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.
Highlights
The centrosome is the dominant microtubule (MT) generation site in animal somatic cells, acentrosomal MTs play critical roles in many aspects of cell biology, such as cell division and polarization, in a variety of cell types (Bartolini and Gundersen, 2006; Goshima and Kimura, 2010)
This study aimed to elucidate the mechanism of endoplasmic MT generation using protonemal cells of P. patens
MT arrays, in which fixed-angled branching nucleation and severing efficiently generate parallel bundled MTs, cytoplasmic nucleation and variably oriented branching nucleation were frequently observed at seemingly random locations
Summary
The centrosome is the dominant microtubule (MT) generation site in animal somatic cells, acentrosomal MTs play critical roles in many aspects of cell biology, such as cell division and polarization, in a variety of cell types (Bartolini and Gundersen, 2006; Goshima and Kimura, 2010). Land plants, which have lost centrosomes during evolution, generate MTs independent of centrosomes. They serve as an ideal model system for studying the mechanism of acentrosomal MT generation. The MTs, which are required for cellulose synthase deposition, are nucleated at the cortex, bundled in parallel, bound to the plasma membrane, and arranged transverse to the elongation axis of the cell (Dixit and Cyr, 2004; Lloyd and Chan, 2008). Live-cell microscopy, gene perturbation experiments, and mathematical modeling have elucidated a detailed mechanism for this process (Murata et al, 2005; Chan et al, 2009; Nakamura and Hashimoto, 2009; Wasteneys and Ambrose, 2009; Nakamura et al, 2010; Fishel and Dixit, 2013; Lindeboom et al, 2013a).
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