Summary 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 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.

We confirm Landauer's 1961 hypothesis that reducing the number of possible macroscopic states in a system by a factor of 2 requires work of at least kTln2. Our experiment uses a colloidal particle in a time-dependent, virtual potential created by a feedback trap to implement Landauer's erasure operation. In a control experiment, similar manipulations that do not reduce the number of system states can be done reversibly. Erasing information thus requires work. In individual cycles, the work to erase can be below the Landauer limit, consistent with the Jarzynski equality.

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 those expected by the theory of dynamical scaling for semi-dilute polymer solutions. The crossover concentration from the unentangled to entangled regime ce is found to be ∼9c∗ in a good solvent, in accord with other experiments based on the relaxation of a single molecule, the diffusion coefficient, and the solution viscosity measurements. We also test the universality of the concentration dependence of the longest relaxation time for both flexible and semi-flexible polymers in both good and Θ solvents in the dilute and semi-dilute regimes by analyzing literature data sets.

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. molecular motors | macromolecular assemblies | mitosis | cancer N ucleotide triphosphates are the fuel that powers the cell's machinery. Conversion of this fuel into mechanical work, i.e., mechanochemistry, depends on individual machines and the functional context in which they have evolved. Indeed, elucidation of the mechanochemistry of a particular machine provides critical insight into both its functions and modes of regulation. Kinesins are a superfamily of motors that use ATP to undertake microtubule (MT)-based work. Kinesins operate throughout the cell cycle in many contexts and can generate force toward the MT plus or minus end and also depolymerize MTs (1). The kinesin mechanochemical engine-the motor domain (MD)-is highly conserved, and conformational changes in the active site during the motor's ATPase cycle are transmitted to other parts of the MD to generate force (2). Most of our current knowledge about kinesin mechanochemistry comes from studies of the superfamily founding member, the transport motor kinesin-1 (K1) (2). However, accumulating evidence suggests that small modifications within kinesin MDs have profound effects on their cellular function. The molecul...

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.pseudorabies | virus | heterogeneity | asymmetry | point-spread function H erpesviruses are responsible for a broad range of diseases in humans and other animals. The herpesvirus virion consists of four components: (i) the linear dsDNA genome, (ii) a 125-nm diameter T = 16 icosahedral capsid, (iii) a tegument consisting of more than 20 proteins that surround the capsid, and (iv) a lipid bilayer envelope studded with viral glycoproteins. The fully assembled particle is ∼200-250 nm in diameter and is referred to as the heavy particle (H-particle) (1). The capsid has been solved to 8.5 Å resolution and a fraction of the tegument that is symmetrically bound to the capsid surface has been solved to 20 Å resolution by cryo-electron microscopy (cryo-EM) reconstruction (2-4). The detailed resolution afforded by cryo-EM results from the averaging of many particles that are aligned in silico, based on icosahedral symmetry. However, such studies provide an incomplete picture of herpesvirus virions because unlike some smaller enveloped viruses that project icosahedral symmetry into the envelope proteins, the herpesvirus envelope, and the majority of the tegument mass lack radial symmetry and are not "seen" by cryo-EM (5-7). Our understanding of these variable structural layers predominantly comes from single-particle imaging methods that do not use symmetry-based averaging. In particular, cryo-electron tomography (cryo-ET) has provided insight into the general structure of the outer virion layers, but has not yielded sufficient detail to resolve the organization of the constituent protein components (8).Extracellular herpes virions are metastable structures that are triggered by interactions between virion membrane surface proteins and corresponding receptors on the cell, culminating in membrane fusion (9). Although tegument proteins are critical for postfusion steps in nuclear delivery (10-14), their perceived role before cell entry has been as rigid structural elements that bridge the capsid shell to the tails of envelope membrane proteins (15, 16). However, recent evidence indicates that the tegument may reorganize before membrane fusion (17)(18)(19). A de...

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.

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 particle's 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.

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.