Benedict Hebert

Sequence information can be obtained from single DNA molecules.

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.

Spatial mapping of integrin interactions and dynamics during cell migration by Image Correlation Microscopy

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of α5-integrin in migrating cells. α5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is ∼1.4 times more concentrated, ∼4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are ∼900 integrins μm–2 (about fivefold higher than surrounding regions). Interestingly, α5-integrin and α-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, α-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 μm min–1), while all of the paxillin diffuses away rapidly.

Actin–myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility

We have analyzed the spontaneous symmetry breaking and initiation of actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the actin network flow was inwards and radially symmetric. Immediately before motility initiation, the actin network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in actin network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted actin network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale actin–myosin network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.

Probing the integrin-actin linkage using high-resolution protein velocity mapping.

Cell migration is regulated in part by the connection between the substratum and the actin cytoskeleton. However, the very large number of proteins involved in this linkage and their complex network of interactions make it difficult to assess their role in cell migration. We apply a novel image analysis tool, spatio-temporal image correlation spectroscopy (STICS), to quantify the directed movements of adhesion-related proteins and actin in protrusions of migrating cells. The STICS technique reveals protein dynamics even when protein densities are very low or very high, and works in the presence of large, static molecular complexes. Detailed protein velocity maps for actin and the adhesion-related proteins α-actinin, α5-integrin, talin, paxillin, vinculin and focal adhesion kinase are presented. The data show that there are differences in the efficiency of the linkage between integrin and actin among different cell types and on the same cell type grown on different substrate densities. We identify potential mechanisms that regulate efficiency of the linkage, or clutch, and identify two likely points of disconnect, one at the integrin and the other at α-actinin or actin. The data suggests that the efficiency of the linkage increases as actin and adhesions become more organized showing the importance of factors that regulate the efficiency in adhesion signaling and dynamics.

Characterization of blinking dynamics in quantum dot ensembles using image correlation spectroscopy

Quantum dots (QDs) are being increasingly applied as luminescent labels in optical studies for biophysical and cell biological applications due to their unique spectroscopic properties. However, their fluorescence “blinking” characteristics that follow power law statistics make it difficult to use QDs in some quantitative biophysical applications. We present image correlation spectroscopy (ICS) in combination with total internal reflection fluorescence microscopy as a tool to characterize blinking dynamics in QDs. We show that the rate of decay of the ICS measured ensemble correlation function reflects variation in blinking dynamics and can be used to distinguish different blinking distribution regimes. To test and confirm our hypothesis, we also analyze image time series simulations of ensembles of point emitters with set blinking statistics. We show that optimization of the temporal sampling and the number of QDs sampled is essential for detecting changes in blinking dynamics with ICS. We propose that t...

Chapter 7 Single-Molecule Fluorescence Microscopy and its Applications to Single-Molecule Sequencing by Cyclic Synthesis

Abstract Single-molecule DNA sequencing (SMDS) had been proposed well before genomic research had advanced to the point where the DNA sequences of a few human individuals became available. Skepticism arose as to whether or not there was a need to replace methods that had been proven to be productive by a new technology. However, DNA information from thousands of individuals is needed to connect genomic information to the function it serves. Direct extensions of current methods are expected to be still much too expensive and slow to collect the amount of DNA and RNA sequence information that is required to enter the next phase in genomic research. Single-molecule techniques show great promise, as the next generation of DNA sequencing methods will allow the required amount of sequence information to be gathered in a timely and inexpensive manner. While several SMDS methods are under development, currently only single-molecule sequencing by cyclic synthesis advanced to the point where sequence information is produced in a massively parallel way directly from single DNA molecules. This sequencing technology relies on incorporation of fluorescently labeled nucleotides by DNA polymerase into complementary strands of DNA that are immobilized to a surface. The individual DNA strands are separated by a few microns and can be monitored as independent entities. The fluorescent signal of each incorporated labeled nucleotide is then sequentially detected using fluorescent microscopy. Because each DNA molecule is sequenced separately there is no need for synchronization between different molecules. Tens of millions of molecules can be sequenced in parallel in single small reaction volume, and thus this method readily produces high-throughput sequencing at a minimal cost. Currently this technique produces short-reading lengths, which make it suitable to re-sequencing applications in which a reference sequence is given. A single reference genome can serve as a template for the thousands of genomes produced by the short DNA fragments. These data can be used to find rare mutations and genetic heterogeneity in multiple target environments with great accuracy, high rates, and low cost. The ability to extract a massive amount of sequence information will equip cancer research with a powerful tool needed to defeat genetic diseases. In this chapter, different aspects of SMDS by cyclic synthesis will be discussed.

Membrane protein dynamics measured by two-photon ring correlation spectroscopy: theory and application to living cells

Many biochemical reactions and processes are regulated by proteins associated with cellular membranes. Trans-membrane proteins play an important role in many aspects of cellular development, cellular migration and signaling, and many diseases. Quantitative measurement of protein dynamics under various experimental conditions can give insights into the mechanisms of interaction and the functionality of the protein. Fluctuation techniques, such as fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS), have been used for such dynamic measurements in membranes. However, FCS is limited to fast dynamics, and ICS works best on a flat 2-dimensional area. We present an alternative way to measure protein transport in spherical (non flat) living cells that combines laser scanning microscopy and image correlation methods: ring correlation spectroscopy (RCS). The RCS analysis is performed on CLSM or two-photon cross-sectional images of labeled proteins in the cell membrane, where the optical sectioning gives a “ring” of fluorescence in the images. We present computer simulations of two dimensional diffusion confined to the surface of a spherical shell, where the RCS analysis can extract the set input parameters from the simulation. As well, we present RCS analysis of two-photon microscopy images of Pre-B leukocytes cells expressing CD44 labeled with EGFP.

Mapping Protein Transport and Interactions with Nonlinear Image Correlation Spectroscopy

The seminar will introduce two-photon image correlation methods and their application for measurements in of adhesion protein transport and interactions in living cells.