Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei.

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

CLASP stabilization of plus ends created by severing promotes microtubule creation and reorientation.

Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP's function in promoting microtubule amplification by severing and array reorientation.

SPR2 protects minus ends to promote severing and reorientation of plant cortical microtubule arrays.

The cortical microtubule arrays of higher plants are organized without centrosomes and feature treadmilling polymers that are dynamic at both ends. The control of polymer end stability is fundamental for the assembly and organization of cytoskeletal arrays, yet relatively little is understood about how microtubule minus ends are controlled in acentrosomal microtubule arrays, and no factors have been identified that act at the treadmilling minus ends in higher plants. Here, we identify Arabidopsis thaliana SPIRAL2 (SPR2) as a protein that tracks minus ends and protects them against subunit loss. SPR2 function is required to facilitate the rapid reorientation of plant cortical arrays as stimulated by light perception, a process that is driven by microtubule severing to create a new population of microtubules. Quantitative live-cell imaging and computer simulations reveal that minus protection by SPR2 acts by an unexpected mechanism to promote the lifetime of potential SPR2 severing sites, increasing the likelihood of severing and thus the rapid amplification of the new microtubule array.

How selective severing by katanin promotes order in the plant cortical microtubule array.

Plant morphogenesis requires differential and often asymmetric growth. A key role in controlling anisotropic expansion of individual cells is played by the cortical microtubule array. Although highly organized, the array can nevertheless rapidly change in response to internal and external cues. Experiments have identified the microtubule-severing enzyme katanin as a central player in controlling the organizational state of the array. Katanin action is required both for normal alignment and the adaptation of array orientation to mechanical, environmental, and developmental stimuli. How katanin fulfills its controlling role, however, remains poorly understood. On the one hand, from a theoretical perspective, array ordering depends on the "weeding out" of discordant microtubules through frequent catastrophe-inducing collisions among microtubules. Severing would reduce average microtubule length and lifetime, and consequently weaken the driving force for alignment. On the other hand, it has been suggested that selective severing at microtubule crossovers could facilitate the removal of discordant microtubules. Here we show that this apparent conflict can be resolved by systematically dissecting the role of all of the relevant interactions in silico. This procedure allows the identification of the sufficient and necessary conditions for katanin to promote array alignment, stresses the critical importance of the experimentally observed selective severing of the "crossing" microtubule at crossovers, and reveals a hitherto not appreciated role for microtubule bundling. We show how understanding the underlying mechanism can aid with interpreting experimental results and designing future experiments.

The microtubule plus-end tracking proteins SPR1 and EB1b interact to maintain polar cell elongation and directional organ growth in Arabidopsis.

The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1 proteins, setting SPR1 apart from most studied +TIPs in animals and fungi. Moreover, we found that the severity of defects in microtubule dynamics in spr1 eb1b mutant hypocotyl cells correlated well with the severity of growth defects. These data indicate that SPR1 and EB1b have complex interactions as they load onto microtubule plus ends and direct polar cell expansion and organ growth in response to directional cues.

A mechanism for reorientation of cortical microtubule arrays driven by microtubule severing.

Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation.

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis.

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is to drive organization of cellulose microfibrils by guiding the trajectories of active cellulose synthase (CESA) complexes in the plasma membrane, thus orienting nascent microfibrils. Here we provide evidence that cortical microtubules also position the delivery of CESA complexes to the plasma membrane and interact with small CESA-containing compartments by a mechanism that permits motility driven by microtubule depolymerization. The association of CESA compartments with cortical microtubules was greatly enhanced during osmotic stress and other treatments that limit cellulose synthesis. On recovery from osmotic stress, delivery of CESA complexes to the plasma membrane was observed in association with microtubule-tethered compartments. These results reveal multiple functions for the microtubule cortical array in organizing CESA in the cell cortex.