Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics.

Renáta Šnaurová,Pavel Křenek,Olga Šamajová,Pavlína Floková,Tereza Vavrdová,Petra Illešová,George Komis,Jozef Šamaj,Miroslav Ovečka

doi:10.3389/fpls.2020.00693

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 were the methods of choice to follow the dynamics of MAP65-2 in bundles of different complexity. Conclusively, the combination of different superresolution methods allowed for inferences on the distribution and dynamics of MAP65-2 within microtubule bundles of living A. thaliana cells.

Highlights

Microtubules are essential components of the plant cytoskeleton and are crucial for fundamental cellular functions, including cell division, growth and morphogenesis
The purpose of our study, was to report the fine structure of microtubule bundles and track the organization and dynamics of fluorescently tagged MAP65-2 within such bundles. We chose for this purpose three methods, namely, Airyscan confocal laser scanning microscopy (ACLSM), widefield 2D structured illumination microscopy (SIM) and total internal reflection (TIRF)-SIM, which were initially characterized in terms of spatial resolution at the settings used for time-lapsed imaging
When comparing the resolution of TUA6-GFP-labeled individual microtubules visualized by means of ACLSM (Figures 1A,B) or 2D SIM (Figures 1E,F), we found significant differences especially after defining the full-width at half maximum (FWHM) of normalized intensity profiles

Summary

Introduction

Microtubules are essential components of the plant cytoskeleton and are crucial for fundamental cellular functions, including cell division, growth and morphogenesis (reviewed in Panteris and Galatis, 2005; Yamada and Goshima, 2017; Eng and Sampathkumar, 2018; Lazzaro et al, 2018; Sapala et al, 2018). Notable examples include members of the plant FORMIN family (e.g., Sun et al, 2017; Wu and Bezanilla, 2018; Kollárová et al, 2020), members of the ARP2/3 actin nucleation complex (Havelková et al, 2015) and motor proteins of either the kinesin or the myosin superfamilies (e.g., Schneider and Persson, 2015) In this respect kinesins with calponin homology domains such as tobacco KCH1 or cotton KCH2 (e.g., Xu et al, 2009; Buschmann et al, 2010), were found to bind to both cytoskeletal filaments and especially in the case of KCH2, to crosslink actin and microtubules (Xu et al, 2009). Some plant myosins have been found to colocalize with microtubular structures, such as the mitotic spindle (e.g., Sun et al, 2018 for MYOSIN XI) or to directly interact with microtubules (Wu and Bezanilla, 2014 for MYOSIN VIII)

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