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Running Head: Superresolution of cortical microtubule dynamics
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Jozef Šamaj: Centre of the Region Haná for Biotechnological and Agricultural Research,
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Department of Cell Biology, Faculty of Science, Palacký University Olomouc, Šlechtitel
ů 11,
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78371 Olomouc, Czech Republic
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Tel.: +420 585 634 978
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Email: jozef.samaj@upol.cz
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Research Area: Cell Biology
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Secondary Research Area: Breakthrough Technologies
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Plant Physiology Preview. Published on March 31, 2014, as DOI:10.1104/pp.114.238477
Copyright 2014 by the American Society of Plant Biologists
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Dynamics and organization of cortical microtubules as revealed by superresolution
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structured illumination microscopy
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George Komis
a
, Martin Mistrík
b
, Olga Šamajová
a
, Anna Dosko
čilová
a
, Miroslav Ove
čka
a
, Peter
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Illés
a
, Ji
ří Bártek
b,c
, and Jozef Šamaj
a,1
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a
Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell
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Biology, Faculty of Science, Palacký University Olomouc, Šlechtitel
ů 11, 78371 Olomouc,
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Czech Republic
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b
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry Palacký
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University Olomouc, Hn
ěvotínská 5, 779 00 Olomouc, Czech Republic
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c
Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen,
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Denmark
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Summary: This is the first quantitative study on dynamic organization of plant cell focused on
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cortical microtubules by using structured illumination superresolution microscopy.
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This work was supported by grant P501/11/1764 from the Czech Science Foundation GA
ČR, by
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grant LO1204 NPU I Sustainable development of research in the Centre of the Region Haná for
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Biotechnological and Agricultural Research and by European Commission (project BioMedReg).
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Corresponding author:
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Jozef Šamaj
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Email: jozef.samaj@upol.cz
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Abstract
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Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for
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cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for
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optimal documentation of Arabidopsis hypocotyl epidermal cells, dynamic cortical microtubules
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labeled with GFP-MBD and GFP-TUA6 markers were comparatively recorded in wild type
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Arabidopsis plants and in mitogen activated protein kinase mutant mpk4 possessing later
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microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased
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abundance and reduced phosphorylation of microtubule-associated protein MAP65-1, thus
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providing a very useful genetic tool to record intrabundle microtubule dynamics at subdiffraction
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level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved
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microtubule branching and release, and finally allowed quantification of individual microtubules
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within cortical bundles. Time-lapsed SIM imaging allowed visualizing subdiffraction, short-lived
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excursions of the microtubule plus end and dynamic instability behavior of both ends during
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free, intrabundle or microtubule-templated microtubule growth and shrinkage. Finally, short,
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rigid and non-dynamic microtubule bundles in the mpk4 mutant were observed to glide along the
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parent microtubule in a tip-wise manner. Conclusively, this study demonstrates the potential of
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SIM for superresolution time-lapsed imaging of plant cells showing unprecedented details
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accompanying microtubule dynamic organization.
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Introduction
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Plant cell growth and differentiation depend on dynamic cortical microtubule organization
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mechanisms (Ehrhardt, 2008) including branched microtubule formation and release (Murata et
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al., 2005; Nakamura et al., 2010; Fishel and Dixit, 2013), microtubule-templated microtubule
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growth (Chan et al., 2009), angle-of-contact microtubule bundling or catastrophe induction
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(Dixit & Cyr, 2004; Tulin et al., 2012), severing at microtubule cross-overs (Wightman and
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Turner, 2007) and unique dynamic behavior between steady-state treadmilling and dynamic
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instability (Shaw et al., 2003).
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Cortical microtubule dynamics have been explored in vivo and in vitro with total internal
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reflection microscopy (TIRFM; Vizcay-Barrena et al., 2011), variable angle emission
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microscopy (VAEM; Wan et al., 2011), spinning disc microscopy (SD; Shaw & Lucas, 2011),
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and confocal laser scanning microscopy (CLSM; Shaw et al., 2003). TIRFM and VAEM provide
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sufficient resolution and speed but at limited depth of imaging (ca. 200 nm; Martin-Fernandez et
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al., 2013) and inevitably a very narrow field of view when used for in vivo studies (Mattheyses et
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al., 2010). Dynamic CLSM imaging suffers from field of view limitations while also introducing
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phototoxicity to the imaged sample. Furthermore, CLSM will require a speed-to-resolution trade
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off which will necessitate computational extrapolation to bring resolution at affordable levels
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(Rosero et al., 2014). Finally, SD can provide sufficient depth and speed but otherwise poor
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resolution owing to aberrations arising from the sample and the properties of the optics
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commonly used (Shaw and Ehrhardt, 2013).
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Microtubule research evolved concomitantly with optical microscopy and the development of
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fluorescent proteins markers, allowing the resolution of microtubule dynamics and organization
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at video rates (Marc et al., 1998; Shaw et al., 2013). However, the bulk of plant cells organized
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in tissues and the optical properties of cell walls hamper microscopic observations, so that the
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definition of fine details of microtubule organization still relies on laborious transmission
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electron microscopy (TEM; Kang, 2010).
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Alternatively, in vitro assays using TIRF or Allen’s video enhanced contrast - differential
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interference contrast microscopy (AVEC-DIC; Allen et al., 1981) with purified components have
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advanced the understanding of microtubule-MAP interactions while providing mechanistic
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