Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics

Abstract

Microtubule bundling is an essential mechanism underlying the biased organization of interphase and mitotic microtubular systems of eukaryotes in ordered arrays. Microtubule bundle formation can be exemplified in plants, where the formation of parallel microtubule systems in the cell cortex or the spindle midzone is largely owing to the microtubule crosslinking activity of a family of microtubule associated proteins, designated as MAP65s. Among the nine members of this family in Arabidopsis thaliana, MAP65-1 and MAP65-2 are ubiquitous and functionally redundant. Crosslinked microtubules can form high-order arrays, which are difficult to track using widefield or confocal laser scanning microscopy approaches. Here, we followed spatiotemporal patterns of MAP65-2 localization in hypocotyl cells of Arabidopsis stably expressing fluorescent protein fusions of MAP65-2 and tubulin. To circumvent imaging difficulties arising from the density of cortical microtubule bundles, we use different superresolution approaches including Airyscan confocal laser scanning microscopy (ACLSM), structured illumination microscopy (SIM), total internal reflection SIM (TIRF-SIM), and photoactivation localization microscopy (PALM). We provide insights into spatiotemporal relations between microtubules and MAP65-2 crossbridges by combining SIM and ACLSM. We obtain further details on MAP65-2 distribution by single molecule localization microscopy (SMLM) imaging of either mEos3.2-MAP65-2 stochastic photoconversion, or eGFP-MAP65-2 stochastic emission fluctuations under specific illumination conditions. Time-dependent dynamics of MAP65-2 were tracked at variable time resolution using SIM, TIRF-SIM, and ACLSM and post-acquisition kymograph analysis. ACLSM imaging further allowed to track end-wise dynamics of microtubules labeled with TUA6-GFP and to correlate them with concomitant fluctuations of MAP65-2 tagged with tagRFP. All different microscopy modules examined herein are accompanied by restrictions in either the spatial resolution achieved, or in the frame rates of image acquisition. PALM imaging is compromised by speed of acquisition. This limitation was partially compensated by exploiting emission fluctuations of eGFP which allowed much higher photon counts at substantially smaller time series compared to mEos3.2. SIM, TIRF-SIM, and ACLSM

GR24, A Synthetic Strigolactone Analog, and Light Affect the Organization of Cortical Microtubules in Arabidopsis Hypocotyl Cells

“…Being produced mainly in the roots (Foo et al, 2013), strigolactones adjust both shoot (Gomez-Roldan et al, 2008;Umehara et al, 2008) and root (Ruyter-Spira et al, 2011) development in vascular plants as well as in moss caulonema (Hoffmann et al, 2014) in response to changing environmental conditions. Early grafting experiments showed that strigolactones are transported from roots to shoot in the xylem of Arabidopsis and tomato, which provided insight into strigolactone signaling regulation via their localization and transport (Kohlen et al, 2011).…”

“…The exogenous application of a synthetic strigolactone, GR24 (Umehara et al, 2008) and an inhibitor of endogenous strigolactone production, TIS108, a potent FIGURE 9 | Hypothetical model of light-dependent strigolactone effects on microtubules in Arabidopsis. (A) Regarding the overall hypocotyl phenotype, the strigolactone treatment inhibits hypocotyl growth and causes radial expansion of epidermal cells in light-grown seedlings; however, the dark-grown ones were more resistant to the changes of strigolactone content.…”

“…Microscopy platform enabling structured illumination microscopy (SIM) (ELYRA PS.1, Carl Zeiss, Germany) with 63×/1.40 NA Plan-Apochromat oil-immersion objective was used for the time-lapse observations of microtubule dynamics. For the excitation of GFP, a BP495–575/LP750 filter was used ( Vavrdová et al, 2020 ). In addition, 4-day-old seedlings were mounted between a microscope slide and a coverslip in 30 μL of liquid MS medium spaced by double-sided sticky tape and narrow Parafilm stripes, and they were extra sealed using liquid petroleum jelly (nail polish) to form a chamber prior to imaging for sample stabilization.…”

“…Strigolactones initiate arbuscular mycorrhizal symbiosis (Akiyama et al, 2005;Taulera et al, 2020), promote nodulation in the legume-rhizobium symbiosis interaction (Foo et al, 2014;van Zeijl et al, 2015;McAdam et al, 2017), and enhance plant resistance to drought, salt and osmotic stresses, and low soil phosphate and nitrate content (Yoneyama et al, 2007;Foo et al, 2013;Ha et al, 2014;Min et al, 2019). The physiological effects of strigolactones on the aboveground plant part include: (i) regulation of plant height (de Saint Germain et al, 2013;Wang et al, 2020b), (ii) control of shoot branching by modulating auxin transport (Kapulnik et al, 2011;Shinohara et al, 2013;Bennett et al, 2016), (iii) suppression of the preformed axillary bud outgrowth (Gomez-Roldan et al, 2008;Umehara et al, 2008;Domagalska and Leyser, 2011), (iv) increased expansion of leaves (Hu et al, 2019) and cotyledons (Tsuchiya et al, 2010), (v) rescue of the dark-induced elongation of rice mesocotyls (Hu et al, 2010;Sun et al, 2018), (vi) promotion of the secondary growth (Agusti et al, 2011), (vii) regulation of meristem cell number in the root (Koren et al, 2013), and (viii) stimulation of leaf senescence (Koltai, 2014;Ueda and Kusaba, 2015). Furthermore, the synthetic strigolactone GR24 acts synergistically with auxins and it is involved in Arabidopsis seed germination (Toh et al, 2012), potato tuber formation, the outgrowth of the axillary stolon buds, and aboveground potato shoot branching (Roumeliotis et al, 2012).…”

Imaging plant cells and organs with light-sheet and super-resolution microscopy

“…Super-resolved 3D visualization showed disturbed organization of MTs in dividing cells of the Arabidopsis ktn1 mutant using the GFP-TUA6 MT marker ( Komis et al, 2017 ; Vyplelová et al, 2018 ). In addition, high-resolution ACLSM images of MAP65-2 labeled with eGFP or tagRFP proved its partial association with MTs visualized with TUA6-GFP or mRFP-BETA-6 TUBULIN (mRFP-TUB6) markers ( Vavrdová et al, 2020 ). ACLSM was used for dynamic localization of PM proteins with divergent functions, such as FLAGELLIN-SENSITIVE 2 (FLS2), PIN-FORMED 3 (PIN3), and BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which were associated to clusters and moved at different rates ( McKenna et al, 2019 ).…”