Dynamics of actin cables in polarized growth of the filamentous fungus Aspergillus nidulans

Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules

Alteration Reaction and Mass Transfer via Fluids with Progress of Fracturing along the Median Tectonic Line, Mie Prefecture, Southwest Japan

We have analyzed mass transfer in the cataclasite samples collected from the Median Tectonic Line, southwest Japan, in which the degree of fracturing is well correlated with the bulk rock chemical compositions determined by the X-ray fluorescence (XRF) analysis. The results of “isocon” analysis indicate not only a large volume increase up to 110% but also the two-stage mass transfer during cataclasis. At the first stage from the very weakly to weakly fractured rocks, the weight percents of SiO2, Na2O, and K2O increase, while those of TiO2, FeO, MnO, MgO, and CaO decrease. At the second stage from the weakly to moderately and strongly fractured rocks, the trend of mass transfer is reversed. The principal component analysis reveals that the variation of chemical compositions in the cataclasite samples can be mostly interpreted by the mass transfer via fluids and by the difference in chemical composition in the protolith rocks to lesser degree. Finally, the changes in the modal composition of minerals with increasing cataclasis analyzed by the X-ray diffraction (XRD) with the aid of “RockJock” software clearly elucidate that the mass transfer of chemical elements was caused by dissolution and precipitation of minerals via fluids in the cataclasite samples

糸状菌をモデルとした極性生長におけるアクチンケーブルの役割

科学研究費助成事業 研究成果報告書：若手研究(B)2015-2017課題番号 : 15K1866

Superresolution and pulse-chase imaging reveal the role of vesicle transport in polar growth of fungal cells

Polarized growth of filamentous fungi requires continuous transport of biomolecules to the hyphal tip. To this end, construction materials are packaged in vesicles and transported by motor proteins along microtubules and actin filaments. We have studied these processes with quantitative superresolution localization microscopy of live Aspergillus nidulans cells expressing the photoconvertible protein mEosFPthermo fused to the chitin synthase ChsB. ChsB is mainly located at the Spitzenkörper near the hyphal tip and produces chitin, a key component of the cell wall. We have visualized the pulsatory dynamics of the Spitzenkörper, reflecting vesicle accumulation before exocytosis and their subsequent fusion with the apical plasma membrane. Furthermore, high-speed pulse-chase imaging after photoconversion of mEosFPthermo in a tightly focused spot revealed that ChsB is transported with two different speeds from the cell body to the hyphal tip and vice versa. Comparative analysis using motor protein deletion mutants allowed us to assign the fast movements (7 to 10 μm s−1) to transport of secretory vesicles by kinesin-1, and the slower ones (2 to 7 μm s−1) to transport by kinesin-3 on early endosomes. Our results show how motor proteins ensure the supply of vesicles to the hyphal tip, where temporally regulated exocytosis results in stepwise tip extension

Comparative genomics reveals the origin of fungal hyphae and multicellularity

Hyphae represent a hallmark structure of multicellular fungi. The evolutionary origins of hyphae and of the underlying genes are, however, hardly known. By systematically analyzing 72 complete genomes, we here show that hyphae evolved early in fungal evolution probably via diverse genetic changes, including co-option and exaptation of ancient eukaryotic (e.g. phagocytosis-related) genes, the origin of new gene families, gene duplications and alterations of gene structure, among others. Contrary to most multicellular lineages, the origin of filamentous fungi did not correlate with expansions of kinases, receptors or adhesive proteins. Co-option was probably the dominant mechanism for recruiting genes for hypha morphogenesis, while gene duplication was apparently less prevalent, except in transcriptional regulators and cell wall - related genes. We identified 414 novel gene families that show correlated evolution with hyphae and that may have contributed to its evolution. Our results suggest that hyphae represent a unique multicellular organization that evolved by limited fungal-specific innovations and gene duplication but pervasive co-option and modification of ancient eukaryotic functions

Effects of Laser Welding on Formability Aspects of Advanced High Strength Steel

Limiting dome height (LDH) tests were used to evaluate the formability of both base metal and laser butt welded blanks of AHSS (including High strength low alloy (HSLA), Dual phase (DP) steels of different grades). Mechanical properties of the base metal and welded blanks were assessed by uniaxial tensile and biaxial LDH tests, and related to measured microhardness distributions across the welds. The formability ratio of laser welded dual phase sheet steels generally decreases with increased base metal strength. A significant decrease of LDH was observed in the higher strength DP steel welded specimens due to the formation of a softened zone in the Heat Affected Zone(HAZ). Softened zone characteristics were correlated to the LDH. Larger softened zones led to a larger reduction in the LDH. HAZ softening has been shown to be a function of the base metal martensite content and the weld heat input. Formability also decreased with increased weld heat input. Both in experiment and numerical simulations strain is localized in the softened HAZ in the uniaxial tensile testing, indicating that strain localization decreases tensile strength and elongation of laser welds in DP980

Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins

Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds

Stress reversal recorded in calcite vein cuttings from the Nankai accretionary prism, southwest Japan

The Nankai Trough subduction zone in southwest Japan is a typical convergent margin where the Philippine Sea plate subducts in the northwest direction beneath the Eurasian plate, and devastating earthquakes have repeatedly occurred in this region in the past. In order to investigate the evolution of the stress state in the subduction zone, we analyzed deformation microstructures and the preferred orientation of calcite grains in two cuttings of calcite veins from Hole C0002F that was drilled through the inner wedge of the Nankai accretionary prism during the Integrated Ocean Discovery Program (IODP) Expedition 338 in 2012. For both samples collected at depths of 1,085.5 and 1,885.5 meters below the sea floor (mbsf), the c-axes of calcite grains are preferentially oriented perpendicular to the vein wall, which is indicative of competitive growth of calcite during the vein opening caused by a vein normal extension. Also, mechanical e-twins were developed in both samples, and these are inferred to have been developed under the same stress field as that responsible for the formation of calcite veins based on the paleostress analyses in grains with e-twins. For the calcite vein retrieved at the depth of 1,885.5 mbsf, kink bands were also developed by the compression in the direction perpendicular to the vein wall, which is indicative of stress reversal after the formation of mechanical e-twins. Although we could not reach a definite conclusion for the cause of the stress reversal, it could have occurred during either fold development or seismic cycles in the Nankai accretionary prism