Daniel Branton - US grants

Knowledge of how the protein constituents of coated vesicles interact is required if we are to understand how the formation of a coated vesicle coordinates the morphological events of endocytosis or membrane flow with the biochemically specific events of receptor mediated uptake or intracellular protein sorting. Using coated vesicles isolated from mammalian tissues, binding assays, competition assays, and electron microscopy will be used to establish the specificity, affinity and structure of the connections that organize the coated vesicle proteins. Starting at the cytoplasmic side of the coated vesicle and extending eventually to the receptors that face the exoplasmic surface, proteins, polypeptides or fragments of polypeptides will be selectively stripped from the vesicles, and the ability of these components to reassociate with themselves in solution or to rebind to the selectively stripped vesicles in the presence of different ionic environments or protein factors will be measured. Because many cellular functions depend on coated vesicle function, perturbation of the normal protein interactions of coated vesicles can induce numerous disease states. Familial hypercholesterolaemia exemplifies one of the best documented diseases related to malfunction of receptor mediated endocytosis. Understanding coated vesicle function will also be important in controlling diseased states. Targeted cell killing using receptor dependent ligand uptake exemplifies one of the more obvious disease control strategies that will rely on knowledge of how coated vesicles function.

Spectrin function will be analyzed in a model system, Drosophila melanogaster. Spectrin is a membrane-associated cytoskeletal protein that plays a key role in the determination of erythrocyte cell shape. spectrin also plays unknown, but essential, roles in non-erythroid cells. As a result of our recent work that has identified alpha-spectrin mutants, we are in the singular position of being able to directly assess the functions of spectrin in non-erythroid cells. Using a combination of biochemical, molecular genetic, and classical genetic analyses, we will test various hypotheses for spectrin function in non-erythroid tissue at the cellular, developmental and organismal level. Beta-spectrin mutants will be produced and these, together with recently identified alpha-spectrin mutants, will be utilized to investigate spectrin function in vivo. Phenotype analysis will determine the effects of the complete absence of functional spectrin; mosaic analysis will determine the effects of spectrin absence at selected developmental stages. This phenotype information will be used to design experiments that rescue null mutants with spectrin-DNA constructs that will test the cellular or developmental role of a specific aspect of spectrin organization, such as membrane binding, or spectrin chain- length. Although spectrin isoforms are present in nearly all human cells, the neurological and general health consequences of defective spectrin expression in non-erythroid cells are only beginning to be explored. The proposed experiments will provide an in-depth understanding of spectrin's role in non-erythroid cells.

A confocal scanning imaging system will be acquired as a multiuser instrument. This instrument will be employed to enhance ongoing research of several investigators whose work concerns specific cellular components and cellular connections in a variety of complex, developing tissue systems, including cytoskeletal and decapentaplegic proteins in Drosophila, Caenorhabditis, synaptic organization in fish retina, cell cycle regulatory proteins in Caenorhabditis and yeast, axonal guidance and outgrowth in mouse, and protein expression in transgenic plants.

9421831 Branton We propose a new method for rapidly sequencing polymers such as DNA or RNA. The method involves measurements of ionic current modulation as the nucleotides of a linear nucleic acid molecule pass through a channel in an artificial membrane. Our immediate goal is to prove that we can draw single stranded DNA or RNA through a continuously open channel in a lipid bilayer and to demonstrate that, during polymer passage through the channel, ionic currents are reduced in a manner that reflects the properties of the polymer (length, nucleotide composition, etc.). Our long term goals are to demonstrate the feasibility of using a patch-clamp- like approach for direct electro-sensing of monomer sequences in a linear polymer of DNA; and develop this as a routine procedure for sequencing. Because the approach proposed eliminates several of the currently required preparatory chemical steps in DNA sequencing and because it depends on inherently rapid, molecular events, successful sequencing with this method will be an extraordinarily important advance that should reduce the time and cost of nucleic acid sequencing by several orders of magnitude. %%% A new method for the rapid sequencing of nucleic acid polymers like DNA and RNA will be developed. The method to be used involves measurements of changes in ionic currents as the individual building blocks of a nucleic acid pass through a channel in an artificial lipid membrane. The immediate goal of this research project is to show that single stranded DNA or RNA can be drawn through a continuously open channel in the membrane (lipid bilayer) and to demonstrate that during passage of the polymer changes in ionic currents are reduced in a manner that reflects the properties of the nucleic acid (i.e., length, composition of the individual nucleotides which make up the nucleic acid). The long term goals are to demonstrate the feasibility of using this patch- clamp-like approach for direct electro-sensing of ind ividual nucleotides (the building blocks of nucleic acids) in the linear sequence of DNA; and to develop this as a routine procedure for DNA sequencing. Because the proposed approach eliminates several of the currently required preparatory chemical steps in DNA sequencing and because it depends on inherently rapid molecular events, successful sequencing with this method will be an important advance that should reduce the time and cost of nucleic acid sequencing by several orders of magnitude.

DESCRIPTION: Based on their work and that of other laboratories, these investigators propose that hDlg is a component, possibly the essential nucleating agent, of a multi-protein complex of plasma membrane- associated signaling molecules. This hypothesis is based on the observed binding of hDlg sequences to APC, Abl, Crk, Lck, a new putative dual-specificity protein kinase called PBK1 (PDZ-Binding Kinase 1), T/SXV motif channel proteins, SH3-containing proteins, polyproline- containing proteins, and/or protein 4.1 oezrin. The proposed research will test this hypothesis by: (1) refining the map of interaction domains between hDlg and 4.1/ERM proteins and the role of this interaction in targeting to the membrane; (2) quantifying and refining the understanding of hDlg association with itself; (3) quantifying and refining the partners; and (4) assessing the implication of the PDZ- mediated association between hDlg and a putative kinase identified by a yeast two-hybrid screen. The long-term goal is to understand better the mode of action and biochemical characteristics of this tumor suppressor protein.

DESCRIPTION: Based on their work and that of other laboratories, these investigators propose that hDlg is a component, possibly the essential nucleating agent, of a multi-protein complex of plasma membrane- associated signaling molecules. This hypothesis is based on the observed binding of hDlg sequences to APC, Abl, Crk, Lck, a new putative dual-specificity protein kinase called PBK1 (PDZ-Binding Kinase 1), T/SXV motif channel proteins, SH3-containing proteins, polyproline- containing proteins, and/or protein 4.1 oezrin. The proposed research will test this hypothesis by: (1) refining the map of interaction domains between hDlg and 4.1/ERM proteins and the role of this interaction in targeting to the membrane; (2) quantifying and refining the understanding of hDlg association with itself; (3) quantifying and refining the partners; and (4) assessing the implication of the PDZ- mediated association between hDlg and a putative kinase identified by a yeast two-hybrid screen. The long-term goal is to understand better the mode of action and biochemical characteristics of this tumor suppressor protein.

DESCRIPTION (provided by applicant): I will develop the technology and basic science needed to use nanopores for high speed SNP detection and haplotyping. The technology depends on driving single polynucleotide molecules through a nanopore coupled to sensitive single channel recording electronics that can provide a direct read-out of the polymer's characteristics. The development of these new molecular diagnostic methods takes advantage of three recent discoveries: (1) A membrane channel, or nanopore, can be used as a high-throughput device that detects and probes single molecules as they translocate through the nanopore; (2) The ionic current through the nanopore is sensitive to local changes in the cross-sectional area of the translocating molecule; (3) A new, planar fabrication method - ion beam sculpting - that allows us to create a single digit nanoscale pore of a desired dimension in robust solid state insulating membranes. Our work will optimize: (a) zinc-finger protein labeling of DNA; (b) the electrical readout of DNA length; (c) the minimum fragment length and number of different zinc-finger proteins needed to achieve reliable SNP identification and high-speed haplotyping. The proposed technologies will impact basic research areas such as development and cancer that must deal with complex sets of genes and mutations, and where existing methods to rapidly examine the linkages between a large number of polymorphic sites on multiple chromosomes in a large number of individuals are limiting. The tools and basic research proposed will open new possibilities for the future development of high sensitivity, information rich diagnostic methods that are critical for early disease detection.

DESCRIPTION (provided by applicant): The long-term objective is a nanopore detector chip for a general utility instrument capable of inexpensive de novo sequencing that can also be used for re-sequencing projects. The instrument directly generates base-dependent electronic signals as multi-kilobase length fragments of single stranded genomic DNA is driven sequentially through nanopores articulated with electrically contacted single walled carbon nanotube probes. The final system is intended to provide a relatively high quality sequence from =6.5-fold coverage of a genome using DNA from fewer than 1 million cells, with no amplification or labeling. The specific aims are: 1) Characterize ungapped nanotube articulated nanopore detectors in ionic solution with and without DNA molecules to establish a device model;2) Control ssDNA binding, translocation, and sliding on the nanotube surface exposed in ungapped nanotube articulated nanopores;3) Study and optimize DNA molecule induced field effect modulation of nanotube electrode conductance in ungapped nanotube articulated nanopores as a function of nanotube bias, gate voltage, and solution properties;4) Analyze and optimize tunneling current modulations between gapped nanotube electrodes in the first generation detector;5) Design and fabricate a second generation detector with embedded 'T'nanotube geometry and achieve 1 Kb/sec sequencing on Kb length strands of DNA;6) Design a third generation nanopore detector for high throughput 10 Kb/sec/nanopore sequencing. If we are able to resolve each base as it passes through a nanopore at the rate of 104 bases/sec as proposed here, an instrument with an array of 100 such nanopores could produce a high quality draft sequence of one mammalian genome in ~20 hours at a cost of approximately $1,000/mammalian genome. Genomic sequencing at these reduced costs would make vital contributions to improved human health on many fronts, including the understanding, diagnosis, treatment, and prevention of disease;environmental science and remediation;and the genetics of human health and disease derived from the understanding of evolution. PROJECT HEALTH RELEVANCE We are developing the core detector of an instrument that could produce a high-quality draft sequence of one mammalian genome in ~20 hours at a cost of approximately $1,000/mammalian genome. Genomic sequencing at these reduced costs would make vital contributions to improved human health on many fronts, including the understanding, diagnosis, treatment, and prevention of disease;environmental science and remediation;and the genetics of human health and disease derived from the understanding of evolution.

Our research has shown that a single layer of graphene is an ideal membrane in which to fabricate high resolution nanopore detectors to sense the presence of single DNA molecules and their nucleobases. Our objective is to develop the tools and procedures needed to realize a scalable nanopore sequencing device which will significantly reduce future de novo sequencing cost by directly identifying the nucleobases on single stranded genomic DNA molecules that are driven sequentially through an array of precisely dimensioned graphene nanopores. The final system is intended to provide a relatively high quality sequence from >6.5-fold coverage of a genome using DNA from fewer than 1 million cells, with no amplification or labeling. The specific aims are to: a) implement a graphene edge-sputtering process to facilitate high precision fabrication of nanopore arrays; b) optimize discrimination between the four nucleotides of DNA using ionic current blockades or in-plane conductivity change when ssDNA polymers are driven through graphene nanopores; and c) support lipid bilayers across graphene apertures to enhance the feasibility of parallel recordings from arrays of protein pores. Successful completion of these aims will provide the key building blocks of a nanopore sequencing device that can accurately sequence an entire human genome at a cost of less than $1,000. The ability to inexpensively and accurately sequence complete genomes has the potential of remarkably improving many facets of human life and society, including the understanding, diagnosis, treatment and prevention of disease.