Julien Berro

Actin-Filament Stochastic Dynamics Mediated by ADF/Cofilin

BACKGROUND The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, we combined evanescent-wave microscopy with a biomimetic system and followed the behavior of single actin filaments in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. RESULTS In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. CONCLUSIONS Our proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells.

Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We report time courses of the accumulation and loss of 16 fluorescent fusion proteins at sites of clathrin-mediated endocytosis in fission yeast. Mathematical modeling shows that dendritic nucleation hypothesis can account for the kinetics of actin assembly in vivo and disassembly requires actin filament severing along with depolymerization.

Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments

We report time courses of the accumulation and loss of 16 fluorescent fusion proteins at sites of clathrin-mediated endocytosis in fission yeast. Mathematical modeling shows that dendritic nucleation hypothesis can account for the kinetics of actin assembly in vivo and disassembly requires actin filament severing along with depolymerization.

Mathematical Models and Simulations of Cellular Processes Based on Actin Filaments

Actin filaments help to maintain the physical integrity of cells and participate in many processes that produce cellular movements. Studies of the processes that depend on actin filaments have progressed to the point where mathematical models and computer simulations are an essential part of the experimental toolkit. These quantitative models integrate knowledge about the structures of the key proteins and the rate and equilibrium constants for the reactions for comparison with a growing body of quantitative measurements of dynamic processes in live cells. Models and simulations are essential because it is impossible to appreciate by intuition alone the properties that emerge from a network of coupled reactions, particularly when the system contains many components, and force is one of the parameters.

Molecular diversity, metabolic transformation, and evolution of carotenoid feather pigments in cotingas (Aves: Cotingidae).

Carotenoid pigments were extracted from 29 feather patches from 25 species of cotingas (Cotingidae) representing all lineages of the family with carotenoid plumage coloration. Using high-performance liquid chromatography (HPLC), mass spectrometry, chemical analysis, and 1H-NMR, 16 different carotenoid molecules were documented in the plumages of the cotinga family. These included common dietary xanthophylls (lutein and zeaxanthin), canary xanthophylls A and B, four well known and broadly distributed avian ketocarotenoids (canthaxanthin, astaxanthin, α-doradexanthin, and adonixanthin), rhodoxanthin, and seven 4-methoxy-ketocarotenoids. Methoxy-ketocarotenoids were found in 12 species within seven cotinga genera, including a new, previously undescribed molecule isolated from the Andean Cock-of-the-Rock Rupicola peruviana, 3′-hydroxy-3-methoxy-β,β-carotene-4-one, which we name rupicolin. The diversity of cotinga plumage carotenoid pigments is hypothesized to be derived via four metabolic pathways from lutein, zeaxanthin, β-cryptoxanthin, and β-carotene. All metabolic transformations within the four pathways can be described by six or seven different enzymatic reactions. Three of these reactions are shared among three precursor pathways and are responsible for eight different metabolically derived carotenoid molecules. The function of cotinga plumage carotenoid diversity was analyzed with reflectance spectrophotometry of plumage patches and a tetrahedral model of avian color visual perception. The evolutionary history of the origin of this diversity is analyzed phylogenetically. The color space analyses document that the evolutionarily derived metabolic modifications of dietary xanthophylls have resulted in the creation of distinctive orange-red and purple visual colors.

Cytokinetic nodes in fission yeast arise from two distinct types of nodes that merge during interphase

Two distinct classes of cortical nodes form separately during interphase in fission yeast cells and then merge at the cell equator by a diffuse-and-capture mechanism to prepare nodes to form the contractile ring for cytokinesis.

Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast

It is shown that, in addition to capping actin filaments, Aip1p and the capping protein subunit Acp2p are also involved in actin patch polarization in interphase, but not in mitosis. In contrast, Acp1p is not involved in cell polarization.

Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method

A temporal superresolution method is proposed to align data sets with a higher temporal resolution than the measurement resolution. Application to endocytic patches shows that the movement of endocytic vesicles is diffusive and impeded by the actin cytoskeleton. New tools are also proposed to count actin patches and study their polarization.

Mycofumigation through production of the volatile DNA-methylating agent N-methyl-N-nitrosoisobutyramide by fungi in the genus Muscodor

Antagonistic microorganisms produce antimicrobials to inhibit the growth of competitors. Although water-soluble antimicrobials are limited to proximal interactions via aqueous diffusion, volatile antimicrobials are able to act at a distance and diffuse through heterogeneous environments. Here, we identify the mechanism of action of Muscodor albus, an endophytic fungus known for its volatile antimicrobial activity toward a wide range of human and plant pathogens and its potential use in mycofumigation. Proposed uses of the Muscodor species include protecting crops, produce, and building materials from undesired fungal or bacterial growth. By analyzing a collection of Muscodor isolates with varying toxicity, we demonstrate that the volatile mycotoxin, N-methyl-N-nitrosoisobutyramide, is the dominant factor in Muscodor toxicity and acts primarily through DNA methylation. Additionally, Muscodor isolates exhibit higher resistance to DNA methylation compared with other fungi. This work contributes to the evaluation of Muscodor isolates as potential mycofumigants, provides insight into chemical strategies that organisms use to manipulate their environment, and provokes questions regarding the mechanisms of resistance used to tolerate constitutive, long-term exposure to DNA methylation.

Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9

Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe. Copyright