title: Cytoplasmic nucleation and atypical branching nucleation generate endoplasmic microtubules in Physcomitrella patens.
abstract: The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants.
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan Marine Biological Laboratory, Woods Hole, Massachusetts 02543.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Nakaoka
authors.affiliation: Marine Biological Laboratory, Woods Hole, Massachusetts 02543 National Institute of Genetics, Mishima 411-8540, Japan.
authors.forename: Akatsuki
authors.initials: A
authors.lastname: Kimura
authors.affiliation: Marine Biological Laboratory, Woods Hole, Massachusetts 02543.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Tani
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan Marine Biological Laboratory, Woods Hole, Massachusetts 02543 goshima@bio.nagoya-u.ac.jp.
authors.forename: Gohta
authors.initials: G
authors.lastname: Goshima
mesh_terms: D019068:Bryopsida / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
chemical_list: D014404:Tubulin