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- Plant Physiology Preview. Published on March 31, 2014, as DOI:10.1104/pp.114.238477
- Dynamics and organization of cortical microtubules as revealed by superresolution
- Introduction
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Running Head: Superresolution of cortical microtubule dynamics 1 Jozef Šamaj: Centre of the Region Haná for Biotechnological and Agricultural Research, 2 Department of Cell Biology, Faculty of Science, Palacký University Olomouc, Šlechtitel ů 11, 3 78371 Olomouc, Czech Republic 4 Tel.: +420 585 634 978 5 Email: jozef.samaj@upol.cz 6 Research Area: Cell Biology 7 Secondary Research Area: Breakthrough Technologies 8
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George Komis a , Martin Mistrík b , Olga Šamajová a , Anna Dosko čilová a , Miroslav Ove čka a , Peter 13 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 15 Biology, Faculty of Science, Palacký University Olomouc, Šlechtitel ů 11, 78371 Olomouc, 16
Czech Republic 17
b Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry Palacký 18 University Olomouc, Hn ěvotínská 5, 779 00 Olomouc, Czech Republic 19
c Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen, 20 Denmark
21 Summary: This is the first quantitative study on dynamic organization of plant cell focused on 22 cortical microtubules by using structured illumination superresolution microscopy. 23
24 This work was supported by grant P501/11/1764 from the Czech Science Foundation GA ČR, by
25 grant LO1204 NPU I Sustainable development of research in the Centre of the Region Haná for 26 Biotechnological and Agricultural Research and by European Commission (project BioMedReg). 27 Corresponding author: 28 Jozef Šamaj 29 Email: jozef.samaj@upol.cz 30
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33 Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for 34 cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for 35 optimal documentation of Arabidopsis hypocotyl epidermal cells, dynamic cortical microtubules 36 labeled with GFP-MBD and GFP-TUA6 markers were comparatively recorded in wild type 37 Arabidopsis plants and in mitogen activated protein kinase mutant mpk4 possessing later 38 microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased 39 abundance and reduced phosphorylation of microtubule-associated protein MAP65-1, thus 40 providing a very useful genetic tool to record intrabundle microtubule dynamics at subdiffraction 41 level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved 42 microtubule branching and release, and finally allowed quantification of individual microtubules 43 within cortical bundles. Time-lapsed SIM imaging allowed visualizing subdiffraction, short-lived 44 excursions of the microtubule plus end and dynamic instability behavior of both ends during 45 free, intrabundle or microtubule-templated microtubule growth and shrinkage. Finally, short, 46 rigid and non-dynamic microtubule bundles in the mpk4 mutant were observed to glide along the 47 parent microtubule in a tip-wise manner. Conclusively, this study demonstrates the potential of 48 SIM for superresolution time-lapsed imaging of plant cells showing unprecedented details 49 accompanying microtubule dynamic organization. 50
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54 Plant cell growth and differentiation depend on dynamic cortical microtubule organization 55 mechanisms (Ehrhardt, 2008) including branched microtubule formation and release (Murata et 56 al., 2005; Nakamura et al., 2010; Fishel and Dixit, 2013), microtubule-templated microtubule 57 growth (Chan et al., 2009), angle-of-contact microtubule bundling or catastrophe induction 58 (Dixit & Cyr, 2004; Tulin et al., 2012), severing at microtubule cross-overs (Wightman and 59 Turner, 2007) and unique dynamic behavior between steady-state treadmilling and dynamic 60 instability (Shaw et al., 2003). 61 Cortical microtubule dynamics have been explored in vivo and in vitro with total internal 62 reflection microscopy (TIRFM; Vizcay-Barrena et al., 2011), variable angle emission 63 microscopy (VAEM; Wan et al., 2011), spinning disc microscopy (SD; Shaw & Lucas, 2011), 64 and confocal laser scanning microscopy (CLSM; Shaw et al., 2003). TIRFM and VAEM provide 65 sufficient resolution and speed but at limited depth of imaging (ca. 200 nm; Martin-Fernandez et 66 al., 2013) and inevitably a very narrow field of view when used for in vivo studies (Mattheyses et 67 al., 2010). Dynamic CLSM imaging suffers from field of view limitations while also introducing 68 phototoxicity to the imaged sample. Furthermore, CLSM will require a speed-to-resolution trade 69 off which will necessitate computational extrapolation to bring resolution at affordable levels 70 (Rosero et al., 2014). Finally, SD can provide sufficient depth and speed but otherwise poor 71 resolution owing to aberrations arising from the sample and the properties of the optics 72 commonly used (Shaw and Ehrhardt, 2013). 73 Microtubule research evolved concomitantly with optical microscopy and the development of 74 fluorescent proteins markers, allowing the resolution of microtubule dynamics and organization 75 at video rates (Marc et al., 1998; Shaw et al., 2013). However, the bulk of plant cells organized 76 in tissues and the optical properties of cell walls hamper microscopic observations, so that the 77 definition of fine details of microtubule organization still relies on laborious transmission 78 electron microscopy (TEM; Kang, 2010). 79 Alternatively, in vitro assays using TIRF or Allen’s video enhanced contrast - differential 80 interference contrast microscopy (AVEC-DIC; Allen et al., 1981) with purified components have 81 advanced the understanding of microtubule-MAP interactions while providing mechanistic 82
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