Publications

The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

The swimming direction of biological or artificial microscale swimmers tends to be randomised over long time-scales by thermal fluctuations. Bacteria use various strategies to bias swimming behaviour and achieve directed motion against a flow, maintain alignment with gravity or travel up a chemical gradient. Herein, we explore a purely geometric means of biasing the motion of artificial nanorod swimmers. These artificial swimmers are bimetallic rods, powered by a chemical fuel, which swim on a substrate printed with teardrop-shaped posts. The artificial swimmers are hydrodynamically attracted to the posts, swimming alongside the post perimeter for long times before leaving. The rods experience a higher rate of departure from the higher curvature end of the teardrop shape, thereby introducing a bias into their motion. This bias increases with swimming speed and can be translated into a macroscopic directional motion over long times by using arrays of teardrop-shaped posts aligned along a single direction. This method provides a protocol for concentrating swimmers, sorting swimmers according to different speeds, and could enable artificial swimmers to transport cargo to desired locations.

Over the past decade, the Rosetta biomolecular modeling suite has informed diverse biological questions and engineering challenges ranging from interpretation of low-resolution structural data to design of nanomaterials, protein therapeutics, and vaccines. Central to Rosetta’s success is the energy function: a model parametrized from small-molecule and X-ray crystal structure data used to approximate the energy associated with each biomolecule conformation. This paper describes the mathematical models and physical concepts that underlie the latest Rosetta energy function, called the Rosetta Energy Function 2015 (REF15). Applying these concepts, we explain how to use Rosetta energies to identify and analyze the features of biomolecular models. Finally, we discuss the latest advances in the energy function that extend its capabilities from soluble proteins to also include membrane proteins, peptides containing noncanonical amino acids, small molecules, carbohydrates, nucleic acids, and other macromolecules.

The instant invention provides methods and related compositions for identifying polypeptides with improved stability and/or enzymatic activity in comparison to native forms, wherein the identified polypeptides comprise one or more non-natural amino acids. In certain embodiments, the present invention relates to novel phosphotriesterase enzymes comprising one or more non-natural amino acids. In a particular embodiment, the instant invention provides novel phosphotriesterase enzymes with greater stability and/or enhanced activity in comparison to native forms of the enzyme. The present invention also relates to compositions comprising novel phophotriesterase enzymes, such as prophylactics, decontaminants, animal feedstocks, and assay kits.

The proper positioning of mitotic spindle in the single-cell Caenorhabditis elegans embryo is achieved initially by the migration and rotation of the pronuclear complex (PNC) and its two associated astral microtubules (MTs). Pronuclear migration produces global cytoplasmic flows that couple the mechanics of all microtubules, the PNC, and the cell periphery with each other through their hydrodynamic interactions (HIs). We present the first computational study that explicitly accounts for detailed HIs between the cytoskeletal components and demonstrate the key consequences of HIs on the mechanics of pronuclear migration. First we show that, because of HIs between the MTs, the cytoplasm-filled astral MTs behave like a porous medium with its permeability decreasing with increasing the number of MTs. We then directly study the dynamics of PNC migration under various force-transduction models, including the pushing or pulling of MTs at the cortex, and the pulling of MTs by cytoplasmically-bound force generators. While achieving proper position and orientation on reasonable time-scales does not uniquely choose a model, we find that each model produces a different signature in its induced cytoplasmic flow. We suggest then that cytoplasmic flows can be used to differentiate between mechanisms.

Differential binding of transcription factors (TFs) at cis-regulatory loci drives the differentiation and function of diverse cellular lineages. Understanding the regulatory interactions that underlie cell fate decisions requires characterizing TF binding sites (TFBS) across multiple cell types and conditions. Techniques, e.g. ChIP-Seq can reveal genome-wide patterns of TF binding, but typically requires laborious and costly experiments for each TF-cell-type (TFCT) condition of interest. Chromosomal accessibility assays can connect accessible chromatin in one cell type to many TFs through sequence motif mapping. Such methods, however, rarely take into account that the genomic context preferred by each factor differs from TF to TF, and from cell type to cell type. To address the differences in TF behaviors, we developed Mocap, a method that integrates chromatin accessibility, motif scores, TF footprints, CpG/GC content, evolutionary conservation and other factors in an ensemble of TFCT-specific classifiers. We show that integration of genomic features, such as CpG islands improves TFBS prediction in some TFCT. Further, we describe a method for mapping new TFCT, for which no ChIP-seq data exists, onto our ensemble of classifiers and show that our cross-sample TFBS prediction method outperforms several previously described methods.

We analyze one of the simplest active suspensions with complex dynamics: a suspension of immotile "Extensor" particles that exert active extensile dipolar stresses on the fluid in which they are immersed. This is relevant to several experimental systems, such as recently studied tripartite rods that create extensile flows by consuming a chemical fuel. We first describe the system through a Doi-Onsager kinetic theory based on microscopic modeling. This theory captures the active stresses produced by the particles that can drive hydrodynamic instabilities, as well as the steric interactions of rod-like particles that lead to nematic alignment. This active nematic system yields complex flows and disclination defect dynamics very similar to phenomenological Landau-deGennes Q-tensor theories for active nematic fluids, as well as by more complex Doi-Onsager theories for polar microtubule/motor-protein systems. We apply the quasi-equilibrium Bingham closure, used to study suspensions of passive microscopic rods, to develop a non-standard Q-tensor theory. We demonstrate through simulation that this "BQ-tensor" theory gives an excellent analytical and statistical accounting of the suspension's complex dynamics, at a far reduced computational cost. Finally, we apply the BQ-tensor model to study the dynamics of Extensor suspensions in circular and bi-concave domains. In circular domains, we reproduce previous results for systems with weak nematic alignment, but for strong alignment find novel dynamics with activity-controlled defect production and absorption at the boundaries of the domain. In bi-concave domains, a Fredericks-like transition occurs as the width of the neck connecting the two disks is varied.

We describe a modular approach to identify and inhibit protein–protein interactions (PPIs) that are mediated by protein secondary and tertiary structures with rationally designed peptidomimetics. Our analysis begins with entries of high-resolution complexes in the Protein Data Bank and utilizes conformational sampling, scoring, and design capabilities of advanced biomolecular modeling software to develop peptidomimetics.

Membrane proteins are underrepresented in structural databases, which has led to a lack of computational tools and the corresponding inappropriate use of tools designed for soluble proteins. For membrane proteins, lipid accessibility is an essential property. Although programs are available for sequence-based prediction of lipid accessibility and structure-based identification of solvent-accessible surface area, the latter does not distinguish between water accessible and lipid accessible residues in membrane proteins.

Type 1 regulatory T cells (Tr1 cells) are induced by interleukin-27 (IL-27) and have critical roles in the control of autoimmunity and resolution of inflammation. We found that the transcription factors IRF1 and BATF were induced early on after treatment with IL-27 and were required for the differentiation and function of Tr1 cells in vitro and in vivo. Epigenetic and transcriptional analyses revealed that both transcription factors influenced chromatin accessibility and expression of the genes required for Tr1 cell function. IRF1 and BATF deficiencies uniquely altered the chromatin landscape, suggesting that these factors serve a pioneering function during Tr1 cell differentiation.