Biomedical subjects

Hazen P Babcock

Publications and source records attributed to Hazen P Babcock.

8 recordsLinked to original sources

PubMed2004

Using single-particle tracking to study nuclear trafficking of viral genes.

The question of how genetic materials are trafficked in and out of the cell nucleus is a problem of great importance not only for understanding viral infections but also for advancing gene-delivery technology. Here we demonstrate a physical technique that allows gene trafficking to be studied at the single-gene level by combining sensitive fluorescence microscopy with microinjection. As a model system, we investigate the nuclear import of influenza genes, in the form of ribonucleoproteins (vRNPs), by imaging single vRNPs in living cells in real time. Our single-particle trajectories show that vRNPs are transported to the nuclear envelope by diffusion. We have observed heterogeneous interactions between the vRNPs and nuclear pore complexes with dissociation rate constants spanning two orders of magnitude. Our single-particle tracking experiments also provided new insights into the regulation mechanisms for the nuclear import of vRNPs: the influenza M1 protein, a regulatory protein for the import process, downregulates the nuclear import of vRNPs by inhibiting the interactions between vRNPs and nuclear pore complexes but has no significant effect on the transport properties of vRNPs. We expect this single-particle tracking approach to find broad application in investigations of genetic trafficking.

Active Transport, Cell Nucleus↗

PubMed2003

Observation of polymer conformation hysteresis in extensional flow.

Highly extensible Escherichia coli DNA molecules in planar extensional flow were visualized in dilute solution by fluorescence microscopy. For a narrow range of flow strengths, the molecules were found in either a coiled or highly extended conformation, depending on the deformation history of the polymer. This conformation hysteresis persists for many polymer relaxation times and is due to conformation-dependent hydrodynamic forces. Polymer conformational free-energy landscapes were calculated from computer simulations and show two free-energy minima for flow strengths near the coil-stretch transition. Hysteresis cycles may directly influence bulk-solution stresses and the development of stress-strain relations for dilute polymer flows.

Biopolymers↗

PubMed2003

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 cell's 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.

Actins↗

PubMed2003

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 ribozyme's 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.

Dose-Response Relationship, Drug↗

PubMed2002

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.

Adenosine Triphosphatases↗

PubMed2002

Dynamics and configurational fluctuations of single DNA molecules in linear mixed flows.

We examine the dynamics of DNA molecules in mixed flows where the ratio of vorticity to strain rate may be slightly above or below unity via Brownian dynamics simulation. We find that the chain dynamics in these flows are dramatically different than those found for simple shear flow. When the strain rate exceeds vorticity, the dynamics are found to be driven by the extra amount of straining. For vorticity-dominated flows, a periodicity in chain extension is observed with considerable chain deformation.

Chemical Phenomena↗

PubMed2002

Correlating structural dynamics and function in single ribozyme molecules.

We have studied the correlation between structural dynamics and function of the hairpin ribozyme. The enzyme-substrate complex exists in either docked (active) or undocked (inactive) conformations. Using single-molecule fluorescence methods, we found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also found substrate cleavage to be rate-limited by a combination of conformational transitions and reversible chemistry equilibrium. The complex structural dynamics quantitatively explain the heterogeneous cleavage kinetics common to many catalytic RNAs. The intimate coupling of structural dynamics and function is likely a general phenomenon for RNA.

Carbocyanines↗

PubMed2001

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.

Animals↗