Yonggun Jun

Concentration dependence of the longest relaxation times of dilute and semi-dilute polymer solutions

The longest relaxation times of polymer solutions of semi-flexible T4 DNA and flexible 18 M molar mass polyacrylamide (PAAm) in dilute and semi-dilute concentration range are studied by the polymer extension relaxation of stretched single DNA molecules and by the stress relaxation of PAAm solutions measurements. For both polymer solutions, the longest relaxation time normalized by the value at infinite dilution with the same solvent viscosity τ/τ0 increases with increasing concentration. In the dilute regime, the longest relaxation time increases just slightly with increasing concentration as τ/τ0=[1+cA−2(cA)1.5+2(cA)2] as well as the empirical relation of τ/τ0=exp(cA) up to c∼3c∗ with A≈0.5[η], where c∗ is the overlap concentration, in accord with the theory and previous experiments. For the semi-dilute solutions, the scaling of τ/τ0 with concentration shows two different exponents in two concentration regions, corresponding to the unentangled and entangled regimes. The exponents are consistent with thos...

Calibration of Optical Tweezers for In Vivo Force Measurements: How do Different Approaches Compare?

There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties. We evaluate the relative merits of these methods and directly compare two of them, a refractive index matching method, and a light-momentum-change method. Since in vivo forces are frequently relatively high (e.g., can exceed 15 pN for lipid droplets), a high-power laser is employed. We discover that this high-powered trap induces local temperature changes, and we develop an approach to compensate for uncertainties in the magnitude of applied force due to such temperature variations.

Invariance of Initiation Mass and Predictability of Cell Size in Escherichia coli

It is generally assumed that the allocation and synthesis of total cellular resources in microorganisms are uniquely determined by the growth conditions. Adaptation to a new physiological state leads to a change in cell size via reallocation of cellular resources. However, it has not been understood how cell size is coordinated with biosynthesis and robustly adapts to physiological states. We show that cell size in Escherichia coli can be predicted for any steady-state condition by projecting all biosynthesis into three measurable variables representing replication initiation, replication-division cycle, and the global biosynthesis rate. These variables can be decoupled by selectively controlling their respective core biosynthesis using CRISPR interference and antibiotics, verifying our predictions that different physiological states can result in the same cell size. We performed extensive growth inhibition experiments, and we discovered that cell size at replication initiation per origin, namely the initiation mass or unit cell, is remarkably invariant under perturbations targeting transcription, translation, ribosome content, replication kinetics, fatty acid and cell wall synthesis, cell division, and cell shape. Based on this invariance and balanced resource allocation, we explain why the total cell size is the sum of all unit cells. These results provide an overarching framework with quantitative predictive power over cell size in bacteria.

Comprehensive Structural Model of the Mechanochemical Cycle of a Mitotic Motor Highlights Molecular Adaptations in the Kinesin Family.

Significance Kinesins are a superfamily of ATP-dependent motors that are important for a wide variety of microtubule-based functions in eukaryotic cells. Kinesins have evolved to allow variable tuning of their motor properties, but the link between molecular variation and motor function is largely unknown. To understand this link, we have studied an essential mitotic kinesin, kinesin-5, which is the target of anticancer drugs. We used cryo-electron microscopy to visualize directly sequential conformational changes of structural elements during the motor ATPase cycle. We have identified the contribution of kinesin-5–specific variations to motor function indicating that kinesins indeed are precisely tuned according to cellular function. This insight will be important in designing kinesin-specific inhibitors in different disease contexts. Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin—the transport motor kinesin-1—is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor’s structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture

Significance Neuroinvasive herpesviruses cause diseases in humans ranging from cold sores to central nervous system infections. Unlike most icosahedral viruses, herpesvirus capsids are surrounded by protein layers that lack polyhedral architecture. The outer layers are critical for herpesvirus infectivity. Although the disorganized layers are visible by electron microscopy, the protein topography of these layers remains unclear. We fused fluorophores to virus proteins and pinpointed their positions within virions at nanometer resolution. The findings reveal a complex and variable asymmetric architecture, shed light on herpesvirus assembly, and provide a foundation for visualizing viral particle dynamics during herpesvirus infection. The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection.

The Structural Kinetics of Switch-1 and the Neck Linker Explain the Functions of Kinesin-1 and Eg5

Significance The kinesins are molecular motors that couple ATP binding to movement. Although crystallographic and cryo-EM methods have identified the structural changes that occur in several kinesins, the images they generate are static pictures that provide no insight into how dynamic these conformations are or how they are coupled together to generate force. We have addressed this through a novel combination of time-resolved fluorescence and transient-state kinetics to measure the conformational equilibria between two key domains in two functionally distinct kinesins: kinesin-1 and Eg5. Our results are significant because they provide a unique insight into how conformational dynamics vary between two kinesins with different functions, and explain the distinct enzymologies these two kinesins have. Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction—including switch-1 and the neck linker—how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as “structural kinetics,” to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.

Mixing of passive tracers in the decay Batchelor regime of a channel flow

We report detailed quantitative studies of passive scalar mixing in a curvilinear channel flow, where elastic turbulence in a dilute polymer solution of high molecular weight polyacrylamide in a high viscosity water-sugar solvent was achieved. For quantitative investigation of mixing, a detailed study of the profiles of mean longitudinal and radial components of the velocity in the channel as a function of Wi was carried out. Besides, a maximum of the average value as well as a rms of the longitudinal velocity was used to determine the threshold of the elastic instability in the channel flow. The rms of the radial derivatives of the longitudinal and radial velocity components was utilized to define the control parameters of the problem, the Weissenberg Wiloc and the Peclet Pe numbers. The main result of these studies is the quantitative test of the theoretical prediction about the value of the mixing length in the decay Batchelor regime. The experiment shows large quantitative discrepancy, more than 200 t...

tCRISPRi: tunable and reversible, one-step control of gene expression

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

Virtual potentials for feedback traps

The recently developed feedback trap can be used to create arbitrary virtual potentials, to explore the dynamics of small particles or large molecules in complex situations. Experimentally, feedback traps introduce several finite time scales: There is a delay between the measurement of a particles position and the feedback response, the feedback response is applied for a finite update time, and a finite camera exposure integrates motion. We show how to incorporate such timing effects into the description of particle motion. For the test case of a virtual quadratic potential, we give the first accurate description of particle dynamics, calculating the power spectrum and variance of fluctuations as a function of feedback gain, testing against simulations. We show that for small feedback gains, the motion approximates that of a particle in an ordinary harmonic potential. Moreover, if the potential is varied in time, for example by varying its stiffness, the work that is calculated approximates that done in an ordinary changing potential. The quality of the approximation is set by the ratio of the update time of the feedback loop to the relaxation time of motion in the virtual potential.

Elastic turbulence in a curvilinear channel flow.

We report detailed quantitative studies of elastic turbulence in a curvilinear channel flow in a dilute polymer solution of high molecular weight polyacrylamide in a high viscosity water-sugar solvent. Detailed studies of the average and rms velocity and velocity gradients profiles reveal the emergence of a boundary layer associated with the nonuniform distribution of the elastic stresses across the channel. The characteristic boundary width is independent of the Weissenberg number Wi and proportional to the channel width, which is consistent with the findings our early investigations of the boundary layer in elastic turbulence in different flow geometries. The nonuniform distribution of the elastic stresses across the channel and appearance of the characteristic spatial scales of the order of the boundary layer width of both velocity and velocity gradient in the correlation functions of the velocity and velocity gradient fields in a bulk flow may suggest that excessive elastic stresses, concentrated in the boundary layer, are ejected into the bulk flow similar to jets observed in passive scalar mixing in elastic turbulence observed recently. Finally, the experimental results show that one of the main predictions of the theory of elastic turbulence, namely, the saturation of the normalized rms velocity gradient in the bulk flow of elastic turbulence contradicts the experimental observations both qualitatively and quantitatively in spite of the fact that the theory explains well the observed sharp power-law decay of the velocity power spectrum. The experimental findings call for further development of theory of elastic turbulence in a bounded container, similar to what was done for a passive scalar problem.