Throughout evolution, the human genome has been attacked by retrotransposons, parasitic DNA elements that spread through our genome by a copy-paste activity. I previously showed that SVA elements, the youngest class of retrotransposons in our genome, harbour a strong gene-regulatory potential, which is normally repressed by KRAB zinc finger protein ZNF91 (Jacobs et al., 2014, Nature). However, for reasons unknown, repression of retrotransposons is much less efficient in neurons, resulting in the activation of the hidden enhancer potential of SVA elements spread throughout the human genome. The importance of these SVA insertions for the evolution of human neural gene-regulatory networks, and how many genes have come to depend on SVA's regulatory influence, remains elusive. This research program uses 'cortical organoids’; 3-dimensional brain tissues derived from human and primate stem cells, to investigate how recent SVA insertions have impacted human neuronal gene expression. Furthermore, I will investigate how changes of the epigenetic landscape in neurons affect the activity of retrotransposons and the influence they have on nearby neural genes. Finally, I will explore the possibility that loss of epigenetic silencing of retrotransposons is responsible for dysregulation of genes associated with neurological diseases. Preliminary findings suggest a potential role for retrotransposons in susceptibility loci for Alzheimer's and Parkinson's disease. Finding further support for this notion in the current research program, will form the basis of a novel concept that can explain how changes in the epigenetic landscape can uncover a dormant genetic predisposition to disease.

The drive to seek rewards controls almost every aspect of our behavior, from stereotypic reflexive responses to complex voluntary actions. Thus it comes as no surprise that the symptomatology of neurological disorders that interfere with reward include deficits in the capacity to make even simple movements. To drive behavior the brain uses two major subcortical networks, the basal ganglia and the cerebellum. Experimental data and computational models suggest that the basal ganglia and cerebellum play different roles in the control of behavior. In the basal ganglia, reward signals guide action selection, but how reward signals are translated into behavior remains unclear. The cerebellum uses sensory error signals to optimize motor commands; however, whether the signaling in the cerebellum depends on rewards and how reward might be incorporated into the motor commands have yet to be deciphered.

We will explore whether and how reward signals interact with motor commands in the basal ganglia and cerebellum. We will take advantage of primate smooth pursuit eye movements as a powerful system to study the relationship between reward processing and motor behavior. We will record and manipulate activity in both the basal ganglia and cerebellum while animals are involved in novel behavioral tasks. The tasks extend the current state of the art concerning what is known about sensory or motor processing to study how reward is translated into behavior. The overarching goal is to unify research on the basal ganglia and cerebellum to understand the transformations and computations that translate reward drive into action and their implementations in the basal ganglia and cerebellum.

To understand how brains integrate sensory information and generate behavioural responses is a central goal of systems neuroscience. Larval Zebrafish have emerged as a very promising vertebrate model system to address this challenge because their small size and transparency enable optical access to the majority of neurons within the brain at cellular resolution. Important insights on circuit function have already been gained by two-photon imaging of neuronal populations in restrained larvae behaving in virtual environments – however, critical limitations of virtual environments as a replacement for real environments are widely acknowledged and remain a barrier to progress. An ideal solution to this problem would be to image larvae during natural, unrestrained behaviour, and this is the goal we aim to achieve in this project. It has so far not been possible to track naturally moving zebrafish, nor any other vertebrates, while imaging at cellular resolution. Here we propose to overcome this limitation by forming a novel collaboration across disciplines and three different laboratories. We will pool our collective expertise in optical systems design, wavefront-shaping, electrical engineering, and zebrafish neuroscience to perform an experiment that has until now been impossible: imaging comprehensive neural activity in a freely moving, untethered vertebrate. We will collectively design and validate a real-time optical tracking and imaging system to measure brain activity with single neuron resolution in a freely moving vertebrate. This will allow us to monitor, with unprecedented detail, the population activity of the reticulospinal system in larval zebrafish and understand how this population of ~ 300 supraspinal neurons combinatorially codes for the full range of locomotor and postural behaviors.

Microscopy of biological specimens is often performed at low levels of illumination to avoid damage. Under these conditions images are dominated by shot noise, the statistical fluctuations in the number of detected probe particles (e.g., photons or electrons). Better images can be obtained when correlated probe particles are used, which allows extracting more information per interaction with the sample. A way to do so is to pass the illumination multiple times through the specimen [1, 2]. We propose to design and construct a microscope where the specimen is placed at the center of a self-imaging cavity, in which the probe particles interact with the sample multiple times. An optical setup will be used to demonstrate the main benefits of a multipass microscope: contrast enhancement, sub-shot noise imaging, and imaging at low damage. Fast electro-optical switching techniques will be employed to, for the first time, show sub-shot noise imaging without postselection of the probe particles. Various imaging modalities will be explored, such as phase, absorption and polarization microscopy in both bright- and darkfield configurations. Finding the right imaging modality for specific specimens (e.g., living cells) will be at the focus of the project. In parallel, the feasibility of a multipass electron microscope will be explored using charged particle tracing simulations. We will construct and test a prototype as a first step towards the imaging of organic molecules and proteins with atomic resolution. [1] Giovannetti V, Lloyd S and Maccone L, Nat Photon 5, 222-229 (2011). [2] Higgins B L, Berry D W, Bartlett S D, Wiseman H M and Pryde G J, Nature 450, 393-396 (2007).

Both wild and crop plants must contain pathogen infections to survive. Prevailing theories on the evolution and spread of plant pathogens are based primarily on observations of nearly clonal hypervirulent pathogen lineages in agricultural systems, which are characterized by dense stands of genetically uniform individuals and a paucity of other species. Surprisingly little is known about how pathogens evolve in natural settings, where emergence of hypervirulence appears to be much less prevalent.

The central goals of this proposal are to reveal (i) the dynamics of bacterial pathogen colonization in natural plant populations, and (ii) the influence of several factors—pathogen diversity, host genetics and host microbiomes—on pathogen infection dynamics. My specific plan is to investigate the evolution and spread of the endemic plant pathogen Pseudomonas syringae in wild populations of Arabidopsis thaliana, using innovative high-throughput methods for culturing and genome comparisons of bacterial lineages. These observations will reveal associations of pathogen spread with geographic isolation of hosts, host microbiome composition, host genetics, and pathogen genome diversity. I will then test causal relationships for a subset of these associations through controlled laboratory infections. A unique aspect of my proposal is the hierarchical approach—identifying infection dynamics at the leaf, individual plant, and population level—and the integration of experiments in the laboratory with high-throughput investigation of natural infections.

Innate behaviors such as aggression and reproduction are essential for the survival of animals. Neuropeptides are implicated in the regulation of various innate behaviors by changing their basal arousal state and motivation level via modulating neural activity. Oxytocin (OXT) and vasopressin (AVP) are involved in various social behaviors such as aggression, mating, pair-bonding and social recognition. However, how these peptides exert their functions at the neural circuit level is not well understood.
I plan to study the role of OXT/AVP on the neural circuit of aggressive behaviors by addressing the following questions: (1) Whether if OXT/AVP is actually released onto the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl), which was previously shown to control both the appetitive and consummatory phases of fighting and mating? (2) What is the role of the neural activity patterns responding to OXT/AVP release during aggression? (3) How is the activity of OXT / AVP neurons controlled during aggression and from where do the OXT/AVP neurons receive their input? (4) Whether if OXT/AVP control other social behaviors in VMHvl in addition to aggressive behaviors?
In contrast to my previous research experience in neuroendocrinology, the proposed research will focus on how neuropeptides modulate innate behaviors at the circuit level. Therefore, this will be a good opportunity to extend my training from endocrinological to behavioral neuroscience.

The time dependent regulation of genetic activity in eukaryotes is one of the most complex challenges of modern science. Multiple biomolecular factors constantly interact to maintain the necessary expression levels of various genes and functional RNAs. In this project, we propose to utilize the dramatic advances in live cell fluorescent microscopy and stochastic physics to study a canonic nuclear system – X-chromosome inactivation (XCI). This crucial process is necessary for female embryo development and brings about the transcriptional silencing of a whole X chromosome. Yet despite XCI’s centrality, we still lack an understanding of its temporal regulation mechanisms. The spatial dynamics of the nuclear machinery, including the noncoding Xist RNA and its partners, their interaction with chromatin and the dynamic processes leading to the loss of the transcriptional apparatus from genes during XCI, are all unknown. To answer these questions, we will track the diffusion of Xist RNA together with essential nuclear factors, in live female mouse embryonic stem cells, using super resolution microscopy (SIM, dPALM), and stochastic techniques (FCS, FRET). This includes imaging of Xist associated proteins, silenced and activated genetic loci, and transcriptional machinery. Diffusion will be analyzed using advanced mathematics to extract dynamic characteristics of tracked factors; characteristics which are crucial to fully understand XCI. From these, a biophysical model will be developed to describe the interactions, energetic landscapes and as an outcome, the mechanisms which control this fundamental genetic process.

Collective cell migration is essential in biological processes such as angiogenesis, organogenesis, and wound healing. In various cancer types deregulated migration is implicated in the formation of metastasis. Collective cell migration is meditated by leader cells, which sense multiple parameters and execute movement into the open space, followed by the cell sheet. Cells constantly monitor their environment and the sensing of nutrients has been identified as an important determinant of proliferation. The primary objective of this proposal is to investigate the impact of nutrient supply on cell migration. Thus I aim to identify the metabolic switch, a change in nutrient availability and production, sufficient to alter the migratory properties. More specifically, building on the previous findings and tools of the host laboratory, I aim to first characterize cell speed, direction, and persistence of human endothelial cells in the context of altered nutrient supply using a high-throughput live cell imaging platform. Second, by utilizing specifically designed fluorescent probes, I will dissect the spatio-temporal regulation of nutrient-sensing signaling pathways including the activation and repression of mTOR by PI3K/AKT, PLD/PA, or LKB1/AMPK, which leads to altered cytoskeleton arrangements. Finally, the integration of these findings will aid the generation of a model describing how nutrient supply alters cell migration. This model will be validated using chemical inhibitors, inducible gene deletion, or targeted perturbation. The results of this study contribute to our understanding of how the metabolic environment of a cell dictates its properties, functions, and decisions.

Lymph nodes (LNs) are situated at junctures of the blood vascular and the lymphatic system where antigens drain from peripheral tissues via afferent lymphatics. During the development of malignant breast cancer, new LNs emerge within the glandular tissues that are normally devoid of LNs. However, we do not understand the mechanism of development of these LNs and their role in antitumor immunity. We hypothesize that de novo LNs form in the vicinity of mammary carcinomas as an accessory “base camp” for the initiation and maintenance of antitumor immunity. Here, we combine and leverage our expertise in immunology, vascular biology, and bioengineering to address the hypothesis in three specific aims: (1) To molecularly dissect the pathways of tumor-induced development of accessory LNs with a particular emphasis on lymphovasculokines. (2) To assess to which extent accessory LNs support antitumor immunity. (3) To stimulate formation of accessory LNs in mammary tissues to foster antitumor immunity. The potential impact of this project is high as a deeper understanding of the interplay between accessory LNs and tumors may lead to a novel cancer treatment approach.

Cancer cells need to overcome the onerous task of adapting to cellular stress during the multi-step process of cancer development. Several studies suggest that remodeling of gene expression at the level of translation control, and in particular the major initiation complex, eIF4F, and its cap-binding subunit eIF4E, provides cancer cells with a rapid mechanism to alter expression of key transcripts that are required for this process. In a seminal study, the Ruggero group generated the first eIF4E haploinsufficient mouse and demonstrated that these mice containing 50% less eIF4E are strikingly phenotypically normal, but resistant to cancer formation. Genome-wide translational profiling experiments identified not only downregulated but surprisingly also upregulated genes. However, the role of the proteins encoded from these upregulated genes in cellular transformation remains unknown. It is the aim of my project to characterize the molecular and functional features of these mRNAs that are surprisingly selectively upregulated at the translation level upon eIF4E haploinsufficiency during oncogenic insult. I will test different hypotheses to understand translational control mechanisms of these specific mRNAs regulated by the expression level of eIF4E. In addition to the molecular characterization, I will also identify their role in cellular transformation. In summary, this project will provide invaluable information into post-transcriptional regulation underlying oncogenic transformation.

Life as we know it depends on the membrane compartmentalization of the cell. Membrane proteins and their organizing scaffolding proteins maintain the flow of information and materials across the membrane. This project will advance the characterization of membrane and membrane associated proteins by developing novel electron microscopy and biochemical experiments enabled by new nanomaterials.
We focus on gephyrin, the major scaffolding protein in inhibitory synapses, and the glycine receptor (GlyR), which is one of the channels that gephyrin anchors to the synapse. Currently there is no detailed molecular mechanism to how gephyrin polymerizes and forms a scaffold, although the proteins and post-translational modifications that influence clustering in cells have been identified. A major puzzle is the variability and apparent geometric inconsistency of the scaffold. For instance, gephyrin’s hexagonal/trimeric binding geometry does not match the geometry of the binding sites within GlyR, which are arranged within a pentamer. GlyR dependent synapses have important regulatory functions in the brain stem and pain pathway and thus are potential drug targets. The emergence of a recent structure of a GlyR homopentamer has left us with questions about the structure of a physiologically relevant heteropentamer channel, the cytosolic part of the protein, and most importantly about conformational states of the channel that have not been visualized. These gaps in knowledge have hampered efforts to understand gephyrin-GlyR binding as well as to design new GlyR drugs.
This project will develop i) a nanosheet synthetic scaffold that can present gephyrin with its binding motif in a precisely defined geometry (Lau:Materials Science) and ii) nanoparticles functionalized with GlyR drug targets that provide high electron contrast labeling and bi-functional crosslinkers for the structure-function measurements (Yameen:Organic Chemistry). These nanomaterials will enable assays of gephyrin scaffold formation and GlyR complex formation, and electron microscopy functional mapping of GlyR (Mim:Structural Biology). The proposed research will provide insight into gephyrin-GlyR structure-function relationship and demonstrate new tools for biological characterization.

New genetic diversity originates in each generation through genome misreplication during germline development. Despite the fundamental nature of this process, observations of germline mutation have been indirect until recently, with the advent of coupled parent-offspring sequencing. However, because germline mutations are rare, and families are inherently limited in size, statistically robust estimates of germline mutation rates need to aggregate data from hundreds of distinct pedigrees, potentially integrating over biologically meaningful inter- and intra-individual mutational heterogeneity. Here, we propose a novel approach to study the individualistic component of germline mutation, using single-cell sequencing of gametes from a single individual. To provide proof of principle, will employ the model organism Ciona intestinalis, a hermaphroditic marine invertebrate with a small genome, which enables us to observe, with unprecedented depth, mutations occurring in both the male and female germlines in a single genetic background. By also sequencing offspring from the same individual, we will test the hypothesis that gamete sequencing can be used to measure mutations entering subsequent generations. Large samples of gametes will also enable us to study intra-individual mutational heterogeneity. Mutations shared by numerous gametes will be analyzed phylogenetically to provide a portrait of the lineage tree of germline stem cells, and of the dynamic nature of mutational processes along this tree. By extending this approach to the population scale, it should be possible to examine the patterns, and ultimately, the causes of inter-individual variation in germline mutation.

Essential ecosystem functioning and services are often the product of a stable community of interacting species. The degree of stability of these communities is to a large extent determined by the network structure of interactions between the species. Until now, research has focused on understanding how the static network structure influences the stability and robustness of the community in light of biotic and abiotic perturbations. These studies are generally limited to static snapshots of the interaction network. Yet, real ecological communities are in constant flux, and a complete understanding of long-term community dynamics must take into account the intrinsic ability of species to evolve and thereby modify their local interactions, thus changing the interaction network of the community.

This project addresses both theoretical and experimental challenges to bring together ecological and evolutionary dynamics in the study of complex ecological interaction networks. My work will offer predictions of how the network within ecological communities forms as species interacting in various ways come together in different environments. I will also validate and further expand these predictions using a recently developed microbial laboratory system. In this system I will be able to measure population dynamics, network structure as well as genetic change of high-dimensional microbial interaction networks. I expect my results will have broad implications for our understanding of how ecosystems adapt in ecological and evolutionary time to the increasing pressure of anthropogenic change.

The proper progression of development and differentiation relies on complex spatiotemporal regulation of gene expression. DNA sequences, i.e. enhancers, governing this regulation, reside at varying—including distal—locations relative to their regulated genes, where often multiple enhancers regulate the same gene. However, our understanding of the dynamics of enhancer-promoter interactions, and their causal relation to transcription, is highly limited.

To address these fundamental questions, I will exploit advantageous properties of the early Drosophila embryo, where spatially-dependent expression forms a blueprint for the adult fly, across thousands of synchronized nuclei.
I propose to combine genomic approaches with the employment and further development of live-imaging microscopy, to:
1) Identify a large repertoire of enhancer–promoter interactions in the early Drosophila embryo, by conducting a 4C-seq assay for tens of key patterning genes, 2) Characterize the quantitative contribution of enhancer-multiplicity to the transcriptional outcome for select endogenous loci across single-nuclei, using recently developed MS2-based live-imaging and genome editing, 3) Develop experimental and computational capability to simultaneously visualize and quantify enhancer-promoter looping and transcription in living embryos.

The Drosophila embryo will provide a stepping-stone for the application of the developed methods in mammalian cells.
Mechanistic insights from our study promises to advance our understanding of how disease-associated polymorphism within regulatory DNA, that are continuously identified, quantitatively change gene activity and underlie developmental disorders.

Cell polarity is a pre-requirement for many fundamental processes in animal cells, such as asymmetric cell division, axon specification, morphogenesis and epithelial tissue formation. Surprisingly, for all these different processes, polarization is established by the same set of proteins, called partitioning defective (PAR) proteins. Although the PAR proteins are highly conserved and found in all metazoans, it is still not clear how they establish cell polarity and how the spatiotemporal information they provide organizes the cell. This knowledge gap is mainly due to the limited accessibility of the living cell for controlled perturbations and live imaging, which makes a systematic characterization of the underlying molecular processes difficult to achieve. To overcome these limitations, we propose a dual approach combining in vitro and cell extract techniques.
First, we will establish a novel experimental in vitro system, based on cell extracts derived from Drosophila eggs or embryos and membrane-coated microchambers. This assay will allow us to easily manipulate the biochemical content of the system, such as lipid composition of the membrane, the dynamic interactions within the Par network, cytoskeletal components and their interactions with mRNA. In addition, and in contrast to previous approaches, our assay uses microfabricated reaction chambers, which not only mimic the confinement of the cell but also provide us with full control of the reaction geometry. In a parallel approach, we will purify recombinant Par-proteins and quantitatively characterize their interactions to obtain biophysical parameters of the polarizing network which thus far have remained hidden due to the complexity of the cell.
Thus, in one concerted effort, we will gain knowledge about the micrometer scale behavior of the polarizing system as well as the molecular properties of its components. With the help of biophysical modelling, we aim to connect these two different levels of information and obtain a deeper understanding of how this biochemical network establishes spatial information in the living cell.

Animals navigate based on self-motion cues and external references, which serve to correct errors accumulated in path integration. Cells in the medial entorhinal cortex (MEC) encode direction, speed and position, and their output could represent the neural basis for path integration. The vestibular system provides information for angular and linear motion, and lesion studies have suggested that it may be important for path integration-based navigation. Whether and how this is done represents the goal of my project.
In recent years, virtual-reality (VR) has become fashionable in exploring the navigation circuit. Remarkably, two-dimensional (2D) grid properties are compromised in head-fixed rodents experiencing VR. The hypothesis to be tested here is that this occurs because inertial (vestibular) signals are in conflict with locomotion and visual cues – which contrasts with real-world navigation where congruent multisensory cues from vestibular, visual and other modalities all converge to give rise to the sense of motion through space. In VR, only the visual and proprioceptive cues are present, while vestibular cues signal no motion.
I will test the hypothesis that vestibular motion cues are necessary for space coding in the rat MEC. We will monitor grid cell activity during navigation, while rotation and translation inertial cues are independently manipulated using a VR environment mounted on a 3D motion platform. Our findings could revolutionize the understanding of the nature and properties of the spatial code by bridging together diverse expertise and two segregated neuroscience communities (navigation/MEC/hippocampus and vestibular/multisensory).

Control of movement commonly relies on sensory feedback. Similarly, movement influences sensory processing. Vocal production and sound processing are highly integrated for all natural acoustic communication tasks, such as human speech communication; yet these two processes have often been studied separately. This project investigates the audio-vocal integration in the big brown bat, an animal that actively probes the environment by echolocation. The central aim of this project is to test the hypothesis that vocal motor behavior influences the neural activity of inferior colliculus, a central hub in the auditory path way. Neural recordings will be taken from freely behaving bats with chronically implanted electrode arrays, when bats are tracking an approaching food item by echolocation from a resting position on a platform and when bats are flying between two perches. At the same time, sonar vocalizations will be recorded with a light-weight telemetry microphone fixed above the bat’s head. Thus, this project will allow, for the first time, study of neural activity in the inferior colliculus in freely behaving bats over the four periods of echolocation based audio-vocal integration: pre-vocalization, vocalization, vocalization feedback, and echo feedback. Differences in the neural activity between the pre-vocalization/vocalization period and non-vocalization bout (baseline neural activity) reflect the influence of vocal motor behavior on auditory processing. This research project will not only shed light on the mechanisms of audio-motor integration in bat echolocation, but it will also contribute to a broader understanding of sensorimotor feedback mechanisms in mammals.

Mammalian cells developed the ubiquitin system for maintaining a balanced functioning of cellular pathways. It has been recently established that pathogenic bacteria, such as Salmonella, hijack the host ubiquitin system by mimicking the E3 ubiquitin ligases (E3s) for their own benefit. Salmonella E3s are implicated in suppressing the host inflammatory response and assisting the bacteria to escape host defence. However, the mechanism by which bacterial E3s achieve this regulation is currently unknown.
We propose to significantly advance this yet unexplored area of host-pathogen interaction and understand how bacterial E3s subvert cellular environment. By developing an innovative proteomics approach we will investigate the dynamics of ubiquitylation of host proteome covering both early and late time points after bacterial entry. Using functional enrichment analysis we will reveal prominent regulation of the host pathways. In particular we are interested to understand mechanisms by which pathogens suppress host inflammatory response. We aim to learn from bacterial strategies and downregulate chronic inflammation in IBD disease models, much like pathogens suppress inflammation in the host.
Furthermore, this proposal aims to discover novel strategies for antimicrobial therapy. We will develop tools for inducing degradation of bacterial E3s which will enable us to explore the contribution of these enzymes in promoting invasion and proliferation of bacteria in live cells. We anticipate that these findings will be pivotal in developing novel therapeutics to treat life-threatening infections in patients with compromised immunity.

The proposed project aims to dramatically increase the sensitivity of functional ultrasound imaging (fUS) by combining it with another recent breakthrough in ultrasound: genetically encodable acoustic reporters based on gas vesicles (GVs), unique nanostructures from buoyant microorganisms. This work will follow two complementary approaches. In the first approach, I will develop biochemically modified GVs as long-circulating vascular contrast enhancers for fUS. Although they are physically stable structures, wild-type GVs are rapidly cleared from circulation by the reticuloendothelial system. We will extend the circulation half-life of injected GVs in the blood stream through chemical or genetic surface engineering. The second, higher-risk strategy will be to genetically or chemically engineer GV-loaded red blood cells or “echocytes”. The benefit of the echocytes-based approach will be threefold: it is likely to provide the longest enhancement by cloaking GVs; it will confine GVs in cell-like structures to maximize their ultrasound contrast; and it will reduce the dose of contrast agent needed for fUS. These advantages will be assessed through a transcranial imaging study in mice in comparison with existing fUS methods. The significance of enhanced-fUS will be demonstrated by applying it to a real problem in neuroscience: mapping the activation of sensory and reward circuits during and after reward-associative conditioning. Success in either approach is expected to have a major impact in basic neuroscience and potential clinical application by allowing highly-sensitive fUS brain imaging to be performed through intact skulls and in deeper brain areas of larger organisms.

In eukaryotes, most membrane lipids are synthesized in the endoplasmic reticulum and are then delivered to other organelles by both vesicular and non-vesicular transport. Non-vesicular lipid transfer, mediated by lipid transfer proteins (LTPs) that carry lipids through the aqueous cytosolic environment and often act at membrane contact sites (MCSs) regulated by phosphoinositides, help maintain the unique lipid composition of different membranes. For example, cholesterol is synthesized in the endoplasmic reticulum (ER), but most of intracellular cholesterol is localized at the plasma membrane (PM), endosomes and distal Golgi compartments. LTPs that specifically transport cholesterol are thought to play a key role in creating and maintaining these heterogeneity, but the identity of these proteins, their mechanisms of action and their regulation is poorly understood and debated. Recently, a new family of LTPs with StART-like domains and with specificity for cholesterol has been identified. Studies in yeast have proven a key role of some members of this family in the transfer of cholesterol from the ER to the PM at MCSs. The function of three members of this protein family in mammals (GramD1a/b/c) has not been elucidated. In this proposal, I aim to explore the properties of these mammalian proteins with emphasis on cholesterol transport at the ER-PM contact sites. Plans include analysis of the localization and regulated dynamics of genomically tagged GramD1s, analysis of their lipid-transport properties in cell free assays and semi-intact cells, and studies of the impact of these protein on cholesterol distribution using KO and overexpression studies in cells, worms and mice.