John F. Beausang

The promise and challenge of high-throughput sequencing of the antibody repertoire.

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.

Concentration and Length Dependence of DNA Looping in Transcriptional Regulation

In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy

Myosin V is biomolecular motor with two actin-binding domains (heads) that take multiple steps along actin by a hand-over-hand mechanism. We used high-speed polarized total internal reflection fluorescence (polTIRF) microscopy to study the structural dynamics of single myosin V molecules that had been labeled with bifunctional rhodamine linked to one of the calmodulins along the lever arm. With the use of time-correlated single-photon counting technology, the temporal resolution of the polTIRF microscope was improved ~50-fold relative to earlier studies, and a maximum-likelihood, multitrace change-point algorithm was used to objectively determine the times when structural changes occurred. Short-lived substeps that displayed an abrupt increase in rotational mobility were detected during stepping, likely corresponding to random thermal fluctuations of the stepping head while it searched for its next actin-binding site. Thus, myosin V harnesses its fluctuating environment to extend its reach. Additional, less frequent angle changes, probably not directly associated with steps, were detected in both leading and trailing heads. The high-speed polTIRF method and change-point analysis may be applicable to single-molecule studies of other biological systems.

Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns

As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue.

Elementary Simulation of Tethered Brownian Motion

We describe a simple simulation, suitable for an undergraduate project or graduate problem set, of the Brownian motion of a particle in a Hooke’s law potential well. Understanding this physical situation is necessary in many experimental contexts, for instance in single molecule biophysics, and its simulation helps students appreciate the dynamical character of thermal equilibrium. The simulation captures behavior seen in experimental data on tethered particle motion.

Calibration of Tethered Particle Motion Experiments

The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead’s random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation.

Elongation factor G initiates translocation through a power stroke

Significance Elongation factor G (EF-G) uses energy stored in GTP to catalyze movement of transfer RNAs and messenger RNA in the ribosome during the translocation step of prokaryotic protein synthesis. Using single-molecule polarized fluorescence microscopy, three-dimensional rotational motions of individual domains of EF-G were directly captured, for the first time to our knowledge, during normal translocation. Our observations strongly imply a hybrid model, in which the initial steps of translocation are ribosome unlocking driven by a force generated via EF-G–dependent GTP hydrolysis, and further steps of translocation are mainly driven by the energetics of the ribosome itself. These results demonstrate that the ribosome and EF-G make use of power-stroke and Brownian-ratchet mechanisms to ensure efficiency and accuracy of translocation. During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G’s GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

Orientation and Rotational Motions of Single Molecules by Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM)

In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin.

The azimuthal path of myosin V and its dependence on lever-arm length

Myosin V (myoV) is a two-headed myosin capable of taking many successive steps along actin per diffusional encounter, enabling it to transport vesicular and ribonucleoprotein cargos in the dense and complex environment within cells. To better understand how myoV navigates along actin, we used polarized total internal reflection fluorescence microscopy to examine angular changes of bifunctional rhodamine probes on the lever arms of single myoV molecules in vitro. With a newly developed analysis technique, the rotational motions of the lever arm and the local orientation of each probe relative to the lever arm were estimated from the probe’s measured orientation. This type of analysis could be applied to similar studies on other motor proteins, as well as other proteins with domains that undergo significant rotational motions. The experiments were performed on recombinant constructs of myoV that had either the native-length (six IQ motifs and calmodulins [CaMs]) or truncated (four IQ motifs and CaMs) lever arms. Native-length myoV-6IQ mainly took straight steps along actin, with occasional small azimuthal tilts around the actin filament. Truncated myoV-4IQ showed an increased frequency of azimuthal steps, but the magnitudes of these steps were nearly identical to those of myoV-6IQ. The results show that the azimuthal deflections of myoV on actin are more common for the truncated lever arm, but the range of these deflections is relatively independent of its lever-arm length.

Myosin VI lever arm rotation: Fixed or variable?

Reifenberger et al. (1) reported in PNAS that the myosin VI lever arms tilt by 180° on each step. This contrasts with Sun et al. (2), wherein we found variable degrees of axial and azimuthal tilting, consistent with the known variable step size of myosin VI. There are several differences between the two papers, which are listed here and clarified in a web publication (3).