Speakers

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8:40 a.m. - 9:05 a.m.
Quantitative Characterization of Nanomedicine Delivery Systems by Electron Microscopy
Richard Leapman, Ph.D.
National Institute of Biomedical Imaging and Bioengineering (NIBIB), NIH

Electron microscopy provides a method for assessing the degree of monodispersity of nanomedicine delivery systems. Two important parameters of bio-nanoparticles are their total mass and the numbers of specific bound atoms. These quantities can be measured by combining the operation modes of scanning transmission electron microscopy (STEM), and the energy-filtered transmission electron microscopy (EFTEM), respectively. It is shown how these techniques can be applied to characterize PAMAM dendrimers containing Gd MRI contrast agent, which are designed for diagnostic imaging, and single-walled carbon nanotubes containing the anticancer drug cisplatin. Furthermore, by applying the technique of electron tomography, in which an image series is recorded over a range of tilt angles, it is feasible to visualize the distribution of nanoparticles within a cell by reconstructing the 3D volume.

Richard Leapman received his Ph.D. in physics from the University of Cambridge, UK. He did postdoctoral fellowships at the University of Oxford and Cornell University, where he performed research on electron nanospectroscopy. Dr. Leapman then moved to the NIH, where he integrated electron microscopy with spectroscopy to image the elemental composition of organelles in sectioned cells. His current interests include combining quantitative imaging modes based on energy-filtered and scanning-transmission EM with electron tomography to provide 3D structure on a nanoscale.

9:05 a.m. - 9:30 a.m.
Multifunctional Lipid-based Nanocapsules and Triggered Chemotherapy
Robert Blumenthal, Ph.D.
National Cancer Institute (NCI), NIH

The design of multifunctional nanoparticles that deliver their payload into specific organelles and cell interiors is one of the main challenges facing nanotechnologists involved in nanomedicine. My talk will focus on the cell membrane as the frontier between the cell and its environment. The plasma membrane is a lipid-based sheath that envelops a cell, encloses the cytoplasm, and creates a selectively permeable barrier. The lipid bilayers are thin, flexible self-sealing boundaries that are used by cells to create regions of different composition and electrochemical potential. The lipid bilayers are covered with glycoconjugates and interspersed with proteins, which serve as gatekeepers that regulate active and passive transport of molecules essential for cellular functions. The living cell must retain molecules, such as DNA, RNA, and its variety of proteins from dissipating away, while keeping out foreign molecules that might damage or destroy the cell's contents, including molecules essential for life. Our task as cancer nanotechnologists is to find clever ways of selectively destroying diseased (cancer) cells while leaving normal cells intact. In my talk I will discuss our approach to the design of multifunctional lipid-based nanoparticles (Liposomes), which are designed to specifically target and deliver drugs to tumor cells when triggered by heat or light.

Dr. Blumenthal obtained his M.Sc. at the University of Leiden, The Netherlands, and his Ph.D. in physical chemistry at the Weizmann Institute, Israel studying mechanisms of active transport across membranes. Following postdoctoral work at the Institute Pasteur and at Columbia University studying molecular mechanisms of membrane excitability in neurons, he came to the NIH and was ultimately recruited by the NCI. In 1978 he was tenured and in 1980 he became chief of the Section on Membrane Structure and Function. In 2005 he was appointed director of the newly established Center for Cancer Research Nanobiology Program. Dr. Blumenthal has worked in a wide range of areas in membrane biophysics, which includes membrane fusion, membrane transport, cell surface receptors, immune cytotoxic mechanisms, and use of liposomes for delivery of drugs and genes into cells. Dr. Blumenthal's current interest is in the biology of virus and nanoparticle entry into cells and tissues.

9:30 a.m. – 9:55 a.m.
Bridging the Nano Gap: 3D Electron Microscopy of Cells and Viruses at Molecular Resolution
Sriram Subramaniam, Ph.D.
NCI, NIH

Emerging methods in three-dimensional biological electron microscopy provide powerful tools and great promise to bridge a critical gap in imaging in the biomedical size spectrum. This “nano gap” comprises a size range of considerable interest that includes cellular protein machines, giant protein and nucleic acid assemblies, small subcellular organelles and bacteria. These objects are generally too large and/or too heterogeneous to be investigated by high resolution X-ray and NMR methods; yet the level of detail afforded by conventional light and electron microscopy is often not adequate to describe their structures at resolutions high enough to be useful in understanding the chemical basis of biological function. The long-term mission of our research program is to obtain an integrated molecular understanding of cellular architecture by combining novel technologies for 3D biological imaging with advanced methods for image segmentation and computational analysis. I will review our recent progress in imaging and modeling dynamic biological systems, with particular emphasis on applications to HIV/AIDS and cancer.

Dr. Subramaniam received his Ph.D. in Physical Chemistry from Stanford University and completed postdoctoral training in the Departments of Chemistry and Biology at M.I.T. He is chief of the Biophysics Section in the Laboratory of Cell Biology at the Center for Cancer Research, National Cancer Institute, and holds a visiting faculty appointment at the Johns Hopkins University School of Medicine. His current work is focused on the development and application of advanced technologies for imaging viruses and cells at molecular resolution using 3D electron microscopy to address fundamental problems in signal transduction, AIDS and cancer research.

9:55 a.m. - 10:20 a.m.
Single-Molecule Studies on the Mechanism of Myosin V: A Molecular Motor
James Sellers, Ph.D.
National Heart, Lung, and Blood Institute (NHLBI), NIH

Myosin V is an unconventional myosin involved in the transport of various cargoes within cells. Its two-headed structure and ATPase kinetics allow it to move processively along actin filaments taking 36 nm steps in a hand-over-hand mechanism in which a rigid domain of the molecule, termed the neck, acts as a lever arm. The kinetics of the two heads are strongly gated to essentially prevent product release from the lead head as long as the trail head is attached. This insures directionality and aids in processivity. One molecule of ATP is hydrolyzed per step. I will discuss how these facts have been elucidated using primarily single molecule techniques.

Jim Sellers received his PhD degree in Biology with Andrew Szent-Gyorgyi at Brandeis University and then came to NIH to carry out postdoctoral work with Robert Adelstein. After three years of postdoctoral work he started his own research group within NHLBI and is currently the Chief of the Laboratory of Molecular Physiology. He has had a long standing interest in the structure, function and regulation of myosin molecules. Several single molecule techniques are employed by the Sellers Lab and his collaborators to study myosin including optical trapping nanometry, single molecule motility assays utilizing high precision TIRF microscopy, atomic force microscopy and single particle electron microscopy.

10:20 a.m. – 10:40 a.m.
Break

10:40 a.m. - 11:05 a.m.
From Nano-particles to Micofabrication for MRI of Cell Tracking
Alan Koretsky, Ph.D.
National Institute of Neurological Disorders and Stroke (NINDS), NIH

The availability of nanosized dextran-coated iron oxide particles has led to a large interest in using MRI to track cells in animals and humans. Resident macrophages can be imaged in a number of tissues and diseased states after injection of these particles. Furthermore, a large number of cells have been labeled ex-vivo and then migration can be followed in vivo with MRI. It takes over a million nanoparticles in a cell to achieve sufficient detectability by MRI. Recently we have shown that single, micron-sized particles of iron oxide can be detected and used to detect single cells in vivo. These particles have proven useful to study the migration of endogenous neural progenitor cells as they migrate from the subventricular zone to the olfactory bulb. Use of micron sized particles for MRI has opened the possibility of using top-down microfabrication techniques to construct contrast agents for MRI. Structures with interesting MRI properties can be made and may prove useful for cell tracking by MRI.

Alan Koretsky received his PhD in chemistry from UC Berkeley working with Michael Weiner and Mel Klein. He then did a postdoctoral fellowship with Bob Balaban at the NHLBI at NIH. After twelve years as a faculty member at Carnegie Mellon University, Dr. Koretsky returned to NIH in 1999 to start the Laboratory of Functional and Molecular Imaging in NINDS. His group has a long standing interest in developing novel MRI contrast to study brain structure and function.

11:05 a.m. - 11:30 a.m.
Nanomechanics of Intrinsically Disordered Proteins: Coupling Elasticity and Signal Transduction
Kuan Wang, Ph.D.
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH

Natively disordered segments are prevalent in eukaryotic multidomain proteins. These intrinsically unstructured regions link folded domains and are hot spots for protein/RNA/DNA interactions and posttranslational modifications. They play major roles in signaling transduction, cell control, and transcriptional and translational regulation by the characteristic high specificity and low affinity interactions. Nanomechanical and protein interaction studies from our lab on two giant elastic proteins, titin and nebulin, led us to propose that the direct coupling of elasticity and ligand binding of intrinsically disordered protein segments are biologically important attributes for these key players in signaling and stress pathways. This is a novel mechanism for titin and nebulin to sense force and transduce force to biochemical signals in contractile machinery.

Dr. Kuan Wang is the Lab Chief of the Laboratory of Muscle Biology and at NIAMS. Dr. Wang received his Ph.D. degree in Molecular Biochemistry and Biophysics from Yale University. Dr. Wang was professor of Chemistry and Biochemistry at the University of Texas at Austin from 1977 to 1997. Dr. Wang has made significant contributions to the study of muscle biology and cell motility, especially the roles of giant elastic proteins, titin and nebulin, in the assembly, regulation and dysfunction of the cytomatrix in cardiac, skeletal and smooth muscles and in non-muscle cells.

11:30 - 11:55 a.m.
Microscopy studies of the integration of actin dynamics with adhesion in cell migration
Clare Waterman, Ph.D.
National Heart, Lung, and Blood Institute (NHLBI), NIH

Cell migration is mediated by forces generated in the dynamic acto-myosin cytoskeleton and transmitted through mechanosensitive integrin-mediated focal adhesions to the extracellular matrix. We use high-resolution multi-mode and super-resolution light microscopy of cytoskeletal and adhesion proteins together with biophysical measurements of cell-generated forces in living, migrating cells to elucidate the mechanism underlying this process.

Clare Waterman's career has focused on understanding the fundamental basis of cell movement. She was drawn into this question as a master's student in Exercise Physiology at UMASS Amherst where she was first exposed to muscle biology. She then received her PhD from University of Pennsylvania studying the biochemistry of the microtubule motor protein, dynein. She performed post-doctoral training in E.D. Salmon's lab at UNC Chapel Hill, where she was instilled with a love of the light microscope as a quantitative tool for analysis of dynamic protein macromolecular ensembles mediating cell movement. She initiated her independent career at the Scripps Research Institute in 1999 and was tenured in 2005. In 2007, she moved her research program to the intramural program at NHLBI where she is the Chief of the Lab of Cell and Tissue Morphodynamics. She is the recipient of the Raymond & Beverly Sackler International Prize in Biophysics (2007) and the NIH Director’s Pioneer Award (2005)