Hazen P. Babcock

Single-molecule DNA sequencing of a viral genome.

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150× and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

Visualizing infection of individual influenza viruses

Influenza is a paradigm for understanding viral infections. As an opportunistic pathogen exploiting the cellular endocytic machinery for infection, influenza is also a valuable model system for exploring the cells constitutive endocytic pathway. We have studied the transport, acidification, and fusion of single influenza viruses in living cells by using real-time fluorescence microscopy and have dissected individual stages of the viral entry pathway. The movement of individual viruses revealed a striking three-stage active transport process that preceded viral fusion with endosomes starting with an actin-dependent movement in the cell periphery, followed by a rapid, dynein-directed translocation to the perinuclear region, and finally an intermittent movement involving both plus- and minus-end-directed microtubule-based motilities in the perinuclear region. Surprisingly, the majority of viruses experience their initial acidification in the perinuclear region immediately following the dynein-directed rapid translocation step. This finding suggests a previously undescribed scenario of the endocytic pathway toward late endosomes: endosome maturation, including initial acidification, largely occurs in the perinuclear region.

Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays—which do not interfere with biological activity—to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions (‘stalls’) that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein–DNA interactions.

Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton

By combining astigmatism imaging with a dual-objective scheme, we improved the image resolution of stochastic optical reconstruction microscopy (STORM) and obtained <10-nm lateral resolution and <20-nm axial resolution when imaging biological specimens. Using this approach, we resolved individual actin filaments in cells and revealed three-dimensional ultrastructure of the actin cytoskeleton. We observed two vertically separated layers of actin networks with distinct structural organizations in sheet-like cell protrusions.

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.

Exploring the folding landscape of a structured RNA

Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

Single-molecule transition-state analysis of RNA folding

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozymes two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.

The dynamics of partially extended single molecules of DNA

The behaviour of an isolated polymer floating in a solvent forms the basis of our understanding of polymer dynamics,. Classical theories describe the motion of a polymer with linear equations of motion, which yield a set of ‘normal modes’, analogous to the fundamental frequency and the harmonics of a vibrating violin string. But hydrodynamic interactions make polymer dynamics inherently nonlinear, and the linearizing approximations required for the normal-mode picture have therefore been questioned. Here we test the normal-mode theory by measuring the fluctuations of single molecules of DNA held in a partially extended state with optical tweezers. We find that the motion of the DNA can be described by linearly independent normal modes, and we have experimentally determined the eigenstates of the system. Furthermore, we show that the spectrum of relaxation times obeys a power law.

Dynamics of dilute and semidilute DNA solutions in the start-up of shear flow

We have investigated the dynamics of dilute (10−5C*) and semidilute (⩽6C*) DNA solutions both in steady and in the start-up of shear flow by combining fluorescence microscopy, bulk rheological measurements, and Brownian dynamics simulations. First, the microscopic states, i.e., the conformational dynamics of single DNA molecules in solution during the start-up of shear flow, were examined by fluorescence microscopy. To investigate the macroscopic response resulting from the changes in the microscopic state, the bulk shear viscosity of the same DNA solutions was also measured. While the transient dynamics of individual molecules is highly variable, an overshoot in the ensemble-averaged molecular extension is observed above a critical Wi following an overshoot in shear viscosity for both dilute and semidilute DNA solutions. These two overshoots are further analyzed and explained on a physical basis from our simulation findings. Based on the physical picture, we have derived a simple scaling to predict the s...

Developmental mechanism of the periodic membrane skeleton in axons

Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites. DOI: http://dx.doi.org/10.7554/eLife.04581.001