In Drosophila, the medulla part of the optic lobes receives direct color and polarized light information from the R7 and R8 photoreceptors as well as indirect motion information from lamina neurons. 40,000 neurons of 70 different types are processing this information. Expression of the transcription factors that drive this complexity is precisely regulated, spatially and temporally. To understand the role of each transcription factor in determining the characteristics of each of these diverse neurons, I will use supervised learning techniques to develop an algorithm able to predict the phenotype of a differentiated neuron given its “transcription factor code”.
I will first describe the phenotype and profile the transcriptome of most neuronal types in the medulla. To increase the depth of my analysis, I will exploit cutting-edge techniques that allow characterization of the expression profiles of single cells. I will thus create an exhaustive map including detailed phenotypic and molecular characters and transcription factor expression of each of these neuronal types. I will use this map as input for a machine-learning algorithm. I will test the model both in vivo and ex vivo by expressing transcription factor combinations in naïve neuronal precursors and by comparing the phenotype of their progeny with the prediction of the model.
I will use this model to determine transcription factor regulatory networks, study them in Drosophila and in other animal systems to understand the evolution of visual processing centers. The model will also help me program the differentiation of desired neurons from neuronal precursors, contributing on the long term, to regenerative medicine.

Colorectal cancer (CRC) develops during a multi-step process from small lesions of the intestinal epithelium. While genetic mutations in the canonical Wnt signalling pathway are considered to be the initial step of cancer formation, the interaction of the tumour with its microenvironment plays a crucial role in tumour progression. Tumour-reactive immune cells (ICs) such as cytotoxic T lymphocytes (CTLs) are part of the tumour microenvironment and have been shown to be associated with better prognosis. However, cancer cells can escape IC-mediated elimination for example by preventing CTL activation through stimulation of inhibitory receptors on T cells. This process of cancer immunoediting is still poorly understood in many cancer types such as CRC. I therefore aim to study the interaction between cancer cells and tumour-infiltrating ICs to understand how IC anti-tumour responses are repressed. To identify the subtypes of tumour-reactive ICs found in human CRC tissue, I will first perform immunophenotyping. Using different functional assays, I will then assess whether IC heterogeneity is reflected by differential immunocompetency. Subsequently, I am to develop a co-culture system of CRC organoids and tumour-reactive ICs to investigate their interaction under near native conditions, since suitable model systems to study this process in vitro do not exist. Exploiting this new method, I will eventually test different strategies modulating IC activity to reinstate anti-tumour responses against the CRC cells. My work will provide an invaluable tool to investigate IC anti-tumour responses and will give new insights into the role of IC heterogeneity in tumour biology.

Structural characterization of biomacromolecules is essential for understanding cellular processes, investigation of disease mechanisms, and drug development. There is a great demand for characterization methods that can provide low-cost and high-throughput structural information. I propose to develop DNA PUPPETEERED CALIPERS (DPC), which is a biophysical tool for measuring long-range distance restraints of biomacromolecules. DPC employs microbeads that are tethered to a surface via single DNA strands; these strands act as nanoscopic calipers that bind to specific sites on the target macromolecules. A novel REFLECTANCE INTERFERENCE CONTRAST CENTRIFUGAL FORCE MICROSCOPE (RIC-CFM) will enable measurement of a very large number of calipers simultaneously, each with near-angstrom precision. Another key innovation is RECONFIGURABLE LOOPING - a technique that will be used to reversibly (de)activate specific parts of the caliper, which is essential for accurate calibration of the device. This approach does not require large analyte concentrations or elaborate sample preparation. Such a highly sensitive, precise, and massively parallel detection method is unprecedented, and will, for the first time, allow rapid, inexpensive, and high-throughput determination of biomacromolecular structural features. Moreover, DPC will enable the study of molecules under alternating mechanical forces or changing molecular environments (e.g., for the study of protein folding/unfolding/misfolding; or for the detection of allosteric binding events). I anticipate that DPC will become a powerful tool that can revolutionize research in structural biology, bionanotechnology, and biomedicine.

Gene expression is precisely controlled in time and space during the development of metazoan organisms. While numerous studies have established how spatial information is integrated by gene regulatory regions known as enhancers, the mechanisms enabling temporal coordination of gene expression across a field of cells remain vastly unknown. Using Drosophila melanogaster as a model system, my general goal is to understand the developmental consequences of subtle alterations in the timing of gene activation. For example, I have recently shown that pausing of RNA polymerase II at promoters sets a transcriptional synchrony that is required for successful gastrulation. With this project, I propose to discover additional mechanisms involved in the regulation of timing by introducing targeted mutations into various core promoter elements and precisely quantifying the resulting expression temporal dynamics in vivo with single cell sensitivity. We also propose to characterize the mechanisms of transcription memory, whereby transcription activation is inherited through multiple divisions.

Using insights acquired from study of these mechanisms, we will address the important question of the functional role of temporal coordination of gene expression for cell fate specification during development.

The second aim will investigate the existence of, and requirements for, temporal coordination in gene expression in a complex but stereotyped morphogenetic process, cardiogenesis.

Major upstream cardiogenic factors will be examined to document and alter their transcriptional temporal dynamics and address their potential priming through transcription memory.

The discovery of intron-derived RNAs and cytoplasmic intron-retaining transcripts (CIRTs) hints that intronic RNAs, previously regarded as “junk”, play an important role in cellular processes. In order to unravel the dynamics of intron-derived RNA synthesis, transport and function in vivo a novel RNA visualization tool is needed. This is due to the fact that current RNA imaging methods, based either on the fusion of a fluorescent protein (FP) to an RNA-binding protein (RBP) or on fluorescence hybridization, are laborious and limited due to the low signal-to-noise ratio, the need to re-design RNA-binding proteins for each new target RNA, and the inability to detect endogenous RNAs. Thus I propose to develop an innovative, highly sensitive and easy-to-use RNA imaging tool by utilizing the powerful prokaryotic adaptive immunity system CRISPR/Cas and the most modern fluorescent labeling techniques. I will then use the resulting CRIPSR/Cas based RNA visualization tool to investigate CIRTs. To date, neither the mechanisms by which CIRTs are synthesized and processed, nor their functions are known. Thus I will take on a multi-disciplinary approach: apply state-of-the-art CRISPR/Cas RNA imaging and combine it with gene editing technologies and the most advanced tools in biochemistry and structural biology to investigate CIRTs. Hence this interdisciplinary project will set a new milestone towards the development of an easy-to-use endogenous RNA imaging tool in vivo and provide important insights into the localization, interaction, structure, and function of CIRTs by correlating the live cell imaging and structural and functional studies.

Protein organization in cells is classically studied at disparate scales, either through their association in complexes, or by their localization in compartments. We propose to investigate a scale in between, corresponding to micron-scale protein assemblies. While several such assemblies have been characterized recently, as illustrated by P-bodies or protein gels, basic principles related to their formation in vivo remain unknown. Here we propose to design a minimal system that consists of a pair of proteins with the potential to form such large assemblies in vivo. Using this minimal self-assembling system as a model, we will dissect the relationship between its molecular properties (e.g., geometry of the interacting proteins or affinity between them) and its macroscopic properties (e.g., size, regularity, or kinetics of assembly). Aim 1 will be the design of self-assembling protein pairs. We will use homomers as basic building blocks, because the interaction between homomers can result in the formation of infinite and regular structures. We have in fact validated one such design in vivo, and we will expand the range of designs to cover a spectrum of molecular properties. Aim 2 will consist in dissecting the kinetics of self-assembly and disassembly by confocal fluorescence microscopy, enabled by fusing the basic building blocks of the minimal system to fluorescent proteins. Finally, in Aim 3 we will seek to obtain structural information of the assemblies formed by in vivo STEM-tomography. Akin to the first synthetic gene networks, this approach might help single out design principles of otherwise highly complex biological processes.

OCD is a disorder defined by intrusive recurrent thoughts and repetitive compulsive actions. Recent studies in two OCD mouse models have yield contradictory results [6,7], most likely because these studies perturbed the entire corticostriatal circuitry instead of specific cell types. It is therefore important to understand the neural circuits mediating OCD with an appreciation for the heterogenetity of the corticostriatal circuitry. Striatum neurons are composed of anatomically and functionally heterogeneous subpopulations with different cortical inputs. I hypothesize that inhibitory or excitatory signals for certain OCD behaviors are transmitted by different components of the corticostriatal circuitry. In my proposed work, I first plan to dissect the circuitry coding of specific OCD behaviors using different mouse transgenic lines and optogenetic approaches. Cre-dependent, light-activated opsins (ChR2 or NpHR) will be delivered to several GENSAT BAC-Cre driver lines with expression in different cell populations of the corticostriatal pathway. Optogenetic manipulation of these specific corticostriatal pathways will be used to uncover the causation of OCD behaviors. Furthermore, synaptic plasticity of striatal neurons will be examined in genetically identified corticostriatal pathways. Finally, taking advantage of TRAP translational profiling method, I seek to find the molecular mechanism of OCD in different striatum and corticostriatal cell types. For this purpose, I will profile the identified cell types in the two existing mouse models of OCD before and after drug treatment and identify key molecules contributing to OCD behaviors and their amelioration.

The spontaneous generation of living organisms without descents from similar organisms was decisively put to rest with the experiments of Pasteur during the 19th century. As a consequence, the logic of self-reproduction, one of the most fundamental features of biological systems, is very difficult to break down. An approach to this problem consists of synthesizing cell analogs in the laboratory from its basic natural molecular components.
The chemical synthesis of living entities is a standing challenge not achieved yet. Various approaches have been taken to construct synthetic reduced cells in the laboratory, a goal that became experimentally imaginable over the past two decades. The construction of protocells, which explores scenarios of the origin of life, has been the original motivation. With the emergence of the synthetic biology era, bottom-up engineering approaches to synthetic cells are now conceivable. The modular design emerges as the most robust method to construct a minimal cell from natural molecular components. Although significant advances have been made for each piece making this complex puzzle, the integration of the three fundamental parts, information-metabolism-self-organization, into cell-sized liposomes capable of sustained reproduction has failed so far. Our inability to connect these three elements is also a major limitation. New experimental platforms and methods should be taken to accelerate the cell-free synthesis of complex biochemical systems.
Our scientific collaboration, composed of four team members, will address the problem of biological self-reproduction by using an engineering versus evolution approach to cell analogs. While “information-less” protocells will be generated by driving Miller-Urey experiments, a prototype of minimal cell will be constructed by integrating the three indispensable molecular parts making living cells: information, metabolism and self-organization (compartment). Unique experimental platforms developed by the team members will be used to achieve major advances in the synthesis of minimal living systems in the theoretical context pioneered by von Neumann in the 50s. The societal impact of our work includes a deeper comprehension of the emergence of self-reproduction with and without genetic information and the development of powerful platforms for the construction of living entities in vitro.

The development of the retina occurs via the production of over 60 cell types from a pool of multipotent retinal progenitor cells (RPCs). The photoreceptors and horizontal cells (HCs) are part of this complex circuitry, and they are first cells to establish synaptic connection during retina formation. Their connections with bipolar cells form the fundamental unit of the center-surround organization, which is shared among sensory systems. In the chick embryo, HCs come in three distinctive subtypes: H1, H2 and H3. Recent study showed that retinal progenitor cells were producing restricted subsets of horizontal cells and cones only. How specific RPCs make any specific type of neuron is currently not understood. We plan to use these recent observations concerning the genesis of cones and HCs subtypes as an entry point to this question. We propose to investigate the mechanisms of the development and specification of cones and the HC subtypes, made by specific types of RPCs, those that make a pair of H1s, a pair of H3s, or a cone and an H2 cell. To address these questions, we will use expression profiling, electroporation and viral infection in the chick along with morphological and immunohistochemical studies. Specification of each type of HC, which are interneurons, may shed light on the process of diversification of other types of interneurons, which are the most diverse category of neurons in the CNS. In addition, the fact that the HC likely arose early in evolution, may predict that its specification mechanisms were the forerunner of many other types of specification mechanisms.

While many one-to-one plant-microbe interactions have been studied in detail in lab experiments, nature exposes plants to large communities of microbes that are also interacting amongst themselves. Many of these microbes evade or tolerate the plant immune system and colonize leaves, posing a risk to the plant. Because maintaining an immune system is costly, successful plants streamline their defense capabilities. In Arabidopsis thaliana, many putative pathogen resistance genes, as well as pattern recognition receptors that recognize microbial molecules, are uncharacterized. These immune gene families are extremely variable, with great genetic variation maintained even within single field sites. The functional consequences of these immune gene repertoires in wild plants are essentially unknown. Characterization of the plant microbiome using marker genes like 16S rDNA has helped to address these concerns, but most single locus studies bias against the identification of entire domains of microbes and lack the sensitivity to distinguish closely-related pathogens and mutualists. This research explores the evolutionary genetics of plant-microbe interactions in A.thaliana in a wild setting. I will leverage high-throughput sequencing and classification methods to genotype and quantify both eukaryotic and prokaryotic microbes on a sub-species scale, and associate them with plant loci. I will survey wild plants, sow experimental crosses, and sow plants mutated for specific immune genes identified from this work and the literature. These experiments will allow me to address how host genetic diversity affects microbial associations and fitness in nature.

Linking circuits to behavior in mammals is a major challenge of neuroscience. In the mouse, we know which visual circuits control the pupil size or the optokinetic head control, two simple reflexes that require little visual processing. Here, we propose to dissect the visual pathway underlying a more complex innate behavior: the avoidance of looming objects. Mice rapidly escape or freeze when a shadow approaches overhead, which is essential to survive predators. How the visual system can extract such visual feature and trigger motor actions in milliseconds is not established. However, we do know that a type of ganglion cell, named the PV-5, responds specifically to looming stimuli. We think that this retinal channel might mediate the behavior. Given the rapid processing required, we hypothesize that this "looming-sensitive" retinal signal is processed via dedicated small brain nuclei from the accessory optical system, and does not recruit the main pathway going to the visual cortex involved in image formation.
We propose to test this hypothesis with an innovative experimental approach. We will use whole brain ultrasound imaging, a technique that I developed, on a head-fixed mouse to detect all regions specifically activated by looming stimuli. Using the same technique on behaving mice, we will discriminate which of these regions are specifically involved in escape or freezing. Using transynaptic retrograde tracing, we will then identify which ganglion cells project to these nuclei and verify if they are indeed PV-5 cells. Finally using optogenetics, we will specifically activate the PV-5 cells to see on which brain regions it acts and how it affects the mouse behavior.

We propose to apply cell-free synthetic biology to study fundamental aspects of transcriptional regulation, gene regulatory network evolution, and network robustness. Cell-free synthetic biology emerged recently as an in vitro alternative for network engineering. Cell-free synthetic biology implements biological systems in a coupled transcription – translation reaction and therefore is a well-defined environment that is easy to control and interrogate. Since genetic networks can be implemented in vitro as linear dsDNA templates, as opposed to plasmids, it also circumvents time consuming cloning and transformation steps, enabling rapid prototyping of genetic systems. We have recently implemented cell-free systems on a microfluidic microchemostat array, which performs transcription-translation reactions at steady state for 30+hours, allowing us to implement complex genetic networks in vitro. Here we propose to apply this novel concept to study fundamental principles of genetic networks. We will interrogate two different types of oscillators (phase and relaxation) and determine how cis- and trans-changes to the network impact network function and robustness. Specifically, we plan to map the precise cis-binding energy landscape of these networks by characterizing a large number of network variants with modified transcription factor binding sites. To implement trans-element changes we will employ synthetic Zn-finger transcriptional regulators with defined specificity landscapes to determine how changes to the trans-element influence network performance and robustness. Finally, we will also attempt to rationally evolve network function through a promiscuous transcription factor intermediate. To determine how robust these networks are to external perturbations, we will change reaction temperatures, and independently modulate transcription and translation rates. Finally, we will characterize a subset of our networks on the single cell level in vivo to evaluate network robustness in a regime were stochasticity dominates. Gene regulatory networks are complex systems that are difficult to quantitatively characterize in vivo. The in vitro cell-free synthetic biology approach proposed here has the potential to contribute significantly to the mechanistic understanding of complex biological systems.

Callyspongiolide, a unique polyketide-derived macrolide extracted from a Callyspongia species of marine sponge, has recently been shown to possess remarkable antiproliferative properties against cancer cells. Although various cytotoxic substances have been isolated from sponges belonging to the Callyspongia genus, this represents the first macrolide obtained from such species. As it belongs to the family of macrolides, its cytotoxicity could be related to antimitotic properties; however the actual mechanism of action is still unknown. This project aims to determine the mechanism of action of this compound and to develop the first enantioselective total synthesis of Callyspongiolide. A 23-step synthetic pathway has been proposed, starting from low cost and easily accessible starting materials, which should provide a sufficient supply of material to be employed for biological assessment of this new and potentially powerful marine natural product. The synthetic pathway has been designed to allow control over the molecule’s substituents and over the configuration of all chiral stereocenters facilitating analog synthesis.

Aging is one of the most prominent biological processes whose molecular and cellular mechanisms are poorly understood. Several studies demonstrate that aging is a progressive process involving a gradual dynamics of the genome, transcriptome and epigenome, and defects in organelle homeostasis. The current stem cell hypothesis of aging proposes that living organisms are as old as their tissue-specific adult stem cells. The adult mammalian brain contains pools of adult neural stem cells (aNSCs) that are crucial for learning and memory. The pool size and the functionality of aNSCs decrease with age. Although adult neurogenesis plays a central role during aging, the global aged-induced molecular dynamics and key factors that regulate aNSC aging are largely unknown. Here, I propose an integrative research approach that aims at answering basic biological questions regarding aging regulation in mammalians brain. My proposed study includes (1) performing a systematic, genome-wide profiling of aNSCs during brain aging, (2) identifying novel key players that regulate mammalian brain aging in vivo, and (3) modeling human aging using a 3D culture that is derived from human embryonic stem cells. This work will provide considerable information about molecular landscapes during aging of aNSCs, and will explore several key factors that regulate brain aging in mice and humans. Overall, the findings of this research will help to broaden our understanding of the mechanisms that promote or prevent cellular aging, and hopefully, will further provide potential implications for regenerative medicine to help rejuvenate brain cells.

Reprogramming of endogenous glia into iNs by developmentally inspired transcription factors has been recently achieved in vivo. However, little is known about their physiological properties and recruitment into functional neuronal circuits, especially within the context of brain damage. The host lab has recently showed that injury-induced reactive NG2 glia can convert into immature iNs following retrovirus-mediated expression of Sox2 within the adult cerebral cortex. The deficiency in the acquisition of a mature neuronal phenotype may generate functional deficits. Therefore, I will promote the maturation of iNs by combining forced expression of Sox2 with strategies to enhance neuronal maturation: curtailing Sox2 expression, increasing mTOR signaling and pharmacologic ligand-mediated depolarization. I will also study the electrophysiological properties of NG2-derived iNs and compare them to those of native NG2 cells. This is important because NG2 glia even prior to reprogramming receive synaptic inputs from neurons and induced cells may directly inherit some of these connections. I will perform patch-clamp recordings in brain slices from NG2 glia and NG2-derived iNs to reveal changes in excitability, synaptic inputs, and synaptic plasticity following reprogramming. Finally, I will analyze the functional integration of the reprogrammed cells using in vivo 2-photon Ca2+ imaging. I will investigate the recruitment of iNs into functional neuronal circuits in the somatosensory cortex in response to whisker pad stimulation. The proposed study will unveil fundamental data regarding the capacity of NG2 glia-derived iNs to functionally integrate into severely damaged brain tissue.

The nuclear pore complex (NPC) is an essential nuclear transport channel which is functionally maintained in the rat brain through adulthood without being renewed. The aim of this project is to reveal the mechanism by which long-lived cells maintain the scaffold nucleoporins (Nups), essential components for NPC structural integrity, over the lifespan of the animal. Preliminary results suggest that long-lived cells (neurons) may modulate the expression level and phosphorylation status of membrane Nup Pom121 to make scaffold Nups more stably associated with the nuclear envelope, compared to short-lived cells (hepatocytes). To validate this, in Aim1, I will first deplete Pom121 from neurons and examine the effects on the scaffold stability and NPC functions (transport, permeability barrier) using advanced microscopy techniques such as super-resolution microscopy. This will take advantage of a neuron long-term culture system which will allow me to perform experiments on the order of months. Second, using rat brain and liver tissues, I will identify the brain-specific phosphorylation sites of Pom121 by mass spectrometry and examine the role in NPC maintenance by mutating identified sites.
Besides stabilization of the scaffold in the NE, neurons may prevent damage accumulation to scaffold Nups by modulating the expression and phosphorylation of peripheral Nups, which attach to the scaffold and thus may protect scaffold Nups from toxic metabolites. In Aim2, I will investigate the role of peripheral Nup50 in neuronal NPC maintenance as described in Aim1.
In summary, this proposal will provide novel insight into how cells keep their long-lived proteome healthy during aging.

The genetics of Amyotrophic Lateral Sclerosis (ALS) have been intensively explored in the last two decades, identifying multiple disease-related mutations that underlie it. A paradigm shift in understanding ALS pathology has come from discovery of ALS-causing mutations in several genes encoding proteins with fundamental roles in RNA processing pathways. However, the scope, function and significance of RNA metabolism in affected neurons remains elusive, primarily because of the impossibility of examination of the affected neurons throughout disease course. Here, I propose to uncover the extent of aberrant RNA metabolism and its potential contribution to toxicity in ALS pathogenesis using neurons directly “trans-differentiated” from familial-ALS patient’s fibroblasts. I will use a new genome editing approach (CRISPR) to correct ALS-causing mutations and apply genome-wide sequencing technologies on mutated and corrected neurons to determine whether such neurons display a specific profile of altered RNAs. These data, including RNA transcription, splicing and editing, will allow the construction of high-resolution, disease-dependent molecular signatures that underlie the molecular and cellular basis of ALS progression. By exploiting this set of RNA alterations as multi-functional readouts, my final approach will be to perturb ALS-neurons using ~4500 available compounds with known mechanisms of action, thus discovering molecular pathways that reverse an initial RNA signature. Combining the complementary strategies outlined here, this proposal aims to discover and characterize previously unknown mechanisms through which RNA processing abnormalities provoke ALS pathogenesis

iRhoms are members of the rhomboid-like superfamily without any protease activity. In different contexts they either promote ER associated degradation (ERAD) of transmembrane client proteins, or their forward trafficking. To understand more about the molecular mechanisms of these recently discovered regulators, I will screen for proteins that interact with Drosophila iRhom using a new biotin tagging approach called BioID. This technique will systematically identify iRhom interactors in the Drosophila nervous system (where iRhom is highly enriched) . This technique once developed in flies, will be the first of its kind at an organismal level wherein all interactors (including ones which are transient) as well as the constituents of the intracellular neighbourhood of any protein of interest can be characterised. Using this approach, I expect to identify three classes of protein: iRhom clients, regulators, and components of the core ERAD and trafficking machinery. My primary focus will be on ERAD, as this is known to be iRhom’s central function in flies. I hope to understand why iRhom is largely restricted to the nervous system and, specifically, whether the neurons have specific quality control requirements. These interactions will also be explored in mammalian cell lines to understand whether they are conserved through evolution, as well as to address the question of how iRhoms act in different cellular processes. Finally, I will use genetics and a drug based approach in flies to look for a role of iRhom in various neurodegenerative diseases originating from ER stress.

Cellulosomes are protein networks used by anaerobic bacteria to digest cellulosic biomass. The protein networks consist of subunits that are held together by receptor-ligand pairs, some of which have similar affinities to the same binding sites. Intuitively, this structural property of cellulosomal subunits should give rise to a large number of cellulosomal compositions. In nature, however, cellulosomes are found to exhibit only a limited number of preferred compositions. It remains unclear how cells modulate the assembly of cellulosomes. Studies of cellulosomes are hampered by a lack of tools to manipulate genetic components of native cellulosome-producing bacteria. Furthermore, to resolve these issues, studies of cellulosome assembly require nanoscale measurement methods. Our team proposes integrating synthetic biology and single-molecule imaging for the study of cellulosome assembly.
Our long term goal is to integrate synthetic biology and single-molecule methods to understand control mechanisms of cellular dynamics. Based on our combined expertise in synthetic genetic circuits and cellulosomal interactions, we aim to address the hypothesis that architecture of genetic modules can regulate the timing of cellulosomal-subunit expression, thereby modulating the resultant cellulosome compositions. We will test this hypothesis by constructing synthetic genetic modules using cellulosomal subunits from Acetivibrio cellulolyticus and Clostridium thermocellum. We will also use stochastic models to predict the impact of genetic modules on cellulosome assembly. We will then test model predictions at the single-molecule level using cell-free systems and yeast in custom microfluidic devices. Our approach is innovative because it combines synthetic biology and single-molecule imaging to study cellulosome assembly. Our results will establish a foundation toward studying system dynamics of native cellulosome-producing bacteria. Furthermore, our results will unravel general principles of how genetic networks modulate the assembly of biological structures.

The objective of the proposed study is to investigate the molecular mechanisms underlying the regulation of assembly and disassembly axonemal tubulins in the unicellular alga Chlamydomonas reinhardtii. This study we will extend the knowledge on microtubules dynamics in non-mammalian sources. Also, it will provide molecular insight on the flagellar length regulation mechanism. In order to achieve these goals three specific aims were formulated: (i) To characterize the axonemal tubulin properties, including their polymerization and depolymerization processes (i.e. rate and frequency) and to study the effect of post-translational modifications; (ii) To explore the influence and mechanism of three potential microtubule-associated-proteins (CrKin13, CrEB1, FAP256) on the regulation of Chlamydomonas microtubules dynamics; (iii) To develop FRET-TIRF assay to monitor the microtubules dynamic and to improve the resolution of the process by decreasing the fluorescence background. More specifically, I will purify Chlamydomonas axonemal tubulin by using an innovative affinity column that was developed in the host-lab to facilitate the purification of native untagged tubulin from non-mammalian sources. Subsequently, I will investigate the dynamic properties of the axonemal tubulin and which factors influence the process, such as microtubule-associated-proteins or post-translational modifications, by using state-of-the art fluorescence microscopy and other molecular techniques. Also, I will examine the possibility to modify tubulin units with pairs of fluorophores to perform FRET in order to gain a better microscopic insight on microtubule dynamics.