Our knowledge of the world is organized in concepts. Bert and Ernie, for example, are two person-concepts, well known to “Sesame Street” viewers. The ease with which we recognize Ernie and distinguish him from Bert belies the daunting complexity of the computational tasks at hand. This is, first, because the retinal activity patterns generated by an object as complex as Ernie is ever changing. Thus representations need to be computed that lump all the different instantiations of Ernie patterns in the retina, into an activity pattern in the brain that is stable across these different instantiations, yet selective enough to tell one person from another. Second, repeated encounters of the same object need to be grouped together over time and with other information -e.g. the name and the voice- to form a modality-free representation: the concept of “Ernie”. Understanding how the brain achieves these remarkable feats is the goal of this project. Our starting point is the finding of ‘concept cells’ by team member Rodrigo Quian Quiroga, which respond almost like a switch to any representation of one individual (e.g. different pictures, the name), but not to other stimuli. Our central hypothesis is that conceptual knowledge rests on explicit concept cell representations: to understand the neurobiology of knowledge, we need to understand concept cells. Conceptual representation is in fact crucial for perception and memory. We bring in expertise on concept cells in humans (through team member Rodrigo Quian Quiroga), the processing of sensory information in rats (team members Davide Zoccolan and Mathew E. Diamond), face recognition in macaque monkeys (team member Winrich Freiwald), and on theoretical neuroscience (team member Haim Sompolinsky) to tackle the problem of concept representations from different angles. Our goals are to find, localize and understand the functional properties of concept cells in three different species with very different brain sizes; to understand, mechanistically, how concept representations are built from sensory inputs; and to determine how conceptual knowledge might be acquired over time. Exploiting unique advantages of our interdisciplinary approach and the complementary use of three biological systems, our project will be the first concise effort to develop a neuroscience of knowledge.

Many animals can detect and use the Earth’s magnetic field for orientation and navigation functions (Lohmann, 2010; Mouritsen and Ritz, 2005). However, the receptor for magnetoreception in vertebrates remains unknown. Members of our team (Dickman Team) have recently discovered cells in the vestibular brainstem of pigeons that encode the direction, intensity, and polarity of the magnetic field (Wu and Dickman, 2012), as well as a magnetic sense neural pathway that includes regions in the brain known to be involved with spatial orientation and navigation tasks (Wu and Dickman, 2011). In addition, our team has obtained preliminary evidence for biogenic magnetic particles in specialized cellular regions of an inner ear receptor (the lagena) that could function as a magnetoreceptor transduction mechanism. Our preliminary data has suggested that these magnetic particles are made of hematite and maghemite, ferromagnetic compounds with permanent magnet properties. Thus, our primary hypothesis is that the lagena contains cells that perform as a magnetoreceptor. Afferent fibers that innervate those receptor cells are hypothesized to signal magnetic information to the brain. Other team members (Winklhofer Team) have recently developed a magnetic microscope that can identify and isolate cells that contain magnetic particles, then extract these cells for subsequent cellular/molecular and biophysical analyses. In fact, they have identified magnetic cells in the nasal epithelium of fish and performed elemental analyses on the magnetic particles contained within those cells. The primary goal of the proposed project is to determine the nature of the lagena receptor and the neural response properties of primary afferent fibers innervating the receptor cells. The project represents a new collaboration between J. David Dickman (Baylor College of Medicine, USA), a systems neurobiologist and Michael Winklhofer (Munich University, Germany) a biophysicist specializing in biomagnetics. Together, the two research teams will focus on two objectives. The first is to delineate the magnetic receptor mechanism in the avian lagena using state-of-the-art methodologies to isolate and characterize the magnetic particles and their cellular location within the lagena. The second is to characterize the response properties of neural afferents innervating the receptor by using both magnetic and motion stimulation. The findings from these objectives will then be used to develop a novel working model for a new magnetic sense.

Signal transduction pathways enable cells to process information essential to their survival and proliferation, yet the genesis of these pathways remains poorly understood. These pathways are typically assembled using components from a small number of protein families that are organized in different ways to process diverse extra- and intracellular stimuli. We aim to investigate the evolutionary mechanisms that mold the interactions between pathway components following gene duplication or lateral gene transfer, leading to changes in the pathway structure and the emergence of novel signaling functions.
We specifically focus on the evolution of the two-component signaling pathway that drives sporulation initiation in Firmicutes. Sporulation is an ideal model for investigating signaling evolution: the pathways in Bacilli and Clostridiales arose from a common ancestral pathway, have similar components and engender analogous phenotypes, yet are characterized by different network architectures: a two-component system in Clostridiales and a complex phosphorelay in Bacilli.
We propose to reconstruct the evolutionary steps leading to the divergence of these pathways at the level of the protein, domain, interacting interface and active site. We will employ a multidisciplinary approach, combining computational phylogenetics and evolutionary systems biology with genetic manipulations and experimental assays.
A network evolution model will be developed to connect phylogenetic data on sequence evolution directly with the probabilities of gain and loss of interaction. This will allow us to reconstruct the most likely states of the sporulation pathway in key ancestral species and hence to predict network rewiring events: changes in protein dimerization and phosphotransfer specificities. The evolutionary histories produced will allow us to understand how the rewiring of modular proteins supports functional innovation at the network level, for example through the insulation of different signals by the elimination of crosstalk between related signal kinases.
The inferred interaction specificities will be tested in vitro using FRET and phosphotransfer assays. Using ancestral sequence reconstruction, we will probe the viability of the inferred ancestral pathways and the likely sequence of evolutionary transitions leading from one pathway structure to another.
We anticipate that our integrated approach will serve as a blueprint for probing the evolution of other signaling systems.

Light is not only important as an energy source for photosynthesis, but also a critical environmental signal for regulating plant growth and development. Plants have sensory photoreceptors that monitor different parameters of the light environment, including wavelength, intensity, direction and duration, and help plants to adapt their growth pattern to diverse and ever-changing environmental conditions. The photoreceptors and downstream signaling components in lower and higher plants, e.g. in the moss Physcomitrella patens and the seed plant Arabidopsis thaliana, are very similar. However, they respond to their surrounding light environment differently to meet their needs. The long-term goal of our project is to understand how species-specific response patterns to light evolved. In Physcomitrella and Arabidopsis, the phytochrome (PHY) family of photoreceptors, the PHY INTERACTING FACTORS (PIFs) and the nuclear transport protein FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) are critical components of light perception networks that promote photomorphogenesis in response to red/far-red light. Although the topologies of light perception networks, i.e. components (e.g. PHYs and PIFs) and wiring (e.g. PHY-PIF interactions), in higher and lower plants appear to be conserved, their response profiles to light are different. One possibility is that the evolution of species-specific light perception networks may have relied in part on changes in the network topology, which in many cases have been shown to be important for network evolution. Alternatively or in addition, the evolution of species-specific light response modes may have relied on modifications of network parameters, such as RNA/protein synthesis/degradation rates and complex association/dissociation constants. By systematic examination of the light perception networks in Arabidopsis and Physcomitrella using biochemical, genetic and systems biology approaches, we will build mathematical models that will help to distinguish between these possibilities. Using a reiterative method of model based guidance of experimental design and mathematical modeling, these models will identify key steps in the evolution of species-specific light perception networks in terrestrial plants.

One of the most abundant proteins in that structures cell shape and guides its movement by forming filaments, moonlights in the nucleus. This protein, actin, has been studied for years yet precisely because of its dominant life as a filament, little is known how it acts in the cell nucleus. Recent evidence points to it serving in a non-filamentous or globular (G) form in the nucleus. Globular actin forms a complex with other proteins to help remodel chromatin and binds transcription factors to modulate gene expression. Recent data suggest that it directly influences the integrity of the genome once single and double strand damage is incurred. There has been no specific way to visualize or distinguish the filamentous from globular form of actin. Novel chemistry allows selection of circular peptidyl structures that are highly specifc for different forms of protein structure. These bicyclic peptides can be used to study the location, binding partners and function of actin in the nucleus. This program combines the expertise of a chemist, an electron microscopist, a yeast geneticist and a vertebrate protein expert in actin and actin related proteins, to try to solve this problem.

Bacterial biofilms are integrated communities of cells that adhere to surfaces and are fundamental to the ecology and biology of bacteria. The accommodation of a free-swimming cell to a solid surface is more complex than passive cell adhesion. We propose a multi-disciplinary study to investigate the interplay between motility appendages, molecular motors, exopolysaccharide (EPS) production, and hydrodynamics near the surface environment using tools not usually used by bacteriologists. We address two questions in P. aeruginosa.
How do different bacterial motor systems and EPS interact with solid surfaces? How do flagella/TFP adapt and respond to an interaction with a surface, and how do they couple to near-surface hydrodynamics and EPS? We will examine the transition from swimming to attaching for the two principal motor systems, flagella and type IV pili (TFP), using direct force measurements on single motors and correlate with multicell behavior using single cell tracking techniques. In all cases, the different roles of solid versus viscous friction in this low Reynolds number regime will be examined. With these anchoring measurements it will be possible to examine this important cdiGMP signaling network in a new light.
How do bacterial motors, exopolysaccharides, and guiding signals influence early biofilm formation? We track the entire motility history of every cell and make inferences about biofilm initiation at single cell resolution. Single molecule TFP force measurements will be used to examine the possibility of a TFP and flagella sensing function for EPS and EPS-mediated interactions between cells. Results will be interpreted using theoretical techniques developed to study nonlinear feedback mechanisms and ‘jamming’ transitions. We will perform new experiments exploiting cdiGMP signaling mutants that impact motility and/or EPS. Our physical measurements will be combined with techniques to measure cdiGMP levels at the single cell and sub-cellular levels, so that spatiotemporal phenomena in signaling and motility can be directly connected. Thus, the initial events in the association of a bacterium with a surface will be investigated in unprecedented detail by combining genetic techniques with single cell measurements, providing novel mechanistic insights into the earliest steps in biofilm formation.

How do animals process, represent and use multimodal sensory information from the natural environment? Our project will address this fundamental question by mounting ultra-lightweight sensors on free-flying bats to capture auditory, visual and chemical signals received by animals engaged in natural behaviors. The sensor data will lay the foundation for novel perceptual experiments, computational modeling, and biorobotic platforms. Our work will focus on the Egyptian fruit bat, a Neotropical frog-eating bat, a European trawling insectivorous bat, and a North American aerial insectivorous bat. The diversity of bat species and their adaptations to different environments and behavioral tasks make them especially well-suited for this line of inquiry.
Research on sensory processing in bats has, up until now, largely emphasized a single modality, typically echolocation. We will launch a major research effort aimed at uncovering the contribution of multisensory processing to the natural behaviors of free-flying bats. The use of novel on-board sensors will give us access to a bat’s view of the world as it moves in time and space. Sensor recordings will be taken in the field, where bats commute over long distances, negotiate obstacles and find food, and in the laboratory, where animals can be trained to perform tasks in experimentally controlled settings. Lab experiments will include creation of a visual-acoustic virtual reality environment.
Our project is directed at understanding the conditions that give rise to cross-modal enhancement of information, differential weighting of sensory information through separate channels, and behavioral decisions in the face of sensory conflicts. In addition, we propose studies that elucidate the role of spatial memory in a variety of tasks and determine the conditions in which bats switch from sensing modes to memory modes. We will investigate and develop mathematical representations of sensory processing and computational architectures for spatial memory, navigation, learning and adaptive behaviors with a view towards exploiting such architectures in complex robotic navigation systems.
The overarching goal of our project is to advance critical new insights to the multisensory representations and cognitive maps of the environment animals use to make decisions for goal-directed actions. Through integrated field and lab studies, computer modeling and biomimetic robotics, our project will contribute to a deeper understanding of multisensory integration, scene analysis, and spatial navigation in natural settings.

Our research team will investigate how pathogenic bacteria use molecular pumps as part of a mechanism to evade antibacterial drugs. When the drugs enter the bacteria, they can be rapidly expelled again by the action of molecular pumps, before the drugs have an opportunity to harm the organism. The pumps we will study are composed of three different types of proteins, two of which span the protective bacterial membrane, and the third helps to draw the other two together into a functional unit. It is not presently understood how these components of the pump assemble together to make a functioning unit, and how the unit works to recognise and displace the drug to the outside of the cell. The team will attempt to elucidate the three dimensional structure of representative molecular pumps to address these questions. The team will examine how the components interact and how the pump uses energy to displace drugs from the interior of the bacterium out into the medium. We will also examine how the components come together to form a functioning unit in a living bacterium. The information obtained from this work may help us to understand how to combat the growing problem of multidrug resistance in bacterial infections.

Persistent structural and functional modifications of excitatory synapses are suggested to represent the cellular substrate of learning and memory. Dendritic spine morphology, like synaptic function, is dynamically regulated by neuronal activity. Changes in both spine morphology and synaptic efficacy are primarily influenced by actin remodeling. However, how dynamic actin filaments are anchored within dendritic spines and control their morphology and synaptic function remains unknown.
Our working hypothesis is that anchoring of actin filaments in dendritic spines represents a critical determinant of their dynamic and plastic behavior. Anchoring to the plasma membrane (‘peripheral anchor’) likely contributes to shape spine head and hinder the local diffusion of transmembrane proteins whereas anchoring at the base of the spine head (‘central anchor’) is required to generate enlargement forces upon actin polymerization. Elucidating the role of actin anchoring in dendritic spine structure, function and learning-related plasticity therefore represents the scientific aim of this proposal. To this end, we will develop and implement a highly multi-disciplinary approach based on the complementary expertise of the participants and a combination of i) advanced in vitro and in vivo electrophysiology, ii) cutting-edge imaging techniques, molecular tracking and super-resolution microscopy, iii) innovative photo-chemical tools for protein tagging, acute photoinactivation and crosslinking and iv) analysis of network activities and specific cognitive performances in behaving animals.
We will establish the role of peripheral and central anchors of F-actin in dendritic spines. We will combine single cell electrophysiology and molecular imaging techniques to explore the functional consequences of suppressing these anchors either genetically or acutely using protein photo-inactivation (CALI) techniques. Next, we will directly visualize the 3D network of spine actin, its modulation upon long term plasticity induction as well as its alterations upon selectively suppressing peripheral or central anchoring. We will also adapt molecular tagging to reveal the molecular organization of F-actin and its anchors within dendritic spines. We will then further identify the molecular components of peripheral and central anchors of F-actin in spine using new photo-crosslinking strategies to identify the molecular partners of peripheral and central actin anchors. Finally, we will combine our efforts to explore the importance of actin anchoring regulation in learning and memory processes by combining in vivo electrophysiology, photoinactivation and Ca imaging while monitoring cognitive performances measured in behavioral assays.

Calcium is an extremely versatile second messenger, which controls almost all kind of cellular biological events.
As a single atom, calcium regulates biological function by changing its concentration, which is driven by the ~20,000 fold concentration difference between the extracellular environment and the cytoplasm. To avoid catastrophic cytoplasmic calcium induction, cells strictly limit calcium influxes. Activated channels are rapidly shut down and intruding cytoplasmic calcium is swiftly removed to the extracellular space or transferred into inner cellular membrane organelles. Accordingly, the combination of acute ion influx, point source ionic diffusion, and its immediate removal creates an ideal environment for calcium signal oscillations. Here, cardiomyocytes serve as a prime example where a wide range of cellular function is controlled by calcium signaling. Cardiomyocytes develop unique membrane micro domains termed “couplons” which structurally control calcium diffusion. These couplons work to not only couple membrane excitation to cell contractions, but also function as an electric synapse, through which calcium reciprocally controls membrane potentials. Nonetheless, our current understanding of cardiac couplon architecture and its dynamics is significantly lacking. Thus, by using cardiomyocytes as a model system, the goal of this project is to clarify how eukaryotic cells assemble junctional micro membrane domains that are effectively used to generate oscillating calcium signals, and define how these structures spatiotemporally control the information carried by this signal. This goal will be achieved by integrating the diverse expertise of our international team members. We will use 3-dimensional electron microscopy to determine the complete structure of couplons and their clusters, and paint the geometric models with distributions of nodal couplon molecules, determined both by diffraction limited and super-resolution light microscopy. Minimum molecular requirements to synthesize membrane junctions will also be determined and local membrane dynamics will be monitored by the use of newly designed fluorescent bio-reporters. In summary, successful completion of the current project is expected to provide fundamental knowledge of how micro-domain structures and their dynamics control the oscillatory behavior of membrane signaling.

Circadian rhythms are thought to have evolved to enable organisms to organise their activities according to the Earth’s predictable daily cycles but data revealing the benefits of circadian rhythms are scarce. More difficult still is explaining why parasites that exclusively live within the bodies of other organisms have a circadian rhythm. For example, the developmental rhythm of many malaria parasite species is coordinated; parasites invade host red blood cells, replicate, and then release their progeny in a timed, synchronized burst. Similarly, there is increasing evidence of circadian rhythms in the effectors and regulators of host immune responses, but it is not known whether this influences protection from disease. What is lacking is an explicit recognition that rhythms in immune defence and parasite development could provide an evolutionary advantage to hosts, parasites or both, as well as recognition that both parties may control each other’s rhythms. We will integrate different perspectives and methods from evolutionary ecology, chronobiology, parasitology, and immunology to investigate the causes and consequences of rhythms for hosts and parasites. Our project uses an established rodent-malaria model system. We will capitalise on the reciprocal feedback from integrating empirical and theoretical approaches to answer the following questions: (A) How are rhythms in parasite development initiated and maintained? Do parasites use their own time-keeping mechanisms to organise development or do they use cues from the host's circadian rhythms? (B) How do rhythms affect the ability of hosts to cope with infection? Do rhythms maximise immune defence or are they a constraint resulting from competing demands on the immune system? (C) How do rhythms affect the survival and transmission of parasites? Do developmental rhythms enable parasites to avoid host immune responses or facilitate the exploitation of circadian driven resource availability? (D) Are rhythms in parasite development and host immune responses adaptive for parasites, hosts, or neither? Does the evolution of parasite rhythms drive the evolution of host rhythms, or vice-versa? Thus our project breaks new ground by elucidating: the role of rhythms in infections, how rhythms may be exploited for clinical benefit, and the adaptive significance of rhythms for hosts and parasites.

Intracellular recording of the neuronal transmembrane potential has been crucial to our understanding of the biophysical mechanisms underpinning neuronal computation. Existing intracellular recording techniques, however, do not permit long-term measurements of this potential, being limited by electrode-induced changes in neuronal function and the inherent instability of these recording devices. In practice, intracellular electrical recordings are generally limited to less than one hour, but many important processes occur on timescales of hours to days such as learning and plasticity. While in isolated cases repeated recordings from an identified neuron can be a work-around, this remains particularly challenging in vivo.
Here we propose to develop minimally-invasive intracellular recording electrodes for in vivo recordings by combining two complimentary technologies that have been separately developed by the laboratories of Andreas Schaefer (MPI f. medical research, Heidelberg, Germany) and Nick Melosh (Materials Science & Engineering, Stanford, USA). Schaefer group has described solid-conductor intracellular nananelectrodes (SCINEs) that can be inserted into neuronal membranes to record supra- and sub-threshold neuronal potentials without causing membrane damage or diluting cytoplasmic molecules. Unfortunately, these nanoelectrodes only insert into membranes with low probability and limited duration - both, presumably, due to an unstable membrane-electrode interaction. Melosh group has developed technology that allows inorganic nanostructures to insert and fuse spontaneously and stably into lipid bilayers. This is accomplished by creating a 2-10 nm wide hydrophobic band around the hydrophilic metallic electrode, mimicking the internal hydrophobic region of the lipid bilayer. Upon contact with a lipid membrane, this band fuses into the core of the bilayer, thus allowing the nanostructure to traverse the membrane without disrupting it and providing stable adhesion with the cell. Melosh group has verified integration of such “Stealth Probes” into membranes through atomic force microscopy and intracellular cyclic voltammetry.
Together, we plan to develop biomimetically patterned solid-conductor nanoelectrodes, “stealth SCINEs” (sSCINEs), for non-invasive intracellular recording, with Melosh group focusing on design, and testing of membrane-electrode interfaces, and Schaefer group focusing on assessing sSCINEs for physiology and developing in vivo sSCINE recording preparations. First applications will be mapping stability and plasticity of synaptic properties and substhreshold receptive fields in the mouse olfactory bulb — experiments currently impossible due to limited recording duration of current intracellular recording techniques and the high-dimensional input space.

Do cells think? We imagine that thinking is the product of complex nervous systems. Studies by our team and others, has demonstrated that seemingly simple microbes, such as brewers yeast, display signs of having memory and consequently the rudiments of intelligent behavior. Yeasts grown on a particular sugar typically activate genes that code for enzymes to digest that sugar. We found if yeasts are grown for a long time on glucose, they appear to maintain activation of the genes required to digest glucose, and consequently take much longer to switch on the genes required for growth on an alternative sugar once the glucose is used up. This history-dependent behavior can be interpreted as a rational decision because, for yeast, the constant availability of glucose in the past appears to imply that glucose is likely to become available again in the future. In other words, yeast cells somehow "remember" how long they have been consuming a certain sugar, and they adapt their behavior accordingly. These preliminary results show that cells and thus the biochemical machinery – the genes and proteins – that generate their behavior are influenced by the past. We will set up experiments to first quantify history-dependent behavior at the single-cell level and second to identify genes and specific molecular processes involved in memory. We will specifically focus on epigenetic mechanisms, those that do not involve changes to the genes themselves but to changes in the structure of DNA and the inheritance of particular proteins from cell to cell. Further, to identify why history-dependent behavior may have evolved, we will determine environments (sequential appearances of different sugars) that favor and environments that disfavor history-dependent behavior. Our research will not only reveal new insight into microbial physiology, but may also establish general principles into how previous experience shapes future choices for cells. Our efforts may open up new avenues of research, such as whether complex nervous systems may use similar strategies to store information or whether cancer cells “learn” differently from normal cells, perhaps in a way that could be harnessed to selectively kill these cells.

The lack of information on the physiological basis of the neuronal control of mood severely limits the study of mood disorders. Recent evidence suggests that the circadian (~24-hr) timekeeping system in mammals can trigger mania or depression. Circadian mistiming has also been shown in animals or patients showing symptoms of mania or depression. Here, we study how the central circadian pacemaker, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, controls and is regulated by mood. Functionally, the SCN appears homogeneous and most SCN neurons contain the inhibitory neurotransmitter GABA. In reality, however, SCN cells display heterogeneity; many, but not all, SCN neurons contain the intracellular molecular clock and some SCN neurons can be further distinguished by their neuropeptide content. Further, we recently showed that neurons in the SCN show a wide variety of electrical states, from hyperpolarization during the night to a novel depolarized states during the day. Consequently, neurons and subregions in the SCN vary in their key cellular and molecular properties. Here, we use behavioral, genetic, and pharmacological models of mania and depression and determine alterations in SCN timekeeping at molecular, cellular, and network levels. Experimental results will be incorporated into detailed mathematical models, which will then predict future experiments. This will identify which network states of the SCN (or states of subpopulations of SCN neurons) trigger manic or depressive symptoms. We will also determine how mutations in key components of the intracellular circadian clock are linked with mood and lead to changes in the overall state of the SCN. Finally, we will determine if desynchronized timekeeping among SCN neurons or pathological electrical activity, triggers mood phenotypes. In addition to expertise in circadian timekeeping shared by all, our team includes a modeling group at the University of Michigan in the United States, an electrophysiology group at the University of Manchester in the United Kingdom, and a group that studies both animal models for neurological disorders at Hiroshima University in Japan.

Most proteins produced by any genome are processed during their synthesis on ribosomes, including cleavage of the N-terminal methionine, N-terminal acetylation, transport to a membrane channel or association with molecular chaperones for folding. These processing steps are usually specific for the type of protein made and often crucial for cell survival, as their occurrence affects the localization, stability and activity of proteins. These different co-translational activities on nascent chains have to be highly coordinated temporally and spatially to ensure the functionality of the translatome. Most studies have focused on the specificity of processing factors themselves including modifying enzymes, chaperones or transport factors (abbreviated hereafter collectively as NAFs, for Nascent polypeptide-Associated Factors) and little attention has been bestowed on the ribosome as contributing to specificity and coordination. However, recent work suggests the ribosome may have a sensing mechanism that signals the type of the emerging nascent peptide from the interior of the exit tunnel to the exterior of the ribosome for co-translational recruitment of appropriate NAFs. The mechanisms of ribosomal sensing and factor recruitment, however, are unknown.
Here we propose to employ an interdisciplinary approach to analyze how signalling by polypeptides inside the eukaryotic ribosomal tunnel is transmitted to the ribosome exterior for recruitment of the appropriate NAFs. Together, our integrated programme will allow unprecedented insights into how the emerging nascent polypeptide chain is sensed by the ribosome and how it signals to recruit different enzymes, chaperones and transport factors to the ribosome exit tunnel in readiness for their immediate, co-translational modification, folding or transport.

During their life, cells of the immune system experience a large range of mechanical stimuli. These range from shear stresses encountered in blood flow and lymph nodes to irregular topographical cues of extracellular matrix fibers and changes in tension during movement from capillaries to tissue. The process of converting physical cues into biochemical signals and integrating these signals into a cellular response is referred to as mechanotransduction and plays a crucial role in health and disease. To sense the physical cues of its surrounding, cells have specialized mechanosencing proteins arranged on the cellular membrane. While it is accepted that the mechano-chemical signal conversion involves force-induced changes of protein functions, it remains a major mystery how physical and biochemical factors are coupled and integrated to enable mechanical sensing.
The overall goal of this project is to provide mechanistic insight into the role of mechanical stimuli in the regulation of cellular signaling. Specifically, we will determine the influence of applied mechanical forces on the spatial organization of primary mechanosensors called integrins and how these changes alter signaling processes. Thus, our project will explore mechanosensing, mechanotransduction and mechanoresponses. We will define the effects of force and geometry sensing on cellular responses of two types of immune cells, monocytes and dendritic cells.
To achieve this goal, we have assembled a multidisciplinary team that not only brings together expertise in the two cell types, but also integrates biophysics with cell biology and immunology. To address the question of how stress alters membrane organization and signaling at the molecular level, we will use cutting-edge biophysical tools recently developed by team members, including superresolution imaging and nanospectroscopy, hyperspectral microscopy and tailored-made microdevices for mechanical manipulation. The concept that mechanical forces play a role in altering the organization of proteins in the cellular membrane and consequently signal transduction is new in the mechanobiology field. Therefore, our project will provide a significant conceptual advance in our understanding of how the cell interprets external forces that it encounters in its native environment.

As an organism develops from a fertilized egg, cells undergo a sequence of differentiation events, acquiring ever more specific identities. A fundamental problem in biology is to understand how self-renewal and cellular differentiation is carried out by intracellular genetic circuits and cell-cell signaling. Embryonic stem cells, with their remarkable ability to self-renew indefinitely, while retaining the potential to differentiate into a diverse range of specialized cell types upon stimulation (pluripotency) provide a powerful in vitro model system for studying the molecular mechanisms underlying self-renewal and cell fate specification. Addressing this problem, however, has been challenging, because most stem cell populations are inherently heterogeneous, and tools are lacking that can monitor cellular states quantitatively and dynamically at the single-cell level. Here, we employ recent advances in quantitative time-lapse microscopy and stem cell engineering to systematically examine how cellular components are regulated dynamically during pluripotency and differentiation in individual stem cells, and to use this information to understand cell state transitions. To reach this goal, we assembled a team with expertise in stem cells, cell line engineering, quantitative time-lapse microscopy, single-cell dynamics, systems biology, and mathematical modeling. We will use mouse embryonic stem cells as a model system. First, we will generate a library of fluorescently labeled mouse embryonic stem cell lines with endogenous reporters for key signaling pathways, regulatory genes, and other cellular components. Second, using this library, we will use quantitative time-lapse microscopy to analyze the dynamics of proteins in the pluripotent state, studying the temporal and spatial variation in proteins between genetically identical cells. Finally, we will use the same library to study specific cellular differentiation processes, mapping dynamic events that occur during differentiation, and analyzing when and how cell fates become established. A major focus in this work will be the role of heterogeneity, or “noise”, in cellular circuit components. Together, these efforts will enable us to discover new dynamic phenomena concealed by conventional population studies, and enable us to elucidate the effective regulatory architecture of the stem cell.

Human embryonic stem cell (hESC) cultures are heterogeneous, and consist of subpopulations that represent distinct cellular substates of pluripotency. These subpopulations reside within specific spatial domains in stem cell colonies. Much evidence now suggests that intercellular communication between cells in these compartments helps to drive cell fate decisions, just as communication between cells governs fate choice in the peri-implantation embryo. ES cell cultures therefore provide a model for studying how cell interactions govern transitions through state space within a regulatory network. Currently we do not understand how extrinsic signaling governs transitions between cell states, how self renewal and pluripotency are regulated, or how intercellular signaling between different compartments effects spatial patterning. Unraveling these complexities will require a systems biology approach. By combining time-lapse video microscopy and single-cell analytical technologies with mathematical modeling, we will study the spatial and temporal dynamics of signaling pathways that regulate cell fate decisions in hESC culture. Quantitative models that describe the dynamic interplay between signaling pathways will allow us to understand how human embryonic stem cells regulate themselves and how they respond to environmental signals. This knowledge will allow us to predict the optimal combinations, dosage, and time course of exogenous signals to achieve precise and reproducible control of the cell state.

Planar cell polarity (PCP) is an organized differentiation process in which cells within a developing tissue all polarize along one planar direction. In mammals, PCP is crucial for proper development of the inner ear, kidneys, lungs, neocortex, and more. While some of the core mammalian PCP proteins such as Vang2 / Fz3/6 / Dvl2/3 and their regulators have recently been identified, there is still no clear picture of how PCP is generated. Furthermore, this coordinated polarity often involves drastic morphological changes such as convergent-extension (CE), cellular differentiation, and asymmetric cell divisions. It is unclear how these processes interact with PCP patterning. We hypothesize that PCP patterning involves feedback between intercellular signaling and morphology, namely, that intercellular signaling both affects, and is affected by, cellular morphology. Here, we propose a systems-level approach that integrates gene expression, cellular morphology, and asymmetric cell divisions into a comprehensive model of PCP patterning. We will study PCP and its interaction with tissue morphology in two complementary model systems: the inner ear, where PCP is involved in the uniformly oriented differentiation pattern of hair cells and supporting cells; and the neocortex, where PCP is involved in asymmetric division of neuronal progenitors. Our approach combines several unique experimental and theoretical studies. First, we will use state-of-the-art comparative transcriptome analyses on subpopulations of cells (hair and supporting cells for the inner ear; progenitors and differentiated neurons for the neocortex) from wild type and mutant mice for identifying essential regulatory nodes in the PCP network. Second, we will take advantage of the ability to grow live cochlear cultures and neocortex slices in culture dishes to perform real time tracking of PCP development using quantitative time-lapse microscopy. These techniques will be based on the development and characterization of wild type and mutant transgenic mice expressing a combination of fluorescently tagged proteins. Finally, we will employ a novel mathematical modeling strategy that will integrate reaction diffusion models of PCP and cellular differentiation circuits with mechanical vertex models for describing tissue morphology. Overall, these investigations are aimed at obtaining an integrated morphodynamic picture of PCP patterning.

Are roots in the dark? Darwin proposed that the “brain” of a plant resides in its roots. Subsequent research has shown that roots integrate environmental information about resource availability, competitors, etc., to determine critical life history traits of the shoot, such as the timing of resource allocations to reproduction, growth, defense and senescence. During domestication, this environmentally sensitive “brain” is commonly disabled to make crops less environmentally sensitive, easier to transplant, and better synchronized to facilitate harvesting. We propose to explore the importance of a new sensory modality of roots, that of light-perception, in enhancing the ecological performance of a native plant in its native habitat.
Plants use light as both an energy source and a source of signals to alter its physiology to adapt to current or anticipated environmental changes. Forward and reverse genetics have identified photoreceptors and their associated signaling cascades. Surprisingly, some photoreceptors are highly and specifically expressed in roots, which are generally thought to develop in the total darkness of the soil. Light penetration in most soils is limited to a few millimeters close to the soil surface but roots that express a plethora of photoreceptors grow deep in the soil. Since natural selection rapidly winnows non-functional genes from genomes, these root- expressed photoreceptors are likely being maintained in the genomes of higher plants because they play an important role; the goal of this interdisciplinary proposal is to uncover the function of these root-expressed photoreceptors.
While the idea of light-piping through vascular bundle was suggested three decades ago, theoretical and empirical examinations of ‘light-piping’ remains largely unexplored and the question of its relevance for plant function has simply been out of the grasp of single experts working alone or in loose collaborations with other experts. By combining state-of-the-art optical physics and molecular biology, we propose to characterize this transmitted light, uncover how plants respond to this light, and determine if the responses are relevant for the optimization of ecological fitness during the plant’s entire life in the field. Building on the impressive knowledge of light signaling in the model plant, Arabidopsis thaliana community, we will use a wild tobacco, Nicotiana attenuata to uncover the ecological relevance of having “sighted roots” in the plant’s natural habitat. We will employ the versatile molecular and biochemical tools available in Arabidopsis and incorporate the relevant components into an ecological model plant, N. attenuata. We have developed analytical, molecular and ecological platform in N. attenuata to study its rich ecological interaction in the Great Basin Desert of Utah. We propose that the intense light of the desert will illuminate the ecological function of light signaling in roots.