Next to Bacteria and Eukarya, Archaea represent the third domain in the tree of life. Cells from the eukaryotic domain use a cell division machinery, in which actin and myosin play a central role for constriction, while most bacterial and archaeal species use a mechanism based on the tubulin-homologue FtsZ. Some members of a lineage within the archaeal domain, however lack both, an actin/myosin as well as FtsZ-based system. Instead, these species use a protein system closely related to the eukaryotic ESCRT (endosomal sorting complex required for transport) apparatus to perform cytokinesis.
In the proposed project, I will reconstitute archaeal cell division using purified proteins of the ESCRT system from Sulfolobus acidocaldarius and biomimetic membranes prepared from purified archaeal lipids. Using this reconstituted system and advanced fluorescence microscopy, I will be able to get insight into the molecular processes required for archaeal cell division, which will also facilitate our understanding of the mechanistic details of eukaryotic ESCRT mediated vesicle formation and viral budding. Furthermore, these results will shed light on the evolution of binary fission and the relationship between the different domains in the tree of life.

Ubiquitylation of target proteins has been recognized as an eminent regulator of diverse biological processes including apoptosis, the inflammation response, and selective autophagy. The TNF signaling pathway is a signal transduction cascades that, upon specific stimulation of cognate receptors, can stimulate the expression of pro-survival and pro-inflammation factors. Paradoxically, the same stimulus can also lead to caspase-dependent apoptosis. Thus, intricate systems of networks function to switch between different patterns of responses. Failure to properly regulate these networks can lead to sustained inflammation, auto-immunity or aberrant cell death. Autophagy has recently emerged as an ubiquitin-dependent catabolic process that can degrade proteins non-specifically by engulfing cytosolic material during starvation conditions. Selective autophagy, on the other hand, can specifically degrade large protein aggregates, organelles and bacteria. Analogous to proteasomal receptors that bring ubiquitylated proteins to the proteasome for degradation, autophagy receptors recognize ubiquitylated cargo and deliver them to the lysosome for degradation. In this study, I propose to investigate ABIN-1, a protein that functions as a pro-survival factor in NF-kappaB signaling, as a novel autophagy receptor that may function to degrade death-inducing complexes to enable the inhibition of apoptosis.

This project assembles an international team of biologists, mathematicians and engineers to uncover the secrets of the world’s most spectacular animal-built structure: the mound built by the southern African termite Macrotermes. These spectacular structures, long known as the famous “air-conditioned termite mound”, has many new secrets to reveal. The mound does not appear to “air condition” the nest, but is in fact a remarkable organ of extended physiology, essentially the lung for the underground colony. Among the secrets revealed are a novel mechanism for capturing turbulent wind energy to do useful work, and building behavior that is far more sophisticated and complicated than ever realized.
This project seeks to push these new findings into new scientific frontiers, including the physics of using turbulent wind, the largely unexplored biology of the termite brain, and the nature of the collective cognition of the termite colony. From this, we hope to learn new methods for analyzing the adaptation of organisms to their environment, the process of building coherent organs of physiology, new ways to program robot swarms, and perhaps novel ways to exploit wind in climate control of our own buildings.

Cryo-electron tomography is an advanced technique for exploring the structure of cells and sub-cellular compartments at the molecular level in life-like conditions. Here it will be applied to a first in situ ultrastructural analysis of centrosomes in C. elegans embryos. This project is undertaken in a collaborative effort between the groups of Prof. Baumeister at the MPIB in Martinsried and Prof. Hyman at the MPI-CBG in Dresden. State of the art cryo-electron microscopy will be combined with focused ion-beam milling, a unique preparation technique which has only recently been adapted for thinning of biological frozen samples. Correlative cryo-fluorescence microscopy will be essential to localize the centrosome within the large C. elegans single-cell embryos. Cryo-electron tomography on these preparations will provide a high resolution 3-dimensional view of centrosome supramolecular organization. Single particle analysis of expressed and purified structural components of the yet poorly characterized pericentriolar matrix will complement the imaging approaches. Advanced optical microscopy, including super-resolution fluorescence microscopy, will be used to explore localization of functional centrosomal domains and to gain insight into the physico-chemical behaviour of centrosomes as non-membrane bound organelles. This project will provide unprecedented structural data on functional centrosomes and the pericentriolar matrix in particular, in situ and at the nano-scale. The employed interdisciplinary approach represents cutting edge research in both the fields of electron tomography and of basic cell biology.

Modifications in gene regulation play a central role in generating phenotypic diversity. Changes in the timing and conditions under which genes are expressed, and not only modifications in their composition, lead to new phenotypes. Extensive differences in the way genes are regulated have been observed even between closely related species. However, little is known about the intermediary steps that have led to such regulatory diversity and whether these steps are adaptive. The aim of my project is to explore the changes in gene regulation that underlie the diversity in biofilm formation, and thus pathogenesis, of Candida species. The latter as a proxi to understand general principles of regulatory evolution. Biofilm formation in Candida species is an ideal evolutionary model since these species span a wide evolutionary range and show gradual phenotypic differences in pathogenicity. Genome-wide approaches will be used to identify and characterize the regulatory circuits that are responsible for biofilm formation in each species. Interspecific comparison of the transcription factors, their binding affinities and target genes will reveal the evolutionary changes that occurred in each linage. Finally, phenotypic profiling of the regulatory changes will be used to evaluate their adaptive value. Overall the project will shed light on the evolutionary pathways that have led to the diversity observed in gene regulation and on the relative importance of natural selection in these pathways.

The nervous and vascular systems of vertebrates display stereotypical organization of their networks, which are often closely aligned. Abnormalities in these precisely patterned networks lead to a variety of tissue dysfunction and disease. Thus, understanding how these two systems establish their highly patterned networks is important. The central aim of this proposal is to investigate whether and how these two systems cooperate to establish their networks during development and regeneration, a question that remains largely unclear. Here I propose a set of experiments that exploit many attributes of the zebrafish system to define the roles of neurovascular interplay in development and regeneration. First, I will simultaneously monitor the dynamic elongation of blood vessels and different types of nerves during development and regeneration using in vivo time-lapse imaging. Second, I will compare these dynamic behaviors in wild-type and mutant zebrafish, focusing on mutants that lack blood vessels or specific types of nerves. Finally, I will analyze these mutant animals to examine the consequences of the loss of each cell type in the development and regeneration of the other network.

Circadian rhythms are essential for many aspects of human physiology including sleep/wake times, body temperature, hormone secretion, metabolism, glucose homeostasis, and cell cycle progression. The canonical mammalian clock is based on transcriptional activators and repressors intertwined in interlocking feedback loops. Recent work has shown a different type of biological rhythm occurs in mammalian red blood cells, which lack a nucleus and many core clock genes. Specifically, the oxidation state of an antioxidant protein peroxiredoxin oscillates over a 24-hour period. These oscillations do not depend on transcription and are conserved in an ancient eukaryotic alga, which suggests a fundamental role for non-transcriptional oscillators in cells.

During my post-doctoral research, I plan to measure these oscillations using redox-sensitive GFP as a real-time reporter. This will allow me to directly compare how redox oscillations compare to transcription of classical clock genes. By employing RNAi, genetic, and chemical perturbation strategies, I aim to discover components involved in this new oxidation clock and probe how oxidation is coupled to canonical clock components. Furthermore, because red blood cells are an ideal lysate system, I plan to biochemically identify and purify components of the oxidation clock with the ultimate goal of reconstituting an oxidation cycle in vitro.

Circadian rhythms are fundamental to many physiological and behavior processes in wide variety of organisms, so it of interest to understand the mechanisms of this novel oxidation clock and how oxidation couples to classical clock components.

Cancer stem cells (CSCs), like normal somatic stem cells, are defined as cells that can self-renew and produce the progeny. Therefore, if CSC population is survived even after optimal therapy, patients will have relapses. That is why CSCs are considered to be a good target for novel cancer therapies. However, as shown in many reports about CSCs, the important factors such as frequency or specific markers are not consistent. And the mechanisms to cause variation remain obscure. To understand the mechanisms of variation seen in CSCs, it is more reasonable to identify the cells of origin of CSCs and analyze when or how this variation is induced than to isolate cancer stem cells from individual tumors and analyze their variation.
In this study, I will focus on epithelial ovarian cancers (EOCs) which are most common subtype in ovarian cancers and thought to be derived from ovarian surface epithelium (OSE). Morphological and pathological diversity in EOCs is a notable characteristic. Although there is increasing evidence that EOCs contain CSC population like other types of cancer, ovarian CSCs are also heterogeneous and inconsistent. To address this issue, I will reveal a lineage hierarchy within normal OSE and what cells in the hierarchy are responsible for tumor formation showing similar morphology to human ovarian cancers by using a model mouse of human ovarian cancer. Furthermore, to figure out the morphological diversity, I will compare the gene expression profile with the other epithelium in the female reproductive tissues which share the common origin of OSE and identify the responsible genes to give rise to the diversity and heterogeneity in morphology and pathology.

I am interested in exploring the mechanism by which the DNA damaging function of AID is naturally controlled and whether regulation of this mutagenic process can be achieved at the protein level instead of gene manipulation. Currently, no work has been conducted in aiming at directed regulation of AID’s mutagenic potential. In the first part of my work, I would like to investigate the contribution of AID’s self-interacting ability in its functional regulation. I plan to analyze the interaction dynamics in living cells so that I can evaluate the impact of an interaction directly in certain events, such as CSR, SHM or DNA damage condition. The second part of my work aims to isolate synthetic RNA ligand(s) that can bind to AID or its specific domain with high affinity, followed by detailed analysis of the characteristic features and cellular counterpart prediction. I will then examine the most promising candidates for their potential to interfere with the function of AID, including their ability to disrupt AID-AID self-interaction. Successful outcome of this work will open up new dimension in AID research field and will give rise to pioneering work in designing future regulatable molecular device for RNA/DNA editing enzymes.

The emergent morphological properties of metazoan tissues result from the interactions of individual cells, which are animated by intracellular molecular events. A common design in epithelial organs is that of interconnected networks made of a series of tubes. The combination of molecular genetics and live imaging techniques has led to descriptions of the cellular processes involved and the molecular players required for tube morphogenesis (a developmental program also known as branching morphogenesis). The aim of my project is to understand how these processes interact and self-organize to give rise to the ordered structure of branched organs. I will use an innovative approach to develop a predictive model of a paradigmatic example of branching morphogenesis: the HGF induced branching of MDCK cells cysts. To study operatively the program induced by HGF and to follow the information transfer from a cell to the neighbors, I will activate single cells of the cysts by using laser-switchable protein-protein interaction domains that allow the activation of signaling molecules with fine spatial resolution. Thanks to these new kind of perturbative experiments, I will improve current computational models with the intracellular layer to generate multiscale models. Then, I will use the model to build synthetic circuits that will be able to simulate autonomously the endogenous program of morphogenesis; this will reveal the relevance of the components of the program. In sum, this project will use a combination of perturbative, computational, and synthetic approaches with the aim of a predictive understanding of the branching morphogenesis developmental program.

For decades, conscious events and brain states were considered fundamentally different (albeit related): while the former are subjectively experienced, the latter can only be later inferred from external data (e.g., fMRI, EEG). Yet recent neuroscientific developments open new possibilities of making brain states accessible for human subjects, by using a unique online system that intracranially records neural activity and presents it in real time as sensory feedback. To examine the effect of such feedback on subjects' behavior I will use Binocular rivalry, in which subjects' perception alternates between two stimuli presented simultaneously but separately to each eye. Subjects will be asked to focus on one of the images (i.e., try to exert volitional control over rivalry). I hypothesize that sensory feedback about the otherwise unconscious neural activity correlated with the processing of either the dominant or the suppressed image (both typically change towards an alternation), will enhance subjects' ability to delay the alternation. This promises to yield new insights about the widely debated role of consciousness in voluntary control. In addition, I will hold a preparatory experiment to investigate the relations between the content of consciousness and Local Field Potentials (LFPs) evoked by neurons in the Medial Temporal Lobe (MTL). As opposed to spiking activity in the MTL that accompanies conscious percepts only, LFPs might be sensitive also to suppressed images. Such finding will open new venues of research, enabling the acquisition of intracranial information during unconscious processing.

A 14-year old high school student once asked us “Why are microtubules hollow?”. We could have elaborated at length on the central roles played by microtubules in segregating the genetic material and in building tracks for long-range motor movement. Truth to be told, none of us could provide the beginning of a cogent answer to this tremendously fundamental question. In fact, the notion that important events take place inside the lumen of microtubules seemed entirely without basis. To this day, the only known molecular event that takes place inside the microtubule lumen is acetylation of alpha-tubulin on lysine 40, a major post-translational modification whose functional consequences remain elusive. While a correlation between tubulin acetylation and microtubule stability is well-accepted, the causal relationship between stability and acetylation is still being debated and a function for microtubule acetylation remain speculative. In this context, our recent identification of the tubulin acetyltransferase TAT1 provides a much-needed molecular handle on tubulin acetylation and on the biology of the microtubule lumen.
We will now combine our expertise in microtubule biochemistry (Nachury) and in nano-engineering (Théry) to test the interplay between acetylation and the mechanical behavior of microtubules. The proposed research is highly collaborative with most experiments involving reagents from one team and techniques from another team. An important theoretical component will provide an overarching model of microtubule mechanics and a vigorous back-and-forth between modeling and experimental work will ensure that we arrive at an accurate physical understanding of how acetylation influences microtubule mechanics and vice-versa. In the end, we hope to uncover novel molecular paradigms for how events taking place inside the microtubule lumen influence microtubule biology.

Plant cell proliferation and morphogenesis rely on successful and correctly oriented cell division. Eukaryotic cell division is carried out by microtubule-based mechanisms. In plant cells several distinct arrays of microtubules are created and function over the course of the cell cycle: the most obvious are the interphase cortical array, preprophase band, spindle and phragmoplast. All of these arrays are created without benefit of central microtubule organizing body such as a centrosome. As a cell progresses through the cell cycle, microtubule structures are gradually replaced or rearranged from array to the next, but we have little knowledge of the mechanisms that carry out these transitions in microtubule array organization.
Recent studies indicate that gamma tubulin complexes and katanin are required for plant cell division and localized at plant cell spindle poles in a late anaphase. While concentration of nucleation and severing complexes at the spindle poles is expected, the specific functions of these proteins in spindle formation, phragmoplast organization and the reorganization/transition of mitotic arrays are largely unexplored.
We hypothesize the physical or functional interaction between microtubule nucleation factors and microtubule severing factors are of particularly importance for mitotic microtubule array reorganization. The subject of this research is to investigate the spatiotemporal function and relationship of microtubule nucleation and severing during mitotic cells. Specifically, I will apply a new cell biological technique to understand spatiotemporal function of proteins, focusing on microtubule severing function and mechanisms during mitosis.

RNA polymerases are very large enzymes with 0.5-0.7 MDa, which is consisting of 12-17 subunits. These subunit conformational changes enable the interaction with many additional factors, thereby transcribing different classes of genes. Precise regulation mechanism of transcription by multi-subunit RNA polymerases assembly is crucial to all organisms. However, much less is known about the biosynthesis of these enzymes themselves and the nuclear transport pathway remains elusive. This proposal focuses on Iwr1, which is one of a few examples, known to be involved in Pol nuclear import mechanism. The first aim of this proposal is the structure determination of the free form of Iwr1 and Pol II-Iwr1 complex, using several structural method including X-ray crystallography and MD simulation. Such structures would reveal the conformational change of Iwr1 and the interaction surface of Iwr1 on Pol II. In addition, to verify this observation, structure-based mutational analysis of this complex will be carried out. Confocal microscopy of Pol II localization and quantitative immuno-blotting of each Pol II subunits in ?IWR1 strain will be used to obtain functional insight of Iwr1 in vivo. The second aim of this proposal addresses identification and structural characterization of other nuclear transport factors for Pol I and Pol III. To find potential candidates, I will start with a bioinformatics search of NLS containing proteins from the protein interactome data of Pol I and III. To detect the cytoplasmic accumulation of Pol I and Pol III caused by siRNA-mediated depletion of candidate protein, I will identify new nuclear transport factors.

In bacteria, gene expression is subject to large fluctuations among single cells (noise), even when genetically equal cells are exposed to identical conditions. Stochastic processes underpin a variety of bacterial behaviors suggesting that bacteria can exploit noise to transition between alternative phenotypes. Conversely, other regulatory networks are known to be extraordinarily refractory to noise, enabling synchronized, coordinated population-wide behaviors. I will study how such robust gene expression is achieved in regulatory networks by investigating quorum sensing (QS), a process of bacterial cell-to-cell communication. To date, investigations of QS have focused on steady-state measurements of single transcriptional reporters. Importantly, QS is known to affect gene expression globally, demanding rapid, genome-wide profiling techniques to simultaneously track the time-dependent expression of all affected genes. I will define the complete set of genes controlled by QS, in what order each gene turns on or off, and which QS regulators determine each particular expression pattern. I will also identify which QS-controlled genes exhibit noise and which show robust regulation, and define the key regulatory components that confer these features. I will perform these analyses in both individual cells and in populations. These data will be used to develop theoretical models to predict mechanisms allowing QS-signal integration and network performance. Identifying the components that allow synchronized population-wide performance in bacteria will be crucial for understanding the evolution of multi-cellularity in higher organisms, where coordination among cells is vital.

Decision making is the process resulting in the selection of a course of action among several alternatives. Decisions that are mainly based on the sensory evidence (perceptual decisions) constitute a valid model for studying how and where in the brain this process is accomplished. The neuronal correlates of perceptual decisions have been studied mostly by recording the activity of single cells from different cortical areas while primates perform perceptual tasks. It has been found that the trial-to-trial variability of neuronal activity is correlated with behavioral choices and this finding has been interpreted as evidence of a causal effect of sensory neurons on choice. However, recent findings challenge this interpretation and suggest that the direction of causality could be the opposite. This has fundamental implications for neuronal coding: if correlated random fluctuations in the sensory periphery bias behavioral choices, then noise in neural activity significantly constraints information processing; if correlated fluctuations in sensory areas reflect cognitive operation unknown to the experimenter, then they need not generate any constraint on information processing. To clarify this issue, we propose to record the activity of large neuronal populations simultaneously in the auditory cortex and the medial prefrontal cortex of rats performing auditory discriminations. By recording the activity of large neuronal populations, we will be able to quantify the time-course of decision-related activity in single trials on each area. This will allow us to perform the critical comparison to determine causality: where does decision-related activity appear first?

The last two stages in eukaryotic translation, termination and ribosome recycling, are very distinct from the corresponding, well-characterized stages in bacteria. Thus, eukaryotic termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition and peptidyl-tRNA hydrolysis by eRF1, whereas eukaryotic ribosome recycling is mediated by ABCE1, a unique highly conserved member of the ATP cassette family of proteins that contains an iron-sulfur cluster domain, and which does not exist in bacteria. Moreover, in contrast to bacteria, eukaryotic termination and ribosome recycling are intimately linked because eRF1 plays a key role in both processes. Although these two stages of eukaryotic protein synthesis are of major importance in regulation of eukaryotic translation, and are linked to downstream events such as reinitiation and mRNA surveillance, their mechanisms have only recently begun to attract attention. However, future progress in elucidating the molecular mechanism of eukaryotic termination and ribosome recycling is strongly compromised by the lack of structural data. The primary aim of this proposal is therefore to obtain a comprehensive overview of the architecture of defined intermediate states in termination and ribosome recycling and to model the transition between them. To achieve this goal, we shall integrate the complementary expertise of four leading laboratories in cryo-EM, X-ray crystallography, computational biology and functional studies.
The specific aims of the proposed research include: (i) elucidation of the molecular basis for the omnipotent decoding capacity of eRF1 using X-ray studies of modeled eRF1-associated 80S ribosomal complexes containing different stop codons in the A-site, (ii) determination by cryo-EM of the structures of eukaryotic ribosomal complexes corresponding to different stages of termination, from initial ribosomal attachment of release factors through GTP hydrolysis by eRF3 to the final stage corresponding to post-termination complexes containing deacylated tRNA in the P-site, (iii) visualization by cryo-EM of ABCE1 in post-termination complexes and identification of its critical functional interactions with 80S ribosomes and eRF1, (iv) computational analysis and dynamic simulation of ribosomal transition between individual stages in termination and ribosome recycling based on X-ray and cryo-EM results, and (v) functional investigation of the mechanisms of mammalian reinitiation, regarding its importance as a regulatory post-termination event. In summary, the results that are anticipated to emerge from the proposed studies will for the first time provide an integrated structural and mechanistic outline of the last two stages in eukaryotic translation.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition that targets primarily motor neurons, resulting in progressive paralysis and death within a few years from onset. Despite decades of intensive research, no central pathway or mechanistic basis for this motor neuron degeneration has emerged. Consequently, there is still no successful treatment for ALS, which affects more than 1 in 100,000 individuals each year. Within the past 5 years a paradigm shift in our understanding of neurodegeneration has occurred with the implication of the RNA-binding proteins TDP-43 and FUS, in the pathogenesis of ALS and a broad spectrum of neurodegenerative diseases. TDP-43- or FUS-containing cytoplasmic inclusions are found in ALS, and several other neurodegenerative conditions, accompanied by disturbance of the subcellular localization of these proteins. Mutations in either TDP-43 or FUS cause ALS or, in rare cases, frontotemporal lobar degeneration (FTLD), the second most common type of dementia after Alzheimer’s disease. Emerging evidence highlights the role of prion-like, template-directed misfolding of pathogenic proteins associated with various neurodegenerative diseases, in the spread of protein aggregates, pointing to mechanistic parallels underlying neurodegeneration. I plan to capitalize on my expertise in both prion diseases and ALS in order to study the mechanisms of progressive neurodegeneration and spreading of TDP-43 and FUS protein misfolding. I intend to take advantage of the recently shown prion-like properties of these proteins to develop an innovative ex vivo model of ALS, using mouse organotypic slice cultures.

Understanding the neural basis of sensory percepts has been one of the main driving forces in neuroscience. One traditional approach has been to reduce complex sensory stimuli to its elemental components while monitoring activity from individual neurons. However, it has become clear that in order to fully understand how sensory percepts are created in higher-order brain regions such as the cortex, it is key to study behaviorally relevant sensory stimuli in awake behaving animals while simultaneously recording from large neural ensembles. Olfaction is one of the most dominant senses across many organisms. For rodents, the sense of smell is particularly crucial. The context in which odors are experienced is not only critical for decoding the behavioral significance of the odors but also powerfully influences their perceptual qualities. This proposal aims to understand how contextual information about odors is encoded in the primary olfactory (piriform) cortex. Recent advances in genetic engineering and mouse behavior provide unique and unprecedented opportunities to precisely examine this question. I will create a novel spatial context-dependent olfactory discrimination task in a virtual reality environment for head-restrained behaving mice. I will then perform in vivo extracellular multiunit and patch-clamp recordings with optogenetic manipulations during behavior. In combination with anatomy and brain slice recordings, I aim to determine neural mechanisms for encoding spatial contexts and odor identity in populations of piriform cortical neurons and thus reveal fundamental principles governing the emergence of holistic and integrated sensory percepts in the brain.

The dauer diapause is an alternative stage in Caenorhabditis elegans development that is entered in case of unfavorable environmental conditions. These “dauer larvae” can reside in this stage for many months without feeding or aging and develop into reproductive adults with normal lifespan in case of improved environmental conditions.
A complex signaling network regulates entry and exit of the dauer diapause. This network has been extensively studied for many years and key players like TGF-beta, insulin or steroid signaling as well as their interplay have been identified. As a next step, I propose to image for the first time larvae exiting dauer diapause in their entirety with high spatial and temporal resolution using Selective Plane Illumination Microscopy. I want to characterize morphological changes from tissue level down to the migration of individual cells. Using BAC-recombineering, I will fluorescently label key components of the signaling network, characterize their target cells and correlate their activity to the onset of morphological change. To identify the precise timings of transcriptional activation, I plan to directly detect synthesis of mRNA using the MS2-GFP system, which will be detected by high-resolution confocal microscopy. All measurements will be integrated into one common digital atlas that will be initially created using ion beam scanning electron microscopy. This system will allow a more comprehensive understanding of the complex signaling processes during dauer diapause and how they regulate morphological changes.