This program supports the evaluation of promising but unproven technologies for the detection of single molecules and single molecular events inside cells. Projects include feasibility studies for the development of new imaging probes, evaluation of methods for the delivery and targeting of probes within cells, and basic research on the underlying photophysical properties of probes.
For more information about the program, see the program announcement or contact Dr. James Deatherage, NIGMS Division of Cell Biology and Biophysics, at (301) 594-0828 or deatherj@nigms.nih.gov.
(Note: PAR-06-288 is no longer active and has been replaced by PAR-GM-07-234.)
The following is a list of funded projects.
1. In Vivo Imaging and Dynamics of Single Virus-Like Particles
Abstract (description provided by applicant): It is widely accepted that significant differences between the in-vivo virus cycle and its in-vitro representations exist. In-vitro assays lack the cellular regulatory mechanisms that influence the pathways of viral protein synthesis and self-assembly. We propose to implement a demonstration of a new in-vivo imaging technology, based on virus-like particles (VLPs) and able to bridge the existing gap between in-vitro and in-vivo experiments. Virus-like particles are here hybrid biological/inorganic complexes composed of a viral capsid encapsulating a functionalized nanoparticle instead of nucleic acid. The proposed in vivo imaging technology is of potential high impact since it is not limited to virus assembly but it can be extended to many other self-organizing macromolecular complexes occurring in the confines of a cell. An illustration of the new capabilities is provided through the following proof-of-concept experiments: a) determine real-time disassembly trajectories of individual VLPs in vitro and in vivo with 10 nm spatial resolution and 10 ms time resolution; b) locate individual self-assembled VLPs in cells and their spatial relation with the sites for viral replication. The anticipated results from this proposal will provide a new non-intrusive imaging technology coupled to a means of measuring in-vivo sub-cellular dynamics and will teach us about the rules for virus particle assembly, disassembly, and intracellular transit. We will learn unprecedented basic insight into virus biology. The technology will also broaden the area of future VLP applications and yield a new class of high-output probes that can be adapted to a wide range of targets.
Bogdan Dragnea, Ph.D., Principal Investigator, Indiana University Bloomington
2. A Clonable High-Density for 3-D Electron Microscopy of Cellular Structures
Abstract (description provided by applicant): Recent years have seen a strong resurgence of interest in biological electron microscopy (EM) including cryo-electron tomography. A limitation of EM analysis, in particular in cellular samples, is determining the location of a protein of interest. Our ultimate goal is to develop methods that will combine the reliable preservation of cell structure based on rapid freezing and vitrification with labeling technologies that give sufficient signal-to-noise so that these labels are readily visible by EM, particularly in 3D electron tomograms. We propose to develop a metallothionein gene as a "clonable tag" that will bind gold to enhance its density in a variety of samples for EM. Metallothioneins are small proteins (~6.5 kD) that are avid metal binders and that have been shown to form gold clusters in vitro (Mercogliano & DeRosier 2006. J Mol Biol. 355:211-23). Such a clonable high-density tag would revolutionize the utility of cellular tomography because the 3D position of proteins in complex cellular structures could be determined by tomography at nanometer resolution. The utility of metallothionein as a clonable tag will be explored in two aims. The first aim is to develop metallothionein as a clonable label for cryo-electron microscopy and cryo-electron tomography applied to isolated or in vitro reconstituted macromolecular assemblies. In particular, the microtubule-Eg5 motor complex will be used for qualitative and quantitative assessment of the metallothionein labeling properties. The usefulness of metallothionein as a directly visible density marker in averagable and non-averagable structures will be assayed. Metallothionein-tagged cellular components in vitrified sections of intact cells, likely with the use of silver-enhancement will also be tested. The second aim is to develop metallothionein as a clonable density tag for protein localization in rapidly frozen and freeze-substituted material embedded in plastic. The metallothionein-tagged Eg5 kinesin will be localized spindles assembled in vitro using Xenopus egg extracts, as well as in vertebrate tissue culture cells which are suitable for tomography. Finally, the metallothionein tag will be used in budding yeast on a variety of proteins, including alpha-tubulin in rnicrotubules, the Cin8 kinesin-like motor protein, and Spc42, a very abundant spindle pole component. It is anticipated that the metallothionein clonable density tag will be useful for a variety of EM techniques.
Andreas Hoenger, Ph.D., Principal Investigator, University of Colorado at Boulder
3. Enhanced Delivery of Protein Biosensors: A Combinatorial Library Strategy
Abstract (description provided by applicant): Protein biosensors conjugated with fluorescent dyes play a vital role in imaging key intracellular processes. However current methodologies for the delivery of these biosensors to appropriate subcellular compartments are crude and ineffective. Here we propose a novel and powerful approach to solving this problem. We will use peptide combinatorial libraries expressed in an Adeno Associated Virus vector to screen for peptides (so called 'cell penetrating peptides', CPPs) able to promote efficient intracellular delivery of protein biosensors. We will be particularly interested in peptides that enter cells and accumulate in the cytosol rather than in endomembrane compartments, since many biosensors are designed to function in the cytosol. Candidate peptides (CPPs) emerging from the viral screen will be further tested for innate ability to enter cells and for the ability to effectively deliver dye-based protein biosensors. Promising CPPs will be used to form chimeras with protein biosensors. A variety of techniques will be used to evaluate total cellular accumulation and quantitative subcellular distribution of the CPP/biosensor chimeras. We anticipate that this approach will lead to a cohort of novel CPPs that will have an important impact on the entire field of intracellular delivery of proteins, especially biosensors.
Rudolph L. Juliano, Ph.D., Principal Investigator, University of North Carolina Chapel Hill
4. Probes for Detection of DNA Accessibility in Chromatin
Abstract (description provided by applicant): Changes in DNA accessibility at specific loci in chromatin are important determinants for the regulation of gene activity. The proposed research project will evaluate novel molecular probes capable of detecting changes in DNA accessibility at the single molecule level. The probes will be assembled at specific loci in chromatin, and will generate signals based on bioluminescence. We will construct a novel split luciferase system, which will generate a photon flux high enough to enable single molecule detection. Using mammalian cell culture systems and appropriate DNA transfection vectors, we will evaluate the performance of the molecular probe designs for the purpose of monitoring, though in vivo imaging, changes in chromatin accessibility of specific loci in the genome, as cells undergo normal developmental processes. Evaluation criteria will include signal/noise, signal persistence, spatial resolution, time resolution, and robustness for use in routine biological experiments.
Paul M. Lizardi, Ph.D., Principal Investigator, Yale University
5. Polymer Dot Nanoparticles for Detection of Single Molecules in Live Cells
Abstract (description provided by applicant): The objective of the proposed research is to develop a novel class of fluorescent nanoparticles called "Polymer Dots" and test the feasibility of the nanoparticles for detection of single molecules in live cells using conventional fluorescence microscopy methods. Polymer dot nanoparticles are a promising new fluorescent nanoparticle technology based on fluorescent pi-conjugated polymers which provide substantial improvements in brightness as compared to conventional fluorescent dyes and nanoparticles. The specific aims of the project include optimizing and characterizing relevant figures of merit for single molecule detection, development and testing of bioconjugation strategies for targeting specific biomolecules within cells, and demonstration of single nanoparticle detection in living cells. A range of characterization techniques will be employed, including fluorescence spectroscopy, single molecule spectroscopy, atomic force microscopy, electrophoresis, and epifluorescence microscopy. Improvements in brightness and photostability of a factor of 100 to 10,000 as compared to conventional fluorescent dyes are expected, meeting the requirements for fluorescence-based single molecule detection in living cells. Demonstration of facile, flexible bioconjugation methods for targeting specific biomolecules of interest and detection of individual labeled biomolecules within living cells are also expected. The development of this novel fluorescent nanoparticle platform will facilitate future research involving monitoring transport and biochemical events of single molecules within living cells and could also provide the basis for the development of novel bioassays and biosensors.
Jason D. McNeill, Ph.D., Principal Investigator, Clemson University
6. Probes for Multiplexing Single RNA Molecule Detection in Living Cells
Abstract (description provided by applicant): The goal: To prepare oligonucleotide probes that are coupled to fluorochromes to enter the cell and detect intracellular mRNAs in living cells with single copy sensitivity. This application is dedicated to increase in signal to noise ratio by factors exceeding 20 fold over existing technologies, focused mainly on background reduction approaches. 1. To synthesize fluorochrome coupled oligonucleotide probes using a phosphorothioate linkage to enter the cell and hybridize to RNA. We will then optimize the detection of the hybridized probes by initially using the site of highest signal in the nucleus: where the RNA is being transcribed. Various cellular models will be used with a dynamic range of four orders of magnitude; the highest can generate as many as 48,000 fluorochromes at the transcription site. We will then proceed to quantitatively test improvements in sensitivity by using models with progressively fewer targets. 2. To design a fluorochrome-quencher combination that will result in fluorescence only when the probe hybridizes to its target. We will design a library of chemical compounds to screen using a high-throughput probe fluorescence assay. 3. To design imaging hardware and software to filter out the background generated by unhybridized probes leaving only the hybridized fluorescent signals. 4. To multiplex the probes with different fluorochromes so that we will be able to detect multiple RNA species in the same cell. Ultimately, the approach can be formatted for use in living tissue.
Robert H. Singer, Ph.D., Principal Investigator, Yeshiva University
7. Novel Delivery and Targeting of QDots to Track Single Molecules Inside Live Cells
Abstract (description provided by applicant): Imaging of single molecules inside living cells will provide new and essential insight into key cellular processes such as cellular signaling, physiology, trafficking and cytoskeleton dynamics. Currently, numerous technical hurdles have prevented the achievement of this highly desirable yet elusive aim - the subject of this PAR-06-288. To our knowledge, all classic organic fluorophores (e.g. Cy-dyes) and genetically encoded fluorescent proteins (e.g. eGFP) are at least an order of magnitude too dim to provide single molecule tracking in the cellular milieu, and notwithstanding major advances are unlikely to do so soon. A notable exception is inorganic nanocrystals or quantum dots (QDots), which are exceptionally bright and have already been utilized for extracellular tracking of single molecules. However, technical barriers have severely curtailed extra- and especially inter-cellular use of QDots as a vehicle for imaging single molecules. Major obstacles include: lack of a genetically-encoded tags, difficulty of non-disruptive delivery inside cells, fluorescent 'blinking' and monovalency. In our 3 Specific Aims we will i) use biolistic approaches to rapid delivery QDots inside many cells without aggregation, ii) use a novel biotinylation approach to genetically and specific target QDots to any protein ligand inside cells, iii) use new labeling strategies and encapsulated QDot clusters to respectively circumvent multivalency and 'blinking' problems. Our independent, yet linked aims will provide a deep and innovative long-term strategy to capitalize on the potential of QDots to image single molecules inside cells.
Derek Toomre, Ph.D., Principal Investigator, Yale University
8. Trekking with the Ribognome: Single Molecule Microscopy of Intracellular miRNPs
Abstract (description provided by applicant): Currently, there are no suitable microscopy tools available that would allow researchers to follow the vast number of newly discovered, diverse non-protein coding (nc)RNAs around the cell as they fulfill their numerous biological functions, let alone at the single molecule level. The proposed project will fundamentally overcome this limitation by developing a novel probe concept optimized for detecting single small ncRNA molecules inside living cells. It is expected that our "molecular Christmas tree" probe technology will subsequently be transferable to other biopolymers. When developing a new cell microscopy technique, real-world field testing on a biological system for the purpose of determining performance parameters is essential. Among recently discovered ncRNAs are those associated with the new gene regulatory paradigm of RNA interference (RNAi), where in one pathway micro-RNAs (miRNAs) act to repress endogenous genes in all multicellular eukaryotes, including humans. A founding class member is let-7, or lethal-7, which is an evolutionarily conserved miRNA from C. elegans to humans. It has been found to regulate expression of disease-related transcriptional effectors, among them the High Mobility Group AT-hook 2 (HMGA2) protein involved in transcriptional regulation and associated with various cancers as well as diet-induced obesity. Expression of HMGA2 mRNA is controlled by an unusual seven let-7a-1 binding sites. As a proof-of-principle for our intracellular probe technology we will detect the assembly of let-7 miRNA, HMGA2 mRNA and RNAi proteins into single active micro-RNA-protein (miRNP) complexes, by pursuing the following milestones in collaboration with the groups of Sunney Xie (Harvard U.) and David Bartel (Whitehead Institute/MIT): (1) We will design, synthesize and test single molecule detection in cultured cells on a fully controllable sample. The necessary signal-to-noise threshold (amplification level) for intracellular detection of single assembled miRNP complexes is reached once a sufficient number of labeled let-7a probes are loaded onto a single target and slowly diffuse together in a complex. (2) We will test the developed probe technology on the real-world Let-7a/HMGA2 mRNA probe/target system and uniquely address numerous outstanding questions concerning the cell biology of miRNAs. (3) We will define the scope and limitations of our "molecular Christmas tree" probe technology. That is, in parallel to Aims 1 and 2 we will ask: Is multiplexing (i.e., the detection of multiple targets in parallel) possible? Can multiple tandem repeat sequences in a DNA target be detected? Can protein assembly, particularly that occurring during protein misfolding diseases such as Alzheimer's and prion diseases, be detected by our novel "molecular Christmas tree" probe concept? What are the lower limits for DNA repeat sequence and protein polymerization detection and can these limits be further pushed? Can multiple different biopolymers be detected in parallel and how does this affect detection limits?
Nils G. Walter, Ph.D., Principal Investigator, University of Michigan at Ann Arbor
