Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Summary

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Abstract

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.

Introduction

Plant cells are surrounded by an extracellular matrix of polysaccharides and glycoproteins that mechanically resembles a fiber reinforced composite material capable of dynamically changing its mechanical properties¹. Growth in plant cells is driven by the uptake of water into the cell, which results in a concomitant buildup of tensile forces on the cell wall. In response to such forces, modifications to the physical state of the cell wall allows for cell expansion. Cells with primary walls are capable of undergoing rapid growth compared to secondary cell wall containing cells mainly due to differences in the chemical composition of the polysacc....

Protocol

1. Plant growth

1. Sow Arabidopsis seeds expressing microtubule binding domain fused with green fluorescent protein (MBD-GFP)¹⁰ on soil and keep in long day (16 h day /8 h night), 20 °C/6 °C conditions for 1 week for germination.
2. After germination, transfer seedlings to new pots with sufficient growth space to allow robust vegetative growth. Keep plants in short day (8 h day /16 h night), 20 °C/16 °C conditions for 3-5 weeks.
3. Transfer plants to long day (16 h day /8 h night), 20 °C/16 °C conditions and keep until plants bolt (2-3 weeks). Allow the inflorescence to grow up to 2-....

Results

Figure 1 shows typical projection images obtained from MBD-GFP lines with cells at the center of the dome containing disorganized cortical microtubules, and cells at the periphery having a circumferential distribution (Figure 1A,B), whereas the boundary domain cells contain cortical microtubules aligned parallel to the cell's long axis. These observations show differences in the spatial distribution of cortical m.......

Discussion

The assessment of mechanical signal transduction events is crucial to identify molecular regulators involved in the mechano-perception and transduction pathways. The protocol described here provides a quantitative view of such events by using the cortical microtubule response as a readout for such a process in Arabidopsis SAMs. The procedure described here is routinely used in several studies in various tissue types^16,17,18

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FibrilTool			Boudaoud, A. et al., Nat Protoc. 2014
FIJI			Schindelin, J. et al., Nat Methods. 2012
glycine	Merck	1.04201.1000
Leica SP8 confocal microscope	Leica	DM6000 CS
MAP4-GFP			Marc, J. et al., Plant Cell 1998
micropore tape	Leukopor	02482-00
MorphographX			Strauss, S. et al., Methods Mol Biol. 2019
myo-inositol	Sigma	I5125
N6-benzyladenine	Sigma	B3408
nicotinic acid	Sigma	N4126
plastic hinged box	Electron microscopy sciences	64312
PPM (Plant Preservative Mixture)	Plant Cell Technology	PPM
Propidium iodide	Sigma	P4864
pyridoxine hydrochloride	Sigma	P9755
SURFCUT			Erguvan, O. et al., BMC Biol. 2019
thiamine hydrochloride	Sigma	T4625