Visualization of cortical microtubule networks in plant cells by live imaging and immunostaining

Abstract

Summary Cortical microtubules (CMTs) play pivotal roles during plant cell growth and division. The organization of CMTs undergoes important changes during different cellular and developmental processes. Here, we describe two methods for the visualization of CMT organization in plant cells using confocal laser scanning microscopy. CMT networks in the outer tissue layers can be directly visualized by live imaging of a fluorescent reporter line, and a protocol combining sectioning and immunostaining is applied for visualization of CMTs throughout tissues. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020 ).

Dark‐induced chloroplast relocation depends on actin filaments in the liverwort Apopellia endiviifolia along with the light‐ and cold‐induced relocations

“…To observe microtubules and actin filaments through the fluorescence of Alexa Fluor 488 in the stained thallus sections and transient expression of Lifeact‐Citrine in the transformed cells of A. endiviifolia , an SP8X confocal laser scanning microscope (Leica Microsystems) was used. For Alexa Fluor 488 fluorescence, observations were obtained with excitation at 488 nm and emission from 500 to 530 nm (Du et al, 2021). For Lifeact‐Citrine observation, Citrine fluorescence was obtained with excitation at 513 nm and emission from 517 to 546 nm (Osaki & Kodama, 2017).…”

“…Detached A. endiviifolia thalli were pretreated with the microtubule depolymerisation inhibitor oryzalin. The untreated and inhibitor‐treated thalli were fixed in fixative (Du et al, 2021). The fixed thalli were then dehydrated and embedded in paraffin as described in the previous study (Yong et al, 2021).…”

“…To observe microtubules in A. endiviifolia cells, immunofluorescence staining of the thallus was performed as previously described with ACTIN-DEPENDENT CHLOROPLAST DARK-POSITIONING RESPONSE | 1823 some modifications (Du et al, 2021). Detached A. endiviifolia thalli were pretreated with the microtubule depolymerisation inhibitor oryzalin.…”

“…Here, the cytoskeleton components involved in the dark‐positioning response of A. endiviifolia was investigated. Visualisation of the microtubules and actin filaments in A. endiviifolia was performed through the immunofluorescence staining and localisation of the fluorescent protein probe, respectively (Du et al, 2021; Kimura & Kodama, 2016). The microtubule and actin filament depolymerisation inhibitors oryzalin and latrunculin A (lat‐A) were used to treat A. endiviifolia thalli.…”

Twisting development, the birth of a potential new gene

“…Alpha-tubulin immunolabeling experiments were conducted following the protocol by 71 with some modifications. Hypocotyls were dissected from six-days old Arabidopsis plants and transferred to 24-well plates to perform fixation, permeabilization, antibody incubation and washing steps.…”