Watching biomolecules stride in real time.

A noninvasive imaging technique tracks the motion of single biomolecules in live cells.

Proteomic and functional analyses of the periodic membrane skeleton in neurons.

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of potential candidate MPS-interacting proteins that span diverse functional categories.

Membrane-associated periodic skeleton is a signaling platform for RTK transactivation in neurons.

Actin, spectrin, and related molecules form a membrane-associated periodic skeleton (MPS) in neurons. The function of the MPS, however, remains poorly understood. Using super-resolution imaging, we observed that G protein-coupled receptors (GPCRs), cell adhesion molecules (CAMs), receptor tyrosine kinases (RTKs), and related signaling molecules were recruited to the MPS in response to extracellular stimuli, resulting in colocalization of these molecules and RTK transactivation by GPCRs and CAMs, giving rise to extracellular signal-regulated kinase (ERK) signaling.

βII-spectrin promotes mouse brain connectivity through stabilizing axonal plasma membranes and enabling axonal organelle transport.

βII-spectrin is the generally expressed member of the β-spectrin family of elongated polypeptides that form micrometer-scale networks associated with plasma membranes. We addressed in vivo functions of βII-spectrin in neurons by knockout of βII-spectrin in mouse neural progenitors. βII-spectrin deficiency caused severe defects in long-range axonal connectivity and axonal degeneration.

Junction resolving enzymes use multivalency to keep the Holliday junction dynamic.

Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures.

Visualizing and discovering cellular structures with super-resolution microscopy.

Super-resolution microscopy has overcome a long-held resolution barrier-the diffraction limit-in light microscopy and enabled visualization of previously invisible molecular details in biological systems. Since their conception, super-resolution imaging methods have continually evolved and can now be used to image cellular structures in three dimensions, multiple colors, and living systems with nanometer-scale resolution. These methods have been applied to answer questions involving the organization, interaction, stoichiometry, and dynamics of individual molecular building blocks and their integration into functional machineries in cells and tissues.

Structural organization of the actin-spectrin-based membrane skeleton in dendrites and soma of neurons.

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. In the MPS, short actin filaments, capped by actin-capping proteins, form ring-like structures that wrap around the circumference of neurites, and these rings are periodically spaced along the neurite by spectrin tetramers, forming a quasi-1D lattice structure. This 1D MPS structure was initially observed in axons and exists extensively in axons, spanning nearly the entire axonal shaft of mature neurons.

Prevalent presence of periodic actin-spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species.

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types.

Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells.

Translation is under tight spatial and temporal controls to ensure protein production in the right time and place in cells. Methods that allow real-time, high-resolution visualization of translation in live cells are essential for understanding the spatiotemporal dynamics of translation regulation. Based on multivalent fluorescence amplification of the nascent polypeptide signal, we develop a method to image translation on individual mRNA molecules in real time in live cells, allowing direct visualization of translation events at the translation sites.

Spider Silk Peptide Is a Compact, Linear Nanospring Ideal for Intracellular Tension Sensing.

Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 amino acid peptide repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer.

Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility.

Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side.

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.

An improved surface passivation method for single-molecule studies.

We report a surface passivation method based on dichlorodimethylsilane (DDS)-Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS-Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.

Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA.

Pif1 family helicases are conserved from bacteria to humans. Here, we report a novel DNA patrolling activity which may underlie Pif1's diverse functions: a Pif1 monomer preferentially anchors itself to a 3'-tailed DNA junction and periodically reel in the 3' tail with a step size of one nucleotide, extruding a loop. This periodic patrolling activity is used to unfold an intramolecular G-quadruplex (G4) structure on every encounter, and is sufficient to unwind RNA-DNA heteroduplex but not duplex DNA.

Structural mechanisms of PriA-mediated DNA replication restart.

Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs).

Single molecule analysis of Thermus thermophilus SSB protein dynamics on single-stranded DNA.

Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group.

PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks.

The telomere capping complex CST has an unusual stoichiometry, makes multipartite interaction with G-Tails, and unfolds higher-order G-tail structures.

The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2∶4∶2 or 2∶6∶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex.

The SOSS1 single-stranded DNA binding complex promotes DNA end resection in concert with Exo1.

The human SSB homologue 1 (hSSB1) has been shown to facilitate homologous recombination and double-strand break signalling in human cells. Here, we compare the DNA-binding properties of the SOSS1 complex, containing SSB1, with Replication Protein A (RPA), the primary single-strand DNA (ssDNA) binding complex in eukaryotes. Ensemble and single-molecule approaches show that SOSS1 binds ssDNA with lower affinity compared to RPA, and exhibits less stable interactions with DNA substrates.

Single-molecule analysis of SSB dynamics on single-stranded DNA.

SSB proteins bind to and control the accessibility of single-stranded (ss) DNA generated as a transient intermediate during a variety of cellular processes. For subsequent DNA processing, however, SSB needs to be removed and yield to other proteins while avoiding ssDNA exposure to nucleases. Using single-molecule two- and three-color fluorescence resonance energy transfer (FRET) and fluorescence-force spectroscopy, we recently showed that the SSB/DNA complex is a highly dynamic system and SSB functions as a sliding platform that migrates on ssDNA for recruiting other proteins in DNA repair, replication, and recombination.

Detecting intramolecular conformational dynamics of single molecules in short distance range with subnanometer sensitivity.

Single molecule detection is useful for characterizing nanoscale objects such as biological macromolecules, nanoparticles and nanodevices with nanometer spatial resolution. Fluorescence resonance energy transfer (FRET) is widely used as a single-molecule assay to monitor intramolecular dynamics in the distance range of 3-8 nm. Here we demonstrate that self-quenching of two rhodamine derivatives can be used to detect small conformational dynamics corresponding to subnanometer distance changes in a FRET-insensitive short-range at the single molecule level.

SSB functions as a sliding platform that migrates on DNA via reptation.

SSB proteins bind to and control the accessibility of single-stranded DNA (ssDNA), likely facilitated by their ability to diffuse on ssDNA. Using a hybrid single-molecule method combining fluorescence and force, we probed how proteins with large binding site sizes can migrate rapidly on DNA and how protein-protein interactions and tension may modulate the motion. We observed force-induced progressive unraveling of ssDNA from the SSB surface between 1 and 6 pN, followed by SSB dissociation at ∼10 pN, and obtained experimental evidence of a reptation mechanism for protein movement along DNA wherein a protein slides via DNA bulge formation and propagation.

Forcing a connection: impacts of single-molecule force spectroscopy on in vivo tension sensing.

Mechanical tension plays a large role in cell development ranging from morphology to gene expression. On the molecular level, the effects of tension can be seen in the dynamic arrangement of membrane proteins as well as the recruitment and activation of intracellular proteins. Forces applied to biopolymers during in vitro force measurements offer greater understanding of the effects of tension on molecules in live cells, and experimental techniques involving test tubes and live cells can often overlap.

Force-fluorescence spectroscopy at the single-molecule level.

During the past decade, various powerful single-molecule techniques have evolved and helped to address important questions in life sciences. Yet these techniques would be even more powerful if they would be combined, that is, single-molecule manipulation with an orthogonal single-molecule observation. Here, we present a recently developed approach to combine single-molecule optical tweezers with single-molecule fluorescence spectroscopy.

Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.

Mechanical forces are central to developmental, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy.