4 effectors regulate endocytosis and recycling by connecting Rab5 and Rab4 domains on early endosomes. These results provide support for the hypothesis that Rab proteins and their effectors regulate the compartmental organization and trafficking function of early endosomes

An Optogenetic Method to Modulate Cell Contractility during Tissue Morphogenesis

SummaryMorphogenesis of multicellular organisms is driven by localized cell shape changes. How, and to what extent, changes in behavior in single cells or groups of cells influence neighboring cells and large-scale tissue remodeling remains an open question. Indeed, our understanding of multicellular dynamics is limited by the lack of methods allowing the modulation of cell behavior with high spatiotemporal precision. Here, we developed an optogenetic approach to achieve local modulation of cell contractility and used it to control morphogenetic movements during Drosophila embryogenesis. We show that local inhibition of apical constriction is sufficient to cause a global arrest of mesoderm invagination. By varying the spatial pattern of inhibition during invagination, we further demonstrate that coordinated contractile behavior responds to local tissue geometrical constraints. Together, these results show the efficacy of this optogenetic approach to dissect the interplay between cell-cell interaction, force transmission, and tissue geometry during complex morphogenetic processes

Blockchain and Cryptocurrencies: a Classification and Comparison of Architecture Drivers

Blockchain is a decentralized transaction and data management solution, the technological leap behind the success of Bitcoin and other cryptocurrencies. As the variety of existing blockchains and distributed ledgers continues to increase, adopters should focus on selecting the solution that best fits their needs and the requirements of their decentralized applications, rather than developing yet another blockchain from scratch. In this paper we present a conceptual framework to aid software architects, developers, and decision makers to adopt the right blockchain technology. The framework exposes the interrelation between technological decisions and architectural features, capturing the knowledge from existing academic literature, industrial products, technical forums/blogs, and experts' feedback. We empirically show the applicability of our framework by dissecting the platforms behind Bitcoin and other top 10 cryptocurrencies, aided by a focus group with researchers and industry practitioners. Then, we leverage the framework together with key notions of the Architectural Tradeoff Analysis Method (ATAM) to analyze four real-world blockchain case studies from industry and academia. Results shown that applying our framework leads to a deeper understanding of the architectural tradeoffs, allowing to assess technologies more objectively and select the one that best fit developers needs, ultimately cutting costs, reducing time-to-market and accelerating return on investment.Comment: Accepted for publication at journal Concurrency and Computation: Practice and Experience. Special Issue on distributed large scale applications and environment

Interaction of the Phosphotyrosine Interaction/Phosphotyrosine Binding-related Domains of Fe65 with Wild-type and Mutant Alzheimer's β-Amyloid Precursor Proteins

The two tandem phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domains of the Fe65 protein interact with the intracellular region of the Alzheimer's beta-amyloid precursor protein (APP). This interaction, previously demonstrated in vitro and in the yeast two hybrid system, also takes place in vivo in mammalian cells, as demonstrated here by anti-Fe65 co-immunoprecipitation experiments. This interaction differs from that occurring between other PID/PTB domain-containing proteins, such as Shc and insulin receptor substrate 1, and activated growth factor receptors as follows: (i) the Fe65-APP interaction is phosphorylation-independent; (ii) the region of the APP intracellular domain involved in the binding is larger than that of the growth factor receptor necessary for the formation of the complex with Shc; and (iii) despite a significant similarity the carboxyl-terminal regions of PID/PTB of Fe65 and of Shc are not functionally interchangeable in terms of binding cognate ligands. A role for Fe65 in the pathogenesis of familial Alzheimer's disease is suggested by the finding that mutant APP, responsible for some cases of familial Alzheimer's disease, shows an altered in vivo interaction with Fe65

Unmasking Activation of the Zygotic Genome Using Chromosomal Deletions in the Drosophila Embryo

During the maternal-to-zygotic transition, a developing embryo integrates post-transcriptional regulation of maternal mRNAs with transcriptional activation of its own genome. By combining chromosomal ablation in Drosophila with microarray analysis, we characterized the basis of this integration. We show that the expression profile for at least one third of zygotically active genes is coupled to the concomitant degradation of the corresponding maternal mRNAs. The embryo uses transcription and degradation to generate localized patterns of expression, and zygotic transcription to degrade distinct classes of maternal transcripts. Although degradation does not appear to involve a simple regulatory code, the activation of the zygotic genome starts from intronless genes sharing a common cis-element. This cis-element interacts with a single protein, the Bicoid stability factor, and acts as a potent enhancer capable of timing the activity of an exogenous transactivator. We propose that this regulatory mode links morphogen gradients with temporal regulation during the maternal-to-zygotic transition

Desensitisation of Notch signalling through dynamic adaptation in the nucleus

During embryonic development, signalling pathways orchestrate organogenesis by controlling tissue‐specific gene expression programmes and differentiation. Although the molecular components of many common developmental signalling systems are known, our current understanding of how signalling inputs are translated into gene expression outputs in real‐time is limited. Here we employ optogenetics to control the activation of Notch signalling during Drosophila embryogenesis with minute accuracy and follow target gene expression by quantitative live imaging. Light‐induced nuclear translocation of the Notch Intracellular Domain (NICD) causes a rapid activation of target mRNA expression. However, target gene transcription gradually decays over time despite continuous photo‐activation and nuclear NICD accumulation, indicating dynamic adaptation to the signalling input. Using mathematical modelling and molecular perturbations, we show that this adaptive transcriptional response fits to known motifs capable of generating near‐perfect adaptation and can be best explained by state‐dependent inactivation at the target cis‐regulatory region. Taken together, our results reveal dynamic nuclear adaptation as a novel mechanism controlling Notch signalling output during tissue differentiation