Justin S. Bois

NUPACK: Analysis and design of nucleic acid systems

The Nucleic Acid Package (NUPACK) is a growing software suite for the analysis and design of nucleic acid systems. The NUPACK web server (http://www.nupack.org) currently enables: Analysis: thermodynamic analysis of dilute solutions of interacting nucleic acid strands. Design: sequence design for complexes of nucleic acid strands intended to adopt a target secondary structure at equilibrium. Utilities: evaluation, display, and annotation of equilibrium properties of a complex of nucleic acid strands. NUPACK algorithms are formulated in terms of nucleic acid secondary structure. In most cases, pseudoknots are excluded from the structural ensemble.

Anisotropies in cortical tension reveal the physical basis of polarizing cortical flows

Asymmetric cell divisions are essential for the development of multicellular organisms. To proceed, they require an initially symmetric cell to polarize. In Caenorhabditis elegans zygotes, anteroposterior polarization is facilitated by a large-scale flow of the actomyosin cortex, which directs the asymmetry of the first mitotic division. Cortical flows appear in many contexts of development, but their underlying forces and physical principles remain poorly understood. How actomyosin contractility and cortical tension interact to generate large-scale flow is unclear. Here we report on the subcellular distribution of cortical tension in the polarizing C. elegans zygote, which we determined using position- and direction-sensitive laser ablation. We demonstrate that cortical flow is associated with anisotropies in cortical tension and is not driven by gradients in cortical tension, which contradicts previous proposals. These experiments, in conjunction with a theoretical description of active cortical mechanics, identify two prerequisites for large-scale cortical flow: a gradient in actomyosin contractility to drive flow and a sufficiently large viscosity of the cortex to allow flow to be long-ranged. We thus reveal the physical requirements of large-scale intracellular cortical flow that ensure the efficient polarization of the C. elegans zygote.

Thermodynamic Analysis of Interacting Nucleic Acid Strands

Motivated by the analysis of natural and engineered DNA and RNA systems, we present the first algorithm for calculating the partition function of an unpseudoknotted complex of multiple interacting nucleic acid strands. This dynamic program is based on a rigorous extension of secondary structure models to the multistranded case, addressing representation and distinguishability issues that do not arise for single-stranded structures. We then derive the form of the partition function for a fixed volume containing a dilute solution of nucleic acid complexes. This expression can be evaluated explicitly for small numbers of strands, allowing the calculation of the equilibrium population distribution for each species of complex. Alternatively, for large systems (e.g., a test tube), we show that the unique complex concentrations corresponding to thermodynamic equilibrium can be obtained by solving a convex programming problem. Partition function and concentration information can then be used to calculate equilibrium base-pairing observables. The underlying physics and mathematical formulation of these problems lead to an interesting blend of approaches, including ideas from graph theory, group theory, dynamic programming, combinatorics, convex optimization, and Lagrange duality.

Polarization of PAR proteins by advective triggering of a pattern-forming system.

Patterning of Caenorhabditis elegans zygotes involves passive as well as active mechanisms. In the Caenorhabditis elegans zygote, a conserved network of partitioning-defective (PAR) polarity proteins segregates into an anterior and a posterior domain, facilitated by flows of the cortical actomyosin meshwork. The physical mechanisms by which stable asymmetric PAR distributions arise from transient cortical flows remain unclear. We present evidence that PAR polarity arises from coupling of advective transport by the flowing cell cortex to a multistable PAR reaction-diffusion system. By inducing transient PAR segregation, advection serves as a mechanical trigger for the formation of a PAR pattern within an otherwise stably unpolarized system. We suggest that passive advective transport in an active and flowing material may be a general mechanism for mechanochemical pattern formation in developmental systems.

Turing's next steps: the mechanochemical basis of morphogenesis

Nearly 60 years ago, Alan Turing showed theoretically how two chemical species, termed morphogens, diffusing and reacting with each other can generate spatial patterns. Diffusion plays a crucial part in transporting chemical signals through space to establish the length scale of the pattern. When coupled to chemical reactions, mechanical processes — forces and flows generated by motor proteins — can also define length scales and provide a mechanochemical basis for morphogenesis. forces and flows generated by motor proteins — can also define length scales and provide a mechanochemical basis for morphogenesis.

Topological constraints in nucleic acid hybridization kinetics

A theoretical examination of kinetic mechanisms for forming knots and links in nucleic acid structures suggests that molecules involving base pairs between loops are likely to become topologically trapped in persistent frustrated states through the mechanism of ‘helix-driven wrapping’. Augmentation of the state space to include both secondary structure and topology in describing the free energy landscape illustrates the potential for topological effects to influence the kinetics and function of nucleic acid strands. An experimental study of metastable complementary ‘kissing hairpins’ demonstrates that the topological constraint of zero linking number between the loops effectively prevents conversion to the minimum free energy helical state. Introduction of short catalyst strands that break the topological constraint causes rapid conversion to full duplex.

Control of convergent yolk syncytial layer nuclear movement in zebrafish

Nuclear movements play an essential role in metazoan development. Although the intracellular transport mechanisms underlying nuclear movements have been studied in detail, relatively little is known about signals from surrounding cells and tissues controlling these movements. Here, we show that, in gastrulating zebrafish embryos, convergence movements of nuclei within the yolk syncytial layer (YSL) are guided by mesoderm and endoderm progenitors migrating along the surface of the yolk towards the dorsal side of the developing gastrula. Progenitor cells direct the convergence movements of internal yolk syncytial nuclei (iYSN) by modulating cortical flow within the YSL in which the iYSN are entrained. The effect of mesoderm and endoderm progenitors on the convergence movement of iYSN depends on the expression of E-cadherin, indicating that adhesive contact between the cells and the YSL is required for the mesendoderm-modulated YSL cortical flow mediating nuclear convergence. In summary, our data reveal a crucial function for cortical flow in the coordination of syncytial nuclear movements with surrounding cells and tissues during zebrafish gastrulation.

Interaction between Microtubules and the Drosophila Formin Cappuccino and Its Effect on Actin Assembly

Background: Cappuccino is a formin actin nucleator that regulates cytoskeletal organization during Drosophila oogenesis. Results: Cappuccino binds microtubules through two domains and cannot nucleate actin filaments when bound to microtubules. Conclusion: Actin filament assembly and microtubule binding are mutually exclusive activities of Cappuccino. Significance: We provide mechanistic insight into the role of formins as coordinators of the actin and microtubule cytoskeletons. Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

Phenotypic and Physiological Characterization of the Epibiotic Interaction Between TM7x and Its Basibiont "Actinomyces"

Despite many examples of obligate epibiotic symbiosis (one organism living on the surface of another) in nature, such an interaction has rarely been observed between two bacteria. Here, we further characterize a newly reported interaction between a human oral obligate parasitic bacterium TM7x (cultivated member of Candidatus Saccharimonas formerly Candidate Phylum TM7), and its basibiont Actinomyces odontolyticus species (XH001), providing a model system to study epiparasitic symbiosis in the domain Bacteria. Detailed microscopic studies indicate that both partners display extensive morphological changes during symbiotic growth. XH001 cells manifested as short rods in monoculture, but displayed elongated and hyphal morphology when physically associated with TM7x. Interestingly, these dramatic morphological changes in XH001 were also induced in oxygen-depleted conditions, even in the absence of TM7x. Targeted quantitative real-time PCR (qRT-PCR) analyses revealed that both the physical association with TM7x as well as oxygen depletion triggered up-regulation of key stress response genes in XH001, and in combination, these conditions act in an additive manner. TM7x and XH001 co-exist with relatively uniform cell morphologies under nutrient-replete conditions. However, upon nutrient depletion, TM7x-associated XH001 displayed a variety of cell morphologies, including swollen cell body, clubbed-ends, and even cell lysis, and a large portion of TM7x cells transformed from ultrasmall cocci into elongated cells. Our study demonstrates a highly dynamic interaction between epibiont TM7x and its basibiont XH001 in response to physical association or environmental cues such as oxygen level and nutritional status, as reflected by their morphological and physiological changes during symbiotic growth.

Regulation of the formin Cappuccino is critical for polarity of Drosophila oocytes.

The Drosophila formin Cappuccino (Capu) creates an actin mesh‐like structure that traverses the oocyte during midoogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late‐oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd, and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capus function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN‐expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from coexpression of the N‐terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development.