Bulletin of the American Physical Society
APS March Meeting 2018
Volume 63, Number 1
Monday–Friday, March 5–9, 2018; Los Angeles, California
Session P50: Single Molecule Dynamics Inside and Outside of CellsFocus
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Sponsoring Units: DBIO Chair: Omar Saleh, University of California Santa Barbara Room: LACC 511B |
Wednesday, March 7, 2018 2:30PM - 2:42PM |
P50.00001: Thermal stability of kinesin motors Michael Vershinin, Florence Doval, Katelyn Chase Microtubule-based kinesin-1 motors have a remarkably limited thermal range in vitro, with an onset of degradation already at 30 C. However, the same motors in cells function at significantly higher temperatures. We show that trimethylamine N-oxide (TMAO) extends the temperature range of kinesin up to 50 C. This finding suggests that molecular crowding in cells may be sufficient to significantly extend the thermal range of kinesin. We will further discuss kinesin performance across a wide temeprature range and the implications of our findings for both biology and nano-engineering. |
Wednesday, March 7, 2018 2:42PM - 2:54PM |
P50.00002: Abstract Withdrawn
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Wednesday, March 7, 2018 2:54PM - 3:06PM |
P50.00003: Mechanical Force-Based Regulation of Protein Assemblies Ravi Chawla, Katie Ford, Pushkar Lele The bacterial flagellar motor detects the presence of surfaces and undergoes structural remodeling (Lele et al., PNAS 2013). In response to mechanical cues, the stator builds itself by recruiting additional force-generators. However, the mechanism for such load-dependent self-assembly is currently unknown. Here, we tested a hypothesis that the amount of force generated by each stator-unit modulates its association with the rotor. To do this, we measured stator-binding in mutants strains in which the motors reportedly develop lower torque compared to wild-type motors. Our measurements are consistent with the notion that a unit binds stronger when delivering a higher force to the rotor. An analytical model based on a catch-bond type mechanism was developed that incorporated an exponential dependence of the off-rate for individual units on the force delivered to the rotor. The model provided accurate fits to measurements of stator-rotor binding over a range of loads. It is possible that the tensile forces that develop when a stator-unit delivers a high torque uncover cryptic binding sites that stabilize the stator-rotor association. Our results represent the first steps towards establishing a plausible mechanism for mechanical force-based regulation of the flagellar stator. |
Wednesday, March 7, 2018 3:06PM - 3:18PM |
P50.00004: Regulation of a Viral Packaging Motor’s Grip on DNA Douglas Smith, Mariam Ordyan, Venigalla Rao, Istiaq Alam, Marthandan Mahalingam ATP-powered viral DNA packaging motors are among the most powerful biomotors known. Here, we quantify how nucleotide binding regulates the motor’s grip on DNA via optical tweezers measurements with rapid solution exchange. In the apo state (with no nucleotide) there is almost no detectable grip. With low applied force the DNA usually slips at ~2000 bp/s. In contrast, with non-hydrolyzable ATP mimic bound, the motor grips the DNA strongly. Transient slips that occur when ATP dissociates are notably slower (~40 bp/s) than in the apo state, showing that multiple ATP-bound subunits exert friction on the DNA . Although the grip in the ATP-bound state can be ruptured by application of force, with a 30 pN force, the estimated maximum resistance at high prohead filling, slipping is low enough that the motor can still function. With bound ADP three states are observed: one that grips strongly like in with bound ATP, one with virtually no grip like apo, and one where the DNA slips at an intermediate speed (~740 bp/s). Although in the apo and ADP states the motor usually has little grip, when the end of the DNA is about to exit the capsid slipping suddenly arrests. This unique "clamp" state is highly stable and packaging resumes when ATP is added. |
Wednesday, March 7, 2018 3:18PM - 3:30PM |
P50.00005: Insights on viral DNA packaging motor mechanisms from the effects of motor residue changes on single-molecule packaging dynamics Mariam Ordyan, Damian delToro, Douglas Smith Many dsDNA viruses utilize a molecular motor to translocate DNA into preassembled viral prohead shells. We apply methods we have developed for measuring the dynamics of single DNA molecule packaging with optical tweezers to study the mechanism of the bacteriophage lambda motor. In collaboration with the groups of Michael Feiss and Carlos Catalano we used site-directed mutagenesis to investigate the roles of various motor protein residues in translocation function. We found that residue changes in the proposed Walker A and Walker B ATP binding motif and catalytic glutamate cause varying impairments, ranging from partial impairment to total abrogation of DNA translocation activity. Altogether, we studied 23 mutants and identified 11 with no detectable translocation and 12 having varying impairments. The results support the proposed motif assignments and observed changes in motor velocity and pausing and slipping dynamics for the partly impaired mutants give insights on residues involved in ATP binding and positioning, coupling between ATP binding and DNA gripping, and catalysis of hydrolysis. |
Wednesday, March 7, 2018 3:30PM - 3:42PM |
P50.00006: Biophysical Constraints on Decoding Temporal Dynamics Jackson O'Brien, Anders Hansen, Arvind Murugan Recent advances in visualization of single molecules, such as transcription factors with temporal resolution in single cells, have shown that information is transmitted through time-varying dynamics of components shared between multiple pathways. This phenomenon stands in contrast to the typical paradigm where information is transmitted via structurally specific interactions (e.g. the lock and key model). Consequently, signaling through time dynamics via shared components raises natural questions about how such interactions can effect only the intended response. By analyzing realistic, coarse-grained biochemical networks informed by experimental studies, we explore the response of general network topologies to temporally varying upstream inputs. From these results, we are able to establish theoretical limits on the signaling capacity of such networks as a function of biophysical parameters and network complexity, emphasizing the importance of functional network diversity in decoding the full dimensionality of the input space. |
Wednesday, March 7, 2018 3:42PM - 4:18PM |
P50.00007: Mediator and Pol II clusters co-associate in transcription-dependent dynamic condensates in living stem cells Invited Speaker: Ibrahim Cisse At the onset of transcription, a mediator multiprotein complex assembled at a distal enhancer site contacts the pre-initiating RNA Polymerase II (Pol II) complex to help effect transcription activation. How mediator and Pol II co-organize and interact in the 3D nucleus of living cells remains elusive. Here we use quantitative live cell super-resolution and light sheet imaging to study the organization and dynamics of endogenous mediator and Pol II with unprecedented quantitative detail, directly in living mouse embryonic stem cells. In addition to forming transient clusters with average lifetimes of 11.1 +/- 0.9 s, and 12.1 +/-1.4s respectively, mediator and Pol II also form previously uncharacterized large stable clusters in stem cells (~15 stable clusters per cell). The large mediator and Pol II clusters gradually disappear within hours after induction of stem cell differentiation. Stable mediator and Pol II clusters co-localize with each other, as well as with pluripotency factors. Inhibition of Brd4 bromodomains necessary for enhancer association eliminates both mediator and Pol II stable clusters, and inhibition of transcription elongation selectively eliminates stable Pol II but not stable mediator clusters. Tracking of mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property reminiscent of phase separated droplets. Our study demonstrates mediator and Pol II association in diffraction-sized condensates depending on active transcription in living stem cells. |
Wednesday, March 7, 2018 4:18PM - 4:30PM |
P50.00008: A Jump Distance Based Parameter Inference Scheme for Particulate Trajectories in Biological Settings Rebecca Menssen, Madhav Mani Mean square displacement (MSD) analysis has been the standard for analyzing single molecule or particulate trajectories, where its shortcomings have been overlooked in light of its simplicity. The Jump Distance Distribution (JDD) has been proposed by others in the past as a new way to analyze these trajectories, but has not been sufficiently fleshed out in all dimensions or given a robust analysis on performance and how it compares to MSD analyses. We present the forms of the JDD in 1, 2, and 3 dimensions for three different models of motion: pure diffusion, directed diffusion, and anomalous diffusion. We also discuss how to select between competing models using Bayesian model selection, and verify our method through simulation. Through this, we have a method that is superior to MSD analysis, particularly in the data-poor limit. This method works across a wide range of parameters, which should make it broadly applicable to any system where the underlying motion is stochastic. We finish with an application to the method to ms2 trajectories in the early Drosophila embryo. |
Wednesday, March 7, 2018 4:30PM - 4:42PM |
P50.00009: The Molecular Basis of Triphasic Adhesion in E-selectins Anchoring Leukocytes Under Shear Flow Shamreen Iram, Hannah Goldberg, Shishir Adhikari, Michael Hinczewski One of the primary physiological roles of the selectin class of proteins is the anchoring of leukocytes to blood vessel walls by binding/unbinding with preferred ligands on leukocytes. This is crucial at sites of vascular injury and inflammation as a first line of defense or repair-and-control for the immune system. The hinge like structure of the selectin allows it to switch under shear forces between an extended and compact configuration, marked by differences in affinity for the ligand. This shows up as distinct regimes in the behaviour of the bond lifetime as a function of force: slip regimes where lifetimes decrease with force, and catch regimes where they counter-intuitively increase. E-selectin binding to the ligand PSGL-1 presents a particularly complex example of this- with a slip-catch-slip lifetime trend. We explain this triphasic behaviour using a diffusion-on-an-energy-landscape picture mapped to a three state bound-unbound system. By tying together force spectroscopy data and molecular mechanisms of force regulation in this complex along with robust structural corroboration of model parameters, our theory is the first to provide a detailed mechanistic basis for how such triphasic behaviour arises. |
Wednesday, March 7, 2018 4:42PM - 4:54PM |
P50.00010: Single-Molecule Force Spectroscopy Studies of Hyaluronic Acid Motivate a Simple Model for Flexible Polyelectrolyte Interactions with Multivalent Ions Sarah Innes-Gold, Omar Saleh Hyaluronic acid (HA) is a charged linear polysaccharide abundant in extracellular spaces. Its solution conformation and mechanical properties help structure the environment outside of cells and are of interest for biomaterials development. Trivalent cations are particularly relevant to HA gel materials as they interact strongly with the polymer and can form ionic crosslinks. To investigate the nature of HA-trivalent interactions, we use magnetic tweezers to apply biological-scale stretching forces to individual HA chains in the presence of trivalent cations. Based on these experiments, we develop a simple model which qualitatively predicts the behavior of the HA-trivalent system. We propose that the HA chain wraps around the cation to maximize contact between the anionic carboxylate groups and the trivalent cation. We observe a large decrease in experimentally measured persistence length consistent with the short length-scale bending in the wrapping model. We predict that additional monovalent salt can screen away the interaction in a manner dependent on the ion radius, and confirm the existence of this effect in experiments. The model is extended to describe other flexible polyelectrolytes interacting with ions. |
Wednesday, March 7, 2018 4:54PM - 5:06PM |
P50.00011: Knots modify the coil-stretch transition in linear DNA polymers Beatrice Soh, Vivek Narsimhan, Alexander Klotz, Patrick Doyle We perform single-molecule DNA experiments to investigate the relaxation dynamics of knotted polymers and examine the steady-state behavior of knotted polymers in elongational fields. The occurrence of a knot reduces the relaxation time of a molecule and leads to a shift in the molecule's coil-stretch transition to larger strain rates. We measure chain extension and extension fluctuations as a function of Weissenberg number for unknotted and knotted molecules. The curves for knotted molecules can be collapsed onto the unknot curves by defining an effective Weissenberg number based on the measured knotted relaxation time in the low extension regime, or a relaxation time based on Rouse/Zimm scaling theories in the high extension regime. Because a knot reduces a molecule's relaxation time, we observe that knot untying near the coil-stretch transition can result in the molecule experiencing both a stretch-coil transition with the knot, and coil-stretch transition without the knot, at the same strain rate. |
Wednesday, March 7, 2018 5:06PM - 5:18PM |
P50.00012: Measuring Bending of Intrinsically Bent DNA Xinyue Cai, Lourdes Velazquez, Sebastian Arias, Shelby Vexler, Alex Bevier, Deborah Fygenson Intrinsically bent DNA sequences influence nucleosome positioning, which, in turn, helps regulate gene expression. The iconic example is a run of four or more contiguous adenine bases, known as an A-tract. While details of A-tract structure are well-studied, little is known about the mechanical properties. We seek to measure A-tract stiffness from the angular fluctuations of a short (~ 60 bp) DNA sequence with zero, one, or more A-tracts (repeated in phase with the helix screw). Our approach leverages DNA nanotechnology to embed the A-tract-laden sequence between a pair of microns-long rigid rods, which mechanically magnify the relative orientation of the sequence ends. The angular fluctuations are captured by video fluorescence microscopy, analyzed using circular statistics and compared with published results. |
Wednesday, March 7, 2018 5:18PM - 5:30PM |
P50.00013: Motion of DNA in confinement-induced ionic gradients Saeid Movahed, Zubair Azad, Robert Riehn DNA undergoes conformational changes when confined to small fluidic channels. In particular, confinement of long (> 10 kbp) double-stranded DNA to channels with a cross-section of less than (100 nm)2 causes elongation of DNA. It has commonly been observed that long DNA has a tendency to drift within channels, even without an applied flow or electric field. Here we demonstrate that this effect is due to the non-homogeneouos ionic strength within the channel, where anions are depleted compared to the bulk solution, resulting in a lower ionic strength. We combine finite element modeling with single-molecule experiments to show that the motion of DNA is consistent with a drift of DNA from regions of lower to higher ionic strength. |
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