The trans-Golgi network (TGN) is considerated to be a "sorting center". The vesicular trafficking occuring in this process has implications for many processes ranging from cellular homeostasis to complex organ development and whole animal function. Clathrin-coated vesicles are critical carriers of the traffic between the TGN and endosomes. The live cell imaging methods developed in Dr. Kirchhausen's laboratory are a potentially powerful way to decipher the mechanisms that underlie misunderstood clathrin-mediated traffic between the TGN and the endosomes. For this work, I will use a novel 5D live cell imaging approach recently implemented in Dr. Kirchhausen’s laboratory, which permits the automatic identification and analysis of hundreds of clathrin-mediated events occurring within a single living cell in a period of a few minutes. I will determine (1) What kind of structures do they adopt? (2) Are there carriers of small size only (as seen by electron microscopy), similar in characteristics to endocytic coats, or are there also a large number of long lasting tubular carriers budding from the perinuclear region? (3) Do the same structures contain AP-1 and GGA1, or are these adaptors sorted independently? (4) Where (precisely) do TGN and endosome-derived coated vesicles assemble? (5) What are their dynamics and how are the role of the small ArfGTPase in coat formation? (6) What cargos do they bear? (7) Finally, what is the dynamic behavior of clathrin and Hrs in the postulated organizing platforms found in association with early endosomes and presenting relatively large non-curved arrays of clathrin?

The mammalian cell nucleus contains numerous highly dynamic subdomains. I am particularly interested in the formation of these subnuclear structures and in their biological roles in the cell. I previously studied the intranuclear targeting of RNA molecules, from one nuclear domain to another. I now wish to analyze the dynamics of these nuclear structures using an approach which will provide a quantitative picture of the flux of proteins through nuclear bodies. In consultation with A. Lamond, I have chosen to study Cajal body (CB) proteome dynamics. This will require me to perform a large-scale purification of CBs, followed by an analysis of CB components by Mass Spectrometry. Then I aim to analyze the dynamic properties of the CB proteome, both during the cell cycle and in response to different metabolic perturbations, using the SILAC approach. The resulting data should enhance our understanding of the functions of CBs and their mechanism of assembly. This will be combined with parallel quantitative immunofluorescence and molecular studies on selected interesting CB components. These proteomic and microscopy techniques, which are novel for me, are in routine use in the Lamond laboratory. Thus, this fellowship will provide me with an excellent training in cutting edge techniques, which will help me to develop my future career as a research scientist. Furthermore, I will provide other researchers with access to an online CB database hosted at www.LamondLab.com. I am confident that the combination of quantitative proteomics and fluorescence microscopy, used in concert to characterize dynamic cellular processes, represents an effective approach to study nuclear organization.

The aim of this fellowship is to investigate whether Mnt is a 'master' controller of Myc complexes. Myc is a transcription factor that can promote cell growth, proliferation, transformation and apoptosis and is over-expressed in many human cancers. Conventional wisdom has held that Mad and Mnt transcriptional repressors antagonize Myc activation of target genes. However, recent evidence has suggested instead that Myc relieves Mnt-mediated repression, antagonising Mnt to achieve oncogenicity. Both of these models predict that the level of Mnt will influence the oncogenic potential of Myc. Recent results from the Cory laboratory with transgenic mice emphasize the importance of Myc levels in the development of hematopoietic malignancy. Using these mouse strains as sensitive indicators of Myc-induced tumorigenesis, this project aims to investigate the impact of loss of Mnt on Myc-induced tumor incidence and phenotype. I will bring the skills and techniques to investigate the contribution of Mnt to Myc-induced tumorigenesis at the transcriptional level. However, I have no experience in generating or characterizing mouse strains to assess the in vivo importance of such events in development and disease. The Cory laboratory has a proven history in defining Myc-induced tumorigenesis in mice. It is the perfect environment to investigate the role of Mnt in Myc induced malignancy whilst acquiring in vivo experimental experience. This combination will give me a strong base from which to launch my independent research into the molecular basis of tumorigenesis upon completion of this fellowship.

The cytoskeleton is a diverse, multi-protein framework that plays a fundamental role in many cellular activities including cell division and migration. I am interested in studying the mechanisms allowing the dialogue between two components of the cytoskeleton, the Microtubules (MTs) and Filamentous Actin (F-actin) during cell migration and cytokinesis. These two processes are critical in tumor invasion and metastasis and the understanding of their regulation may help developing appropriate therapies for cancer. Combining in vivo approaches and live analysis in cellulo, I have shown that Dmoesin, the sole orthologue of the ERM family in Drosophila melanogaster, was required prior to cytokinesis completion by controlling cortex plasticity during mitosis to position the actomyosin cytokinesis ring. Interestingly, I have also observed that Dmoe inactivation in cells leads to adhesion defects, depending on F-actin and MT dynamics leading to the hypothesis that some of the mechanisms allowing MTs to regulate Actin during these processes are conserved. The aim of this project is to provide a better understanding of the crosstalk between MTs and actin in cytokinesis and cell migration. This study will explore several complementary axes: - A precise in cellulo and in vivo analysis of Dmoe and identified partners in cytokinesis. - A functional screen aimed to identify new components of the crosstalk between MTs and F-Actin during cytokinesis. - The test of the hypothesis that regulatory mechanism between cytokinesis and migration are conserved. The CDA will provide support for carrying out the study on cytokinesis and will allow the development of the parallel study on cell migration.

Telomeres protect the ends of eukaryotic chromosomes from fusion and degradation. They are maintained by the ribonucleoprotein enzyme telomerase. Most human somatic cells do not express telomerase and as a result, telomere length progressively decreases with each round of cell division, limiting their replicative potential. Telomerase is ectopically active in 80-90% of all tumors and may be important for tumor cells to escape replicative senescence. Thus cancer researchers have studied it extensively, as telomerase and its regulators are promising anti-cancer drug targets. My Ph.D. work focused on the DNA damage response, defects in which can lead to genomic instability, a hallmark of cancer cells. Therefore moving to a lab that studies telomere regulation will allow me to explore a different field in cancer research. Elucidating the molecular mechanisms behind telomere length homeostasis has been hindered by the lack of assays that delineate the non-uniform telomere extension events of single chromosome molecules. The Lingner lab has recently described a system to measure elongation of single telomeres in vivo at nucleotide resolution. I will utilize this novel system to analyze known mutants that affect telomere length homeostasis. Using purified proteins under defined reaction conditions, I plan to perform in vitro telomerase assays to determine whether these factors directly influence telomerase activity. The proposed research plan will greatly advance our understanding of the mechanism and regulation of chromosome end maintenance. Given Dr. Lingner’s prominence in the telomerase field, his lab provides an excellent environment to conduct such a study.

The sense of taste provides crucial information about the nature and palatability of food sources. One of the most important questions in sensory and gustatory neurobiology is to understand how the taste system encodes taste quality both peripherally and centrally. Recent molecular and genetic studies have significantly advanced our understanding of peripheral events in taste reception and discrimination. However, in order to define the neural principles that govern taste, it is necessary to identify the neuronal pathways that relay and process peripheral sensory information into the relevant representations in the brain. In this proposal, I propose to utilize various neuro-anatomical tracing methods to define the connectivity pattern between identified taste receptor cells on the tongue and neurons in the geniculate and petrosal ganglia. This fellowship will provide me the unique opportunity to change of my focus from cellular physiology to molecular genetic approach and system neurobiology, which I have had no opportunity to learn in China. This fellowship experience will undoubtedly provide me with new ideas, broaden my expertise, and help me to achieve my scientific goal independently. Furthermore, it will provide me the chance to join one of the leading labs studying the molecular genetics of taste perception in the world, which I believe will surely help me to head my own research laboratory in the field of rodent sensory neuroethology in future.

During embryogenesis, non-canonical Wnt/planar cell polarity (PCP) signaling directs the polarized intercalation and rearrangement of mesoderm and ectoderm cells, which shapes the basic vertebrate body plan at gastrulation. At neurulation, defects in PCP signaling are thought to cause spina bifida and anencephaly - neural tube closure defects that affect 1 in every 1000 human births. PCP signaling has also been implicated in skin development, polarization of the inner ear sensory epithelium, cardiac development and cancer, yet little is known about how PCP signals ultimately regulate these cellular processes. Using zebrafish as a model system for embryological and genetic study, I intend to investigate fundamental questions regarding the establishment and maintenance of PCP signaling during vertebrate embryonic development. Forward genetic approaches will be used to screen for novel regulators and modifiers of vertebrate PCP signaling. I will also utilize an innovative germ line-replacement technology in zebrafish to examine the largely uncharacterized maternal effects of zygotic lethal PCP mutations. Using these PCP signaling mutants and newly developed in vivo fluorescent markers of PCP, I will also perform an intricate series of cell-transplantation studies to visualize, in real time, the effects that local perturbations to PCP signaling have on the establishment, direction and maintenance of planar polarity. An HFSP CDA award would afford me the unique opportunity to apply the reagents and expertise obtained during my HFSP fellowship towards this research, providing me with the financial and scientific independence, tools and support needed to make this work possible.

Our capacity to form memories is believed to require a property of neurons called synaptic plasticity -the ability to specifically and dynamically alter the strength of connections between neurons. LTD and LTP, the long term potentiation and depression of excitatory synaptic transmission, are wide-spread phenomena of synaptic plasticity expressed at possibly every excitatory synapse in the brain. The focus of my post-doctoral research is to elucidate the mechanism underlying LTD of NMDA receptor (NMDAR)-mediated synaptic transmission in the hippocampus, a poorly defined form of plasticity which may underlie memory storage and retrieval. Specifically, I will address a model whereby disruption of tethering of the NMDAR to the actin cytoskeleton is crucial for LTD of NMDAR-mediated excitatory postsynaptic currents (EPSCs). Anchoring of the NMDAR to actin within the post-synaptic density (PSD) is mediated by interactions with proteins of the spectrin / actinin family as well as adaptor proteins, such as PSD-95. I will address the prospect that the mechanism underlying this form of plasticity involves dissociation of the interaction of the NMDAR with PSD-95 or alpha-actinin, potentially through the action of the protease calpain. I intend to examine this model by modifying the molecular repertoire of a electrophysiologically-monitored postsynaptic cell, utilizing small molecule inhibitors, competitive peptides and viral-mediated gene knockdown or overexpression. This project will involve integration of biochemical and molecular proficiency I gained in my Ph.D. with electrophysiological skills I will acquire under the supervision of Prof. Robert Malenka at Stanford University.

The surface of all mammalian cells is covered with glycolipids and glycoproteins that form a dense and complex array of sugar chains. These chains are usually terminated by a member of the large family of sialic acids. The two most common forms of sialic acid found in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivate N-glycolylneuraminic acid (Neu5Gc). Due to a mutation in the CMP-Neu5Ac-hydroxylase gene that occurred after our common ancestor with the chimpanzee, human cells cannot produce Neu5Gc - instead they carry an increased amount of Neu5Ac, its metabolic precursor. This resulted a marked change in cell surface sialic acid composition, which was suggested by the Varki lab to have a significant role in the divergence of the human linage from the great apes. It is already shown to affect interactions with pathogens, causing resistance or susceptibility to attack. Interestingly, all mammalian brains have markedly reduced levels of Neu5Gc, suggesting an advantage to the complete loss of Neu5Gc in humans. We will study the impact of varying Neu5Gc/Neu5Ac ratios in human cells on the biophysics and biology of cells in a very uncommon approach. We will combine single cell analysis and imaging techniques, which are my area of expertise, with biochemical techniques, developed in the Varki lab, to study the implications on cell adhesion, motility, and cell-cell interactions. Special emphasis will be on neural cells and polysialic acids (known to be involved in neural plasticity). This work will introduce me to the unique experimental approaches for studying sugars. Eventually, I plan to open my own glycobiology laboratory in Israel.