The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology.

In the past years it has become evident that stochastic effects in regulatory networks play an important role, leading to an increasing in stochastic modelling attempts. In contrast, metabolic networks involving large numbers of molecules are most often modelled deterministically. Going towards the integration of different model systems, gen-regulatory networks become part of a larger model system including signalling pathways and metabolic networks. Thus, the question arises of how to efficiently and accurately simulation such coupled or hybrid systems. We present an algorithmic approach for the simulation of hybrid stochastic and deterministic reaction models that allows for adaptive step-size integration of the deterministic equations while at the same time accurately tracing the stochastic reaction events. We present a mathematical derivation of the hybrid system on the stochastic process level, and present numerical examples that outline the power of hybrid simulations.Résumé. Au cours des dernières années, il est devenu clair que les effets aléatoires jouaient un rôle important dans les réseaux de régulation, et les modèles employés aujourd'hui pour décrire ces réseaux sont de nature stochastique. En revanche, les réseaux métaboliques, qui mettent en jeu un grand nombre de molécules, sont le plus souvent décrits par des modèles déterministes. Dans la modélisation de systèmes complexes, réseaux régulateurs de gènes, chemins de signaux et réseaux métaboliques sont intégrés dans un même modèle. Se pose alors la question de simuler efficacement et avec précision de tels modèles couplés (on parle aussi de modèles hybrides). Nous présentons ici une approche pour la simulation de modèles de réactions hybrides stochastiques/déterministes permettantà la fois d'avoir recoursà des pas de temps adaptatifs dans l'intégration deséquations déterministes et de simuler précisément les réactions décrites par des processus stochastiques. Des simulations numériques illustrent la puissance de ces simulations hybrides.

The massive acquisition of data in molecular and cellular biology has led to the renaissance of an old topic: simulations of biological systems. Simulations, increasingly paired with experiments, are being successfully and routinely used by computational biologists to understand and predict the quantitative behaviour of complex systems, and to drive new experiments. Nevertheless, many experimentalists still consider simulations an esoteric discipline only for initiates. Suspicion towards simulations should dissipate as the limitations and advantages of their application are better appreciated, opening the door to their permanent adoption in everyday research.

The existence and nature of an active chromosome segregation apparatus in bacteria has been a long-standing debate. A novel Brownian ratchet-type mechanism of chromosome segregation mediated by the Min system is identified in E. coli.

SmartCell has been developed to be a general framework for modelling and simulation of diffusion-reaction networks in a whole-cell context. It supports localisation and diffusion by using a mesoscopic stochastic reaction model. The SmartCell package can handle any cell geometry, considers different cell compartments, allows localisation of species, supports DNA transcription and translation, membrane diffusion and multistep reactions, as well as cell growth. Moreover, different temporal and spatial constraints can be applied to the model. A GUI interface that facilitates model making is also available. In this work we discuss limitations and advantages arising from the approach used in SmartCell and determine the impact of localisation on the behaviour of simple well-defined networks, previously analysed with differential equations. Our results show that this factor might play an important role in the response of networks and cannot be neglected in cell simulations.

Recent technological and theoretical advances are only now allowing the simulation of detailed kinetic models of biological systems that reflect the stochastic movement and reactivity of individual molecules within cellular compartments. The behavior of many systems could not be properly understood without this level of resolution, opening up new perspectives of using computer simulations to accelerate biological research. We review the modeling methodology applied to stochastic spatial models, also to the attention of non-expert potential users. Modeling choices, current limitations and perspectives of improvement of current general-purpose modeling/simulation platforms for biological systems are discussed.

All the people I worked with gave me incredible support during these years, technical and emotional. From the first day, with my shirt decorated with bananas, to the lasts, when they had to bear my nervous and stressed mood. I thank Anna, Linda and Pierre, for the funny moments, the laughs and the many beers together. And for the precious scientific discussions, of course! Thank you also to Eethan, the living scientific encyclopedia, with (almost) always the right answer to my problems in the lab. Thank you, Enoch, for the discussions, especially those in the microscopy room. Emir, thank you for your help with the cloning, microscopy and the bacteriographs. I like your enthusiasm toward science, do not lose it! Thank you, Mehmet, for the support to the protein function prediction of my project, I appreciated the point of view from someone with a "dry biology" background and the comments on my thesis. Jeremy, Mirko, Giada, and Jara, thank you for the daily chats and jokes together. The daily routine was lighter with you around. Thank you, Dagmar, oh Dagmar, for your patience and your smiles. Lastly, thanks to the grumpy Daniel, my dear deskmate and benchmate, fellow of complains.Vorrei anche ringraziare i miei amici rimasti in Italia. Anche se ci vediamo poco, quando ci troviamo è sempre una festa. Sono fortunato a non averli persi dopo più di 5 anni passati all'estero: è anche grazie a loro che ho mantenuto la tenacia per arrivare fino alla fine. In particolare, Claudio, Stefano e Vito, grazie per la vostra vicinanza nonostante la lontananza. Grazie anche a Laura, la mia supervisor durante il mio tirocinio di tesi magistrale in Unimi, che mi ha insegnato come pensare e come muovermi nell'ambito di un progetto di ricerca: quello che ho imparato in quell'anno mi è stato molto utile durante il dottorato.Un grazie immenso alla mia famiglia e al nonno, senza i quali non sarei potuto arrivare dove sono ora, incoraggiandomi e sostenendomi sempre. E infine, Margherita, grazie per essere sempre stata al mio fianco in questi anni, anche quando non avevo occhi che per la tesi, risollevandomi dai momenti di sconforto e spazzando via i cattivi pensieri. ContributionsLeonard Ernst, a former bachelor student of the Di Ventura lab, cloned a 2-plasmid-based library composed of 5 VVD-AraC fusion proteins under IPTG induction and performed the first explorative experiments to demonstrate the light-responsiveness of the system. Emir Bora Akmeriç, technical assistant in our laboratory, helped me during the last year of my doctorate in the cloning process of some constructs and with the induction and imaging of the genes with poor or unknown function. Moreover, he took care of the Blade Runner bacteriograph, repeating it until he made it flawless.Mehmet Ali Öztürk, postdoc in our laboratory, contributed with structural biology analysis of the I1-I2 sequences rearrangements and collected, interpreted and catalogued the function and localization prediction outputs of the genes with poor or unknown function. He also provided valuable sug...