Dynamics and Organization of Cortical Microtubules as Revealed by Superresolution Structured Illumination Microscopy  

Abstract

Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization.

Response to Comment on “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics”

“…To illustrate, the predicted point spread function (PSF) of a reversible saturable/switchable optical (fluorescence) transitions (RESOLFT) microscope under reported conditions (Li ref. 10) of live-cell imaging has a full width at half maximum of 54 nm (Fig. 1E), yet in simulations (Fig.…”

Superresolution live imaging of plant cells using structured illumination microscopy

“…Taking this into account, we recently developed a protocol that successfully allowed us to decipher the subdiffraction details of plant cortical microtubule organization using SIM in living Arabidopsis hypocotyl epidermal cells 22 . Using high-end optics (i.e., high-numerical-aperture (NA) objective, high-refractive-index immersion oil and low-thickness-tolerance coverslips), we were able to image Arabidopsis seedlings that were stably transformed with genetically encoded microtubule markers (i.e., GFP-MBD 23,24 and GFP-TUA6 (ref.…”

“…25)). This allowed us to monitor the dynamics of labeled microtubules at a reasonable temporal resolution 21,22 .…”

Preparation of plants for developmental and cellular imaging by light-sheet microscopy

“…Etiolated seedlings provide an interesting After inserting the aerial part of the seedling into the glass capillary, it was compressed by the air trapped between the piston and the medium surface When introduction of the air-exposed parts of the plant into the glass capillary is concluded, further insertion of the capillary into the solid medium to accommodate the root must be compensated by pulling out the piston of the capillary at the same speed as the capillary is inserted into the medium 1F(iv),7 Sample position is not stable during imaging Solidified medium slides down or retracts up in the glass capillary or syringe; the glass capillary or syringe was not completely dry before use; the block of medium is too large; there is too much air between the block and the piston, or the piston itself is not tight enough Use thoroughly clean and dry sample holders, and pistons with new seals. Do not prepare long agarose blocks with samples and avoid excessive air space between the sample and the piston in glass capillaries or syringes paradigm for analysis of subcellular dynamics, as exemplified by studies on the cortical microtubules 33 . The resolution of the light-sheet microscopy is sufficient for imaging of microtubules in elongated cells of etiolated hypocotyls (Fig.…”