Dephosphorylation of the MAP kinases MPK6 and MPK3 fine-tunes responses to wounding and herbivory in Arabidopsis.

The Arabidopsis MAP Kinases (MAPKs) MPK6 and MPK3 and orthologs in other plants function as major stress signaling hubs. MAPKs are activated by phosphorylation and are negatively regulated by MAPK-inactivating phosphatases (MIPPs), which alter the intensity and duration of MAPK signaling via dephosphorylation. Unlike in other plant species, jasmonic acid (JA) accumulation in Arabidopsis is apparently not MPK6- and MPK3-dependent, so their role in JA-mediated defenses against herbivorous insects is unclear. Here we explore whether changes in MPK6/3 phosphorylation kinetics in Arabidopsis MIPP mutants lead to changes in hormone synthesis and resistance against herbivores. The MIPPs MKP1, DsPTP1, PP2C5, and AP2C1 have been implicated in responses to infection, drought, and osmotic stress, which all impinge on JA-mediated defenses. In loss-of-function mutants, we found that the four MIPPs alter wound-induced MPK6/3 phosphorylation kinetics and affect the accumulation of the defense hormones JA, abscisic acid, and salicylic acid, as compared to wild type plants (Col-0). Moreover, MPK6/3 misregulation in MIPP or MAPK mutant plants resulted in slight changes in the resistance to Trichoplusia ni and Spodoptera exigua larvae as compared to Col-0. Our data indicate that MPK6/3 and the four MIPPs moderately contribute to wound signaling and defense against herbivorous insects in Arabidopsis.

The power of weak, transient interactions across biology: A paradigm of emergent behavior.

A growing list of diverse biological systems and their equally diverse functionalities provides realizations of a paradigm of emergent behavior. In each of these biological systems, pervasive ensembles of weak, short-lived, spatially local interactions act autonomously to convey functionalities at larger spatial and temporal scales. In this article, a range of diverse systems and functionalities are presented in a cursory manner with literature citations for further details. Then two systems and their properties are discussed in more detail: yeast chromosome biology and human respiratory mucus.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus.

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks.

Modeling and Simulation of Mucus Flow in Human Bronchial Epithelial Cell Cultures - Part I: Idealized Axisymmetric Swirling Flow.

A multi-mode nonlinear constitutive model for mucus is constructed directly from micro- and macro-rheology experimental data on cell culture mucus, and a numerical algorithm is developed for the culture geometry and idealized cilia driving conditions. This study investigates the roles that mucus rheology, wall effects, and HBE culture geometry play in the development of flow profiles and the shape of the air-mucus interface. Simulations show that viscoelasticity captures normal stress generation in shear leading to a peak in the air-mucus interface at the middle of the culture and a depression at the walls. Linear and nonlinear viscoelastic regimes can be observed in cultures by varying the hurricane radius and mean rotational velocity. The advection-diffusion of a drug concentration dropped at the surface of the mucus flow is simulated as a function of Peclet number.

Entropy gives rise to topologically associating domains.

We investigate chromosome organization within the nucleus using polymer models whose formulation is closely guided by experiments in live yeast cells. We employ bead-spring chromosome models together with loop formation within the chains and the presence of nuclear bodies to quantify the extent to which these mechanisms shape the topological landscape in the interphase nucleus. By investigating the genome as a dynamical system, we show that domains of high chromosomal interactions can arise solely from the polymeric nature of the chromosome arms due to entropic interactions and nuclear confinement. In this view, the role of bio-chemical related processes is to modulate and extend the duration of the interacting domains.

DNA loops generate intracentromere tension in mitosis.

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.

Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores.

Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS). In human bronchial epithelial cell cultures (HBECCs), maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h), to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5-2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist-induced mucin secretion.

Polymer models of interphase chromosomes.

Clear organizational patterns on the genome have emerged from the statistics of population studies of fixed cells. However, how these results translate into the dynamics of individual living cells remains unexplored. We use statistical mechanics models derived from polymer physics to inquire into the effects that chromosome properties and dynamics have in the temporal and spatial behavior of the genome. Overall, changes in the properties of individual chains affect the behavior of all other chains in the domain. We explore two modifications of chain behavior: single chain motion and chain-chain interactions. We show that there is not a direct relation between these effects, as increase in motion, doesn't necessarily translate into an increase on chain interaction.

Buckling instability of self-assembled colloidal columns.

Suspended, slender self-assembled domains of magnetically responsive colloids are observed to buckle in microgravity. Upon cessation of the magnetic field that drives their assembly, these columns expand axially and buckle laterally. This phenomenon resembles the buckling of long beams due to thermal expansion; however, linear stability analysis predicts that the colloidal columns are inherently susceptible to buckling because they are freely suspended in a Newtonian fluid. The dominant buckling wavelength increases linearly with column thickness and is quantitatively described using an elastohydrodynamic model and the suspension thermodynamic equation of state.

Micro-heterogeneity metrics for diffusion in soft matter.

Passive particle tracking of diffusive paths in soft matter, coupled with analysis of the path data, is firmly established as a fundamental methodology for characterization of both diffusive transport properties (the focus here) and linear viscoelasticity. For either focus, particle time series are typically analyzed by ensemble averaging over paths, a perfectly natural protocol for homogeneous materials or for applications where mean properties are sufficient. Many biological materials, however, are heterogeneous over length scales above the probe diameter, and the implications of heterogeneity for biologically relevant transport properties (e.g. diffusive passage times through a complex fluid layer) motivate this paper. Our goals are three-fold: first, to detect heterogeneity as reflected by the ensemble path data; second, to further decompose the ensemble of particle paths into statistically distinct clusters; and third, to fit the path data in each cluster to a model for the underlying stochastic process. After reviewing current best practices for detection and assessment of heterogeneity in diffusive processes, we introduce our strategy toward the first two goals with methods from the statistics and machine learning literature that have not found application thus far to passive particle tracking data. We apply an analysis based solely on the path data that detects heterogeneity and yields a decomposition of particle paths into statistically distinct clusters. After these two goals are achieved, one can then pursue model-fitting. We illustrate these heterogeneity metrics on diverse datasets: for numerically generated and experimental particle paths, with tunable and unknown heterogeneity, on numerical models for simple diffusion and anomalous sub-diffusion, and experimentally on sucrose, hyaluronic acid, agarose, and human lung culture mucus solutions.

A biophysical basis for mucus solids concentration as a candidate biomarker for airways disease.

In human airways diseases, including cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), host defense is compromised and airways inflammation and infection often result. Mucus clearance and trapping of inhaled pathogens constitute key elements of host defense. Clearance rates are governed by mucus viscous and elastic moduli at physiological driving frequencies, whereas transport of trapped pathogens in mucus layers is governed by diffusivity. There is a clear need for simple and effective clinical biomarkers of airways disease that correlate with these properties. We tested the hypothesis that mucus solids concentration, indexed as weight percent solids (wt%), is such a biomarker. Passive microbead rheology was employed to determine both diffusive and viscoelastic properties of mucus harvested from human bronchial epithelial (HBE) cultures. Guided by sputum from healthy (1.5-2.5 wt%) and diseased (COPD, CF; 5 wt%) subjects, mucus samples were generated in vitro to mimic in vivo physiology, including intermediate range wt% to represent disease progression. Analyses of microbead datasets showed mucus diffusive properties and viscoelastic moduli scale robustly with wt%. Importantly, prominent changes in both biophysical properties arose at ∼4 wt%, consistent with a gel transition (from a more viscous-dominated solution to a more elastic-dominated gel). These findings have significant implications for: (1) penetration of cilia into the mucus layer and effectiveness of mucus transport; and (2) diffusion vs. immobilization of micro-scale particles relevant to mucus barrier properties. These data provide compelling evidence for mucus solids concentration as a baseline clinical biomarker of mucus barrier and clearance functions.

Individual pericentromeres display coordinated motion and stretching in the yeast spindle.

The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.

Pericentric chromatin loops function as a nonlinear spring in mitotic force balance.

The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.

Multi-scale kinetics of a field-directed colloidal phase transition.

Polarizable colloids are expected to form crystalline equilibrium phases when exposed to a steady, uniform field. However, when colloids become localized this field-induced phase transition arrests and the suspension persists indefinitely as a kinetically trapped, percolated structure. We anneal such gels formed from magneto-rheological fluids by toggling the field strength at varied frequencies. This processing allows the arrested structure to relax periodically to equilibrium--colloid-rich, cylindrical columns. Two distinct growth regimes are observed: one in which particle domains ripen through diffusive relaxation of the gel, and the other where the system-spanning structure collapses and columnar domains coalesce apparently through field-driven interactions. There is a stark boundary as a function of magnetic field strength and toggle frequency distinguishing the two regimes. These results demonstrate how kinetic barriers to a colloidal phase transition are subverted through measured, periodic variation of driving forces. Such directed assembly may be harnessed to create unique materials from dispersions of colloids.